WorldWideScience

Sample records for aptamer-tagged small rna

  1. Small RNA in rice genome

    Institute of Scientific and Technical Information of China (English)

    王凯; 朱小蓬; 钟兰; 陈润生

    2002-01-01

    Rice has many characteristics of a model plant. The recent completion of the draft of the rice genome represents an important advance in our knowledge of plant biology and also has an important contribution to the understanding of general genomic evolution. Besides the rice genome finishing map, the next urgent step for rice researchers is to annotate the genes and noncoding functional sequences. The recent work shows that noncoding RNAs (ncRNAs) play significant roles in biological systems. We have explored all the known small RNAs (a kind of ncRNA) within rice genome and other six species sequences, including Arabidopsis, maize, yeast, worm, mouse and pig. As a result we find 160 out of 552 small RNAs (sRNAs) in database have homologs in 108 rice scaffolds, and almost all of them (99.41%) locate in intron regions of rice by gene predication. 19 sRNAs only appear in rice. More importantly, we find two special U14 sRNAs: one is located in a set of sRNA ZMU14SNR9(s) which only appears in three plants, 86% sequences of them can be compared as the same sequence in rice, Arabidopsis and maize; the other conserved sRNA XLHS7CU14 has a segment which appears in almost all these species from plants to animals. All these results indicate that sRNA do not have evident borderline between plants and animals.

  2. Switching off small RNA regulation with trap-mRNA

    DEFF Research Database (Denmark)

    Overgaard, Martin; Johansen, Jesper; Møller-Jensen, Jakob;

    2009-01-01

    Small non-coding regulatory RNAs in bacteria have been shown predominantly to be tightly regulated at the level of transcription initiation, and sRNAs that function by an antisense mechanism on trans-encoded target mRNAs have been shown or predicted to act stoichiometrically. Here we show that Mic......M, which silences the expression of an outer membrane protein, YbfM under most growth conditions, does not become destabilized by target mRNA overexpression, indicating that the small RNA regulator acts catalytically. Furthermore, our regulatory studies suggested that control of micM expression is unlikely...... to operate at the level of transcription initiation. By employing a highly sensitive genetic screen we uncovered a novel RNA-based regulatory principle in which induction of a trap-mRNA leads to selective degradation of a small regulatory RNA molecule, thereby abolishing the sRNA-based silencing of its...

  3. Small RNA combination therapy for lung cancer

    Science.gov (United States)

    Xue, Wen; Dahlman, James E.; Tammela, Tuomas; Khan, Omar F.; Sood, Sabina; Dave, Apeksha; Cai, Wenxin; Chirino, Leilani M.; Yang, Gillian R.; Bronson, Roderick; Crowley, Denise G.; Sahay, Gaurav; Schroeder, Avi; Langer, Robert; Anderson, Daniel G.; Jacks, Tyler

    2014-01-01

    MicroRNAs (miRNAs) and siRNAs have enormous potential as cancer therapeutics, but their effective delivery to most solid tumors has been difficult. Here, we show that a new lung-targeting nanoparticle is capable of delivering miRNA mimics and siRNAs to lung adenocarcinoma cells in vitro and to tumors in a genetically engineered mouse model of lung cancer based on activation of oncogenic Kirsten rat sarcoma viral oncogene homolog (Kras) and loss of p53 function. Therapeutic delivery of miR-34a, a p53-regulated tumor suppressor miRNA, restored miR-34a levels in lung tumors, specifically down-regulated miR-34a target genes, and slowed tumor growth. The delivery of siRNAs targeting Kras reduced Kras gene expression and MAPK signaling, increased apoptosis, and inhibited tumor growth. The combination of miR-34a and siRNA targeting Kras improved therapeutic responses over those observed with either small RNA alone, leading to tumor regression. Furthermore, nanoparticle-mediated small RNA delivery plus conventional, cisplatin-based chemotherapy prolonged survival in this model compared with chemotherapy alone. These findings demonstrate that RNA combination therapy is possible in an autochthonous model of lung cancer and provide preclinical support for the use of small RNA therapies in patients who have cancer. PMID:25114235

  4. Small regulatory RNA and Legionella pneumophila

    Directory of Open Access Journals (Sweden)

    Sebastien P Faucher

    2011-05-01

    Full Text Available Legionella pneumophila is a gram-negative bacterial species that is ubiquitous in almost any aqueous environment. It is the agent of Legionnaires’ disease, an acute and often under-reported form of pneumonia. In mammals, L. pneumophila replicates inside macrophages within a modified vacuole. Many protein regulators have been identified that control virulence-related properties, including RpoS, LetA/LetS and PmrA/PmrB. In the past few years, the importance of regulation of virulence factors by small regulatory RNA has been increasingly appreciated. This is also the case in L. pneumophila where three sRNAs (RsmY, RsmZ and 6S RNA were recently shown to be important determinants of virulence regulation and 79 actively transcribed sRNAs were identified. In this review we describe current knowledge about sRNAs and their regulatory properties and how this relates to the known regulatory systems of L. pneumophila. We also provide a model for sRNA-mediated control of gene expression that serves as a framework for understanding the regulation of virulence-related properties of L. pneumophila.

  5. Endogenous Small RNA Clusters in Plants

    Institute of Scientific and Technical Information of China (English)

    Yong-Xin Liu; Meng Wang; Xiu-Jie Wang

    2014-01-01

    In plants, small RNAs (sRNAs) usually refer to non-coding RNAs (ncRNAs) with lengths of 20-24 nucleotides. sRNAs are involved in the regulation of many essential processes related to plant development and environmental responses. sRNAs in plants are mainly grouped into microRNAs (miRNAs) and small interfering RNAs (siRNAs), and the latter can be further classified into trans-acting siRNAs (ta-siRNAs), repeat-associated siRNAs (ra-siRNAs), natural anti-sense siRNAs (nat-siRNAs), etc. Many sRNAs exhibit a clustered distribution pattern in the genome. Here, we summarize the features and functions of cluster-distributed sRNAs, aimed to not only provide a thorough picture of sRNA clusters (SRCs) in plants, but also shed light on the identification of new classes of functional sRNAs.

  6. SnoRNA derived small RNA%核仁小RNA源性小RNA

    Institute of Scientific and Technical Information of China (English)

    徐玲; 胡娜

    2010-01-01

    最新研究结果表明,一些与RNA介导基因沉默相关的小RNA由核仁小RNA(small nucleolar RNA,snoRNA)加工产生,这种小RNA被称为核仁小RNA源性小RNA(snoRNA derived small RNA,sdRNA).sdRNA现象分布物种广;涉及的snoRNA种类全,数量多;产生的小RNA分子大小不一,数量、种类多.表明这种小RNA在生物中存任着广泛的普遍性.sdRNA的发现拓展了snoRNA的功能,揭示了snoRNA与RNA介导的基因沉默之间的紧密关系,增强了SnoRNA在RNA调控网络中的重要性,并为进一步研究RNA调控网络开启了一扇门.

  7. Small Rna Regulatory Networks In Pseudomonas Putida

    DEFF Research Database (Denmark)

    Bojanovic, Klara; Long, Katherine

    2015-01-01

    chemicals and has a potential to be used as an efficient cell factory for various products. P. putida KT2240 is a genome-sequenced strain and a well characterized pseudomonad. Our major aim is to identify small RNA molecules (sRNAs) and their regulatory networks. A previous study has identified 37 sRNAs...... in this strain, while in other pseudomonads many more sRNAs have been found so far.P. putida KT2440 has been grown in different conditions which are likely to be encountered in industrial fermentations with the aim of using sRNAs for generation of improved cell factories. For that, cells have been grown in LB...... and harvested in different growth phases, as well as osmotic, membrane and oxidative stress conditions. RNA sequencing data has been analysed with the open source software system Rockhopper, and it has revealed over 180 putative sRNAs. Most of them (86%) seem to be novel and uncharacterized. The majority...

  8. Endogenous Small RNA Clusters in Plants

    Directory of Open Access Journals (Sweden)

    Yong-Xin Liu

    2014-04-01

    Full Text Available In plants, small RNAs (sRNAs usually refer to non-coding RNAs (ncRNAs with lengths of 20–24 nucleotides. sRNAs are involved in the regulation of many essential processes related to plant development and environmental responses. sRNAs in plants are mainly grouped into microRNAs (miRNAs and small interfering RNAs (siRNAs, and the latter can be further classified into trans-acting siRNAs (ta-siRNAs, repeat-associated siRNAs (ra-siRNAs, natural anti-sense siRNAs (nat-siRNAs, etc. Many sRNAs exhibit a clustered distribution pattern in the genome. Here, we summarize the features and functions of cluster-distributed sRNAs, aimed to not only provide a thorough picture of sRNA clusters (SRCs in plants, but also shed light on the identification of new classes of functional sRNAs.

  9. RNA interference and small interference RNA%RNA干涉和小干涉RNA

    Institute of Scientific and Technical Information of China (English)

    张芹; 王勇; 钱凯先

    2005-01-01

    RNA干涉(RNA interference.RNAi)可以抑制诸多真核基因的表达,小干涉RNA(small intmference RNA,siRNA)是RNA干涉的引发物,不同的siRNA可以引导不同水平的RNA干涉,不同种细胞中siRNA的寡核苷酸链的性质也有很大的不同.非编码RNA中的微小RNA(micnoRNA,miRNA)与siRNA在结构及作用方式等方面有很多区别和联系.RNAi的作用机制大致有两种:在Drosophila中的一般作用模式和在C.elegans中的扩大作用模式.利用RNA干涉技术可以通过抑制基因的表达来研究其功能;RNA干涉作为基因治疗的工具,在肝炎和AIDS的治疗过程中有一定的作用.RNAi的高特异性、高效性、放大效应和无细胞特异性等特点在科研中得到了广泛的应用.

  10. SMALL NONCODING RNA AS PERSPECTIVE BIOMARKERS: BIOGENESIS AND THERAPEUTIC STRATIGIES

    Directory of Open Access Journals (Sweden)

    V. V. Tiguntsev

    2016-01-01

    Full Text Available The review presents the opening story, biogenesis and functions of basic groups of human’s small noncoding RNA: microRNA and short interfering RNA. These RNA molecules inhibit gene expression during translation by RNA interference. It was found that microRNA and short interfering RNA circulate in bioliquids and can serve as biomarkers of different human diseases because of its conservative sequences, tissue specificity and resistance to environment factors. The paper considers techniques to study noncoding RNA (cloning, bioinformatics analysis and hybridization methods: northern-blotting, RT-PCR, in situ hybridization, microarray analysis, reporter analysis. Possible noncoding RNA-targeted therapy can suggest delivery microRNA, anti-microRNA, antagomirs, microRNAsponges to target tissue by virus molecules, liposomes or nanoparticles. 

  11. Small catalytic RNA: Structure, function and application

    Energy Technology Data Exchange (ETDEWEB)

    Monforte, J.A.

    1991-04-01

    We have utilized a combination of photochemical cross-linking techniques and site-directed mutagenesis to obtain secondary and tertiary structure information for the self-cleaving, self-ligating subsequence of RNA from the negative strand of Satellite Tobacco Ringspot Virus. We have found that the helical regions fold about a hinge to promoting four different possible tertiary interactions, creating a molecular of similar shape to a paperclip. A model suggesting that the ``paperclip`` and ``hammerhead`` RNAs share a similar three dimensional structure is proposed. We have used a self-cleaving RNA molecule related to a subsequence of plant viroids, a ``hammerhead,`` to study the length-dependent folding of RNA produced during transcription by RNA polymerase. We have used this method to determine the length of RNA sequestered within elongating E. coli and T7 RNA polymerase complexes. The data show that for E. coli RNA polymerase 12{plus_minus}1 nucleotides are sequestered within the ternary complex, which is consistent with the presence of an RNA-DNA hybrid within the transcription bubble, as proposed by others. The result for T7 RNA polymerase differs from E. coli RNA polymerase, with only 10{plus_minus}1 nucleotides sequestered within the ternary complex, setting a new upper limit for the minimum RNA-DNA required for a stable elongating complex. Comparisons between E. coli and T7 RNA polymerase are made. The relevance of the results to models or transcription termination, abortive initiation, and initiation to elongation mode transitions are discussed.

  12. Small catalytic RNA: Structure, function and application

    Energy Technology Data Exchange (ETDEWEB)

    Monforte, J.A.

    1991-04-01

    We have utilized a combination of photochemical cross-linking techniques and site-directed mutagenesis to obtain secondary and tertiary structure information for the self-cleaving, self-ligating subsequence of RNA from the negative strand of Satellite Tobacco Ringspot Virus. We have found that the helical regions fold about a hinge to promoting four different possible tertiary interactions, creating a molecular of similar shape to a paperclip. A model suggesting that the paperclip'' and hammerhead'' RNAs share a similar three dimensional structure is proposed. We have used a self-cleaving RNA molecule related to a subsequence of plant viroids, a hammerhead,'' to study the length-dependent folding of RNA produced during transcription by RNA polymerase. We have used this method to determine the length of RNA sequestered within elongating E. coli and T7 RNA polymerase complexes. The data show that for E. coli RNA polymerase 12{plus minus}1 nucleotides are sequestered within the ternary complex, which is consistent with the presence of an RNA-DNA hybrid within the transcription bubble, as proposed by others. The result for T7 RNA polymerase differs from E. coli RNA polymerase, with only 10{plus minus}1 nucleotides sequestered within the ternary complex, setting a new upper limit for the minimum RNA-DNA required for a stable elongating complex. Comparisons between E. coli and T7 RNA polymerase are made. The relevance of the results to models or transcription termination, abortive initiation, and initiation to elongation mode transitions are discussed.

  13. The evolving world of small RNAs from RNA viruses.

    Science.gov (United States)

    Li, Mei-Ling; Weng, Kuo-Feng; Shih, Shin-Ru; Brewer, Gary

    2016-09-01

    RNA virus infection in plants and invertebrates can produce virus-derived small RNAs. These RNAs share features with host endogenous small interfering RNAs (siRNAs). They can potentially mediate RNA interference (RNAi) and related RNA silencing pathways, resulting in specific antiviral defense. Although most RNA silencing components such as Dicer, Ago2, and RISC are conserved among eukaryotic hosts, whether RNA virus infection in mammals can generate functional small RNAs that act in antiviral defense remains under discussion. Here, we review recent studies on the molecular and biochemical features of viral siRNAs and other virus-derived small RNAs from infected plants, arthropods, nematodes, and vertebrates and discuss the genetic pathways for their biogenesis and their roles in antiviral activity. WIREs RNA 2016, 7:575-588. doi: 10.1002/wrna.1351 For further resources related to this article, please visit the WIREs website. PMID:27046163

  14. Preparation of Multiplexed Small RNA Libraries From Plants

    OpenAIRE

    Gilbert, Kerrigan B.; Fahlgren, Noah; Kasschau, Kristin D.; Chapman, Elisabeth J.; Carrington, James C.; Carbonell, Alberto

    2014-01-01

    High-throughput sequencing is a powerful tool for exploring small RNA populations in plants. The ever-increasing output from an Illumina Sequencing System allows for multiplexing multiple samples while still obtaining sufficient data for small RNA discovery and characterization. Here we describe a protocol for generating multiplexed small RNA libraries for sequencing up to 12 samples in one lane of an Illumina HiSeq System single-end, 50 base pair run. RNA ligases are used to add the 3′ and 5...

  15. Regulation of small RNA stability: methylation and beyond

    Institute of Scientific and Technical Information of China (English)

    Lijuan Ji; Xuemei Chen

    2012-01-01

    As central components of RNA silencing,small RNAs play diverse and important roles in many biological processes in eukaryotes.Aberrant reduction or elevation in the levels of small RNAs is associated with many developmental and physiological defects.The in vivo levels of small RNAs are precisely regulated through modulating the rates of their biogenesis and turnover.2'-O-methylation on the 3' terminal ribose is a major mechanism that increases the stability of small RNAs.The small RNA methyltransferase HUA ENHANCER1 (HEN1) and its homologs methylate microRNAs and small interfering RNAs (siRNAs) in plants,Piwi-interacting RNAs (piRNAs) in animals,and siRNAs in Drosophila.3' nucleotide addition,especially uridylation,and 3'-5' exonucleolytic degradation are major mechanisms that turnover small RNAs.Other mechanisms impacting small RNA stability include complementary RNAs,cis-elements in small RNA sequences and RNA-binding proteins.Investigations are ongoing to further understand how small RNA stability impacts their accumulation in vivo in order to improve the utilization of RNA silencing in biotechnology and therapeutic applications.

  16. Modeling Small Noncanonical RNA Motifs with the Rosetta FARFAR Server.

    Science.gov (United States)

    Yesselman, Joseph D; Das, Rhiju

    2016-01-01

    Noncanonical RNA motifs help define the vast complexity of RNA structure and function, and in many cases, these loops and junctions are on the order of only ten nucleotides in size. Unfortunately, despite their small size, there is no reliable method to determine the ensemble of lowest energy structures of junctions and loops at atomic accuracy. This chapter outlines straightforward protocols using a webserver for Rosetta Fragment Assembly of RNA with Full Atom Refinement (FARFAR) ( http://rosie.rosettacommons.org/rna_denovo/submit ) to model the 3D structure of small noncanonical RNA motifs for use in visualizing motifs and for further refinement or filtering with experimental data such as NMR chemical shifts. PMID:27665600

  17. RNA targeting by small molecules: Binding of protoberberine, benzophenanthridine and aristolochia alkaloids to various RNA structures

    Indian Academy of Sciences (India)

    Gopinatha Suresh Kumar

    2012-07-01

    Studies on RNA targeting by small molecules to specifically control certain cellular functions is an area of remarkable current interest. For this purpose, a basic understanding of the molecular aspects of the interaction of small molecules with various RNA structures is essential. Alkaloids are a group of natural products with potential therapeutic utility, and very recently, their interaction with many RNA structures have been reported. Especially noteworthy are the protoberberines and aristolochia alkaloids distributed widely in many botanical families. Many of the alkaloids of these group exhibit excellent binding affinity to many RNA structures that may be exploited to develop RNA targeted therapeutics. This review attempts to present the current status on the understanding of the interaction of these alkaloids with various RNA structures, mainly highlighting the biophysical aspects.

  18. Unique small RNA signatures uncovered in the tammar wallaby genome

    Directory of Open Access Journals (Sweden)

    Lindsay James

    2012-10-01

    Full Text Available Abstract Background Small RNAs have proven to be essential regulatory molecules encoded within eukaryotic genomes. These short RNAs participate in a diverse array of cellular processes including gene regulation, chromatin dynamics and genome defense. The tammar wallaby, a marsupial mammal, is a powerful comparative model for studying the evolution of regulatory networks. As part of the genome sequencing initiative for the tammar, we have explored the evolution of each of the major classes of mammalian small RNAs in an Australian marsupial for the first time, including the first genome-scale analysis of the newest class of small RNAs, centromere repeat associated short interacting RNAs (crasiRNAs. Results Using next generation sequencing, we have characterized the major classes of small RNAs, micro (mi RNAs, piwi interacting (pi RNAs, and the centromere repeat associated short interacting (crasi RNAs in the tammar. We examined each of these small RNA classes with respect to the newly assembled tammar wallaby genome for gene and repeat features, salient features that define their canonical sequences, and the constitution of both highly conserved and species-specific members. Using a combination of miRNA hairpin predictions and co-mapping with miRBase entries, we identified a highly conserved cluster of miRNA genes on the X chromosome in the tammar and a total of 94 other predicted miRNA producing genes. Mapping all miRNAs to the tammar genome and comparing target genes among tammar, mouse and human, we identified 163 conserved target genes. An additional nine genes were identified in tammar that do not have an orthologous miRNA target in human and likely represent novel miRNA-regulated genes in the tammar. A survey of the tammar gonadal piRNAs shows that these small RNAs are enriched in retroelements and carry members from both marsupial and tammar-specific repeat classes. Lastly, this study includes the first in-depth analyses of the newly

  19. Functionalization of an Antisense Small RNA

    Science.gov (United States)

    Rodrigo, Guillermo; Prakash, Satya; Cordero, Teresa; Kushwaha, Manish; Jaramillo, Alfonso

    2016-01-01

    In order to explore the possibility of adding new functions to preexisting genes, we considered a framework of riboregulation. We created a new riboregulator consisting of the reverse complement of a known riboregulator. Using computational design, we engineered a cis-repressing 5′ untranslated region that can be activated by this new riboregulator. As a result, both RNAs can orthogonally trans-activate translation of their cognate, independent targets. The two riboregulators can also repress each other by antisense interaction, although not symmetrically. Our work highlights that antisense small RNAs can work as regulatory agents beyond the antisense paradigm and that, hence, they could be interfaced with other circuits used in synthetic biology. PMID:26756967

  20. Current preclinical small interfering RNA (siRNA)-based conjugate systems for RNA therapeutics.

    Science.gov (United States)

    Lee, Soo Hyeon; Kang, Yoon Young; Jang, Hyo-Eun; Mok, Hyejung

    2016-09-01

    Recent promising clinical results of RNA therapeutics have drawn big attention of academia and industries to RNA therapeutics and their carrier systems. To improve their feasibility in clinics, systemic evaluations of currently available carrier systems under clinical trials and preclinical studies are needed. In this review, we focus on recent noticeable preclinical studies and clinical results regarding siRNA-based conjugates for clinical translations. Advantages and drawbacks of siRNA-based conjugates are discussed, compared to particle-based delivery systems. Then, representative siRNA-based conjugates with aptamers, peptides, carbohydrates, lipids, polymers, and nanostructured materials are introduced. To improve feasibility of siRNA conjugates in preclinical studies, several considerations for the rational design of siRNA conjugates in terms of cleavability, immune responses, multivalent conjugations, and mechanism of action are also presented. Lastly, we discuss lessons from previous preclinical and clinical studies related to siRNA conjugates and perspectives of their clinical applications. PMID:26514375

  1. Silent no more: Endogenous small RNA pathways promote gene expression

    OpenAIRE

    Wedeles, Christopher J; Wu, Monica Z.; Claycomb, Julie M.

    2014-01-01

    Endogenous small RNA pathways related to RNA interference (RNAi) play a well-documented role in protecting host genomes from the invasion of foreign nucleic acids. In C. elegans, the PIWI type Argonaute, PRG-1, through an association with 21U-RNAs, mediates a genome surveillance process by constantly scanning the genome for potentially deleterious invading elements. Upon recognition of foreign nucleic acids, PRG-1 initiates a cascade of cytoplasmic and nuclear events that results in heritable...

  2. Mapping the small RNA content of simian immunodeficiency virions (SIV.

    Directory of Open Access Journals (Sweden)

    Markus Brameier

    Full Text Available Recent evidence indicates that regulatory small non-coding RNAs are not only components of eukaryotic cells and vesicles, but also reside within a number of different viruses including retroviral particles. Using ultra-deep sequencing we have comprehensively analyzed the content of simian immunodeficiency virions (SIV, which were compared to mock-control preparations. Our analysis revealed that more than 428,000 sequence reads matched the SIV mac239 genome sequence. Among these we could identify 12 virus-derived small RNAs (vsRNAs that were highly abundant. Beside known retrovirus-enriched small RNAs, like 7SL-RNA, tRNA(Lys3 and tRNA(Lys isoacceptors, we also identified defined fragments derived from small ILF3/NF90-associated RNA snaR-A14, that were enriched more than 50 fold in SIV. We also found evidence that small nucleolar RNAs U2 and U12 were underrepresented in the SIV preparation, indicating that the relative number or the content of co-isolated exosomes was changed upon infection. Our comprehensive atlas of SIV-incorporated small RNAs provides a refined picture of the composition of retrovirions, which gives novel insights into viral packaging.

  3. Delivery of small interfering RNA (siRNA) using the sleeping beauty transposon.

    Science.gov (United States)

    Fletcher, Bradley S

    2010-11-01

    RNA interference (RNAi) is an evolutionarily conserved process that silences gene expression through double-stranded RNA species in a sequence-specific manner. Small interfering RNAs (siRNAs) can promote sequence-specific degradation and/or translational repression of target RNA by activation of the RNA-induced silencing complex (RISC). Traditionally, silencing in mammalian cells had been achieved by transfection of synthetically derived siRNA duplexes, resulting in transient gene suppression of the target sequence. As the technology was advanced, inhibitory short-hairpin-shaped RNAs (shRNAs) could be produced by transcription from RNA polymerase-III (pol-III)-driven promoters, such as H1, U6, or cytomegalovirus (CMV)-enhanced pol III promoters. Following transcription, the shRNAs are processed by the enzyme Dicer into active siRNA. This approach allows for the continuous production of siRNA within cells using a DNA template and offers increased options for delivery of the pol-III-driven transcriptional units. A number of different viral vectors, as well as plasmid DNAs, have been utilized to deliver shRNA to mammalian cells. Here, the Tc1/mariner DNA transposon Sleeping Beauty (SB) is used as a tool to deliver shRNA-encoding transcriptional units. The SB transposon system uses a "cut-and-paste" mechanism to insert the transposon into random TA dinucleotides within the target genome. The shRNAs are then processed and used for gene knockdown. PMID:21041394

  4. Transcriptome and small RNA deep sequencing reveals deregulation of miRNA biogenesis in human glioma.

    Science.gov (United States)

    Moore, Lynette M; Kivinen, Virpi; Liu, Yuexin; Annala, Matti; Cogdell, David; Liu, Xiuping; Liu, Chang-Gong; Sawaya, Raymond; Yli-Harja, Olli; Shmulevich, Ilya; Fuller, Gregory N; Zhang, Wei; Nykter, Matti

    2013-02-01

    Altered expression of oncogenic and tumour-suppressing microRNAs (miRNAs) is widely associated with tumourigenesis. However, the regulatory mechanisms underlying these alterations are poorly understood. We sought to shed light on the deregulation of miRNA biogenesis promoting the aberrant miRNA expression profiles identified in these tumours. Using sequencing technology to perform both whole-transcriptome and small RNA sequencing of glioma patient samples, we examined precursor and mature miRNAs to directly evaluate the miRNA maturation process, and examined expression profiles for genes involved in the major steps of miRNA biogenesis. We found that ratios of mature to precursor forms of a large number of miRNAs increased with the progression from normal brain to low-grade and then to high-grade gliomas. The expression levels of genes involved in each of the three major steps of miRNA biogenesis (nuclear processing, nucleo-cytoplasmic transport, and cytoplasmic processing) were systematically altered in glioma tissues. Survival analysis of an independent data set demonstrated that the alteration of genes involved in miRNA maturation correlates with survival in glioma patients. Direct quantification of miRNA maturation with deep sequencing demonstrated that deregulation of the miRNA biogenesis pathway is a hallmark for glioma genesis and progression.

  5. Identification of Bacterial Small RNAs by RNA Sequencing

    DEFF Research Database (Denmark)

    Gómez Lozano, María; Marvig, Rasmus Lykke; Molin, Søren;

    2014-01-01

    Small regulatory RNAs (sRNAs) in bacteria are known to modulate gene expression and control a variety of processes including metabolic reactions, stress responses, and pathogenesis in response to environmental signals. A method to identify bacterial sRNAs on a genome-wide scale based on RNA...

  6. Re-inspection of small RNA sequence datasets reveals several novel human miRNA genes.

    Directory of Open Access Journals (Sweden)

    Thomas Birkballe Hansen

    Full Text Available BACKGROUND: miRNAs are key players in gene expression regulation. To fully understand the complex nature of cellular differentiation or initiation and progression of disease, it is important to assess the expression patterns of as many miRNAs as possible. Thereby, identifying novel miRNAs is an essential prerequisite to make possible a comprehensive and coherent understanding of cellular biology. METHODOLOGY/PRINCIPAL FINDINGS: Based on two extensive, but previously published, small RNA sequence datasets from human embryonic stem cells and human embroid bodies, respectively [1], we identified 112 novel miRNA-like structures and were able to validate miRNA processing in 12 out of 17 investigated cases. Several miRNA candidates were furthermore substantiated by including additional available small RNA datasets, thereby demonstrating the power of combining datasets to identify miRNAs that otherwise may be assigned as experimental noise. CONCLUSIONS/SIGNIFICANCE: Our analysis highlights that existing datasets are not yet exhaustedly studied and continuous re-analysis of the available data is important to uncover all features of small RNA sequencing.

  7. Small RNA transcriptomes of mangroves evolve adaptively in extreme environments.

    Science.gov (United States)

    Wen, Ming; Lin, Xingqin; Xie, Munan; Wang, Yushuai; Shen, Xu; Liufu, Zhongqi; Wu, Chung-I; Shi, Suhua; Tang, Tian

    2016-01-01

    MicroRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs) are key players in plant stress responses. Here, we present the sRNA transcriptomes of mangroves Bruguiera gymnorrhiza and Kandelia candel. Comparative computational analyses and target predictions revealed that mangroves exhibit distinct sRNA regulatory networks that differ from those of glycophytes. A total of 32 known and three novel miRNA families were identified. Conserved and mangrove-specific miRNA targets were predicted; the latter were widely involved in stress responses. The known miRNAs showed differential expression between the mangroves and glycophytes, reminiscent of the adaptive stress-responsive changes in Arabidopsis. B. gymnorrhiza possessed highly abundant but less conserved TAS3 trans-acting siRNAs (tasiRNAs) in addition to tasiR-ARFs, with expanded potential targets. Our results indicate that the evolutionary alteration of sRNA expression levels and the rewiring of sRNA-regulatory networks are important mechanisms underlying stress adaptation. We also identified sRNAs that are involved in salt and/or drought tolerance and nutrient homeostasis as possible contributors to mangrove success in stressful environments.

  8. Global Mapping of Small RNA-Target Interactions in Bacteria.

    Science.gov (United States)

    Melamed, Sahar; Peer, Asaf; Faigenbaum-Romm, Raya; Gatt, Yair E; Reiss, Niv; Bar, Amir; Altuvia, Yael; Argaman, Liron; Margalit, Hanah

    2016-09-01

    Small RNAs (sRNAs) associated with the RNA chaperon protein Hfq are key posttranscriptional regulators of gene expression in bacteria. Deciphering the sRNA-target interactome is an essential step toward understanding the roles of sRNAs in the cellular networks. We developed a broadly applicable methodology termed RIL-seq (RNA interaction by ligation and sequencing), which integrates experimental and computational tools for in vivo transcriptome-wide identification of interactions involving Hfq-associated sRNAs. By applying this methodology to Escherichia coli we discovered an extensive network of interactions involving RNA pairs showing sequence complementarity. We expand the ensemble of targets for known sRNAs, uncover additional Hfq-bound sRNAs encoded in various genomic regions along with their trans encoded targets, and provide insights into binding and possible cycling of RNAs on Hfq. Comparison of the sRNA interactome under various conditions has revealed changes in the sRNA repertoire as well as substantial re-wiring of the network between conditions. PMID:27588604

  9. Small RNA transcriptomes of mangroves evolve adaptively in extreme environments.

    Science.gov (United States)

    Wen, Ming; Lin, Xingqin; Xie, Munan; Wang, Yushuai; Shen, Xu; Liufu, Zhongqi; Wu, Chung-I; Shi, Suhua; Tang, Tian

    2016-01-01

    MicroRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs) are key players in plant stress responses. Here, we present the sRNA transcriptomes of mangroves Bruguiera gymnorrhiza and Kandelia candel. Comparative computational analyses and target predictions revealed that mangroves exhibit distinct sRNA regulatory networks that differ from those of glycophytes. A total of 32 known and three novel miRNA families were identified. Conserved and mangrove-specific miRNA targets were predicted; the latter were widely involved in stress responses. The known miRNAs showed differential expression between the mangroves and glycophytes, reminiscent of the adaptive stress-responsive changes in Arabidopsis. B. gymnorrhiza possessed highly abundant but less conserved TAS3 trans-acting siRNAs (tasiRNAs) in addition to tasiR-ARFs, with expanded potential targets. Our results indicate that the evolutionary alteration of sRNA expression levels and the rewiring of sRNA-regulatory networks are important mechanisms underlying stress adaptation. We also identified sRNAs that are involved in salt and/or drought tolerance and nutrient homeostasis as possible contributors to mangrove success in stressful environments. PMID:27278626

  10. Equilibrium self-assembly of small RNA viruses

    Science.gov (United States)

    Bruinsma, R. F.; Comas-Garcia, M.; Garmann, R. F.; Grosberg, A. Y.

    2016-03-01

    We propose a description for the quasiequilibrium self-assembly of small, single-stranded (ss) RNA viruses whose capsid proteins (CPs) have flexible, positively charged, disordered tails that associate with the negatively charged RNA genome molecules. We describe the assembly of such viruses as the interplay between two coupled phase-transition-like events: the formation of the protein shell (the capsid) by CPs and the condensation of a large ss viral RNA molecule. Electrostatic repulsion between the CPs competes with attractive hydrophobic interactions and attractive interaction between neutralized RNA segments mediated by the tail groups. An assembly diagram is derived in terms of the strength of attractive interactions between CPs and between CPs and the RNA molecules. It is compared with the results of recent studies of viral assembly. We demonstrate that the conventional theory of self-assembly, which does describe the assembly of empty capsids, is in general not applicable to the self-assembly of RNA-encapsidating virions.

  11. Small interfering RNA delivery through positively charged polymer nanoparticles

    Science.gov (United States)

    Dragoni, Luca; Ferrari, Raffaele; Lupi, Monica; Cesana, Alberto; Falcetta, Francesca; Ubezio, Paolo; D'Incalci, Maurizio; Morbidelli, Massimo; Moscatelli, Davide

    2016-03-01

    Small interfering RNA (siRNA) is receiving increasing attention with regard to the treatment of many genetic diseases, both acquired and hereditary, such as cancer and diabetes. Being a high molecular weight (MW) polyanion, siRNA is not able to cross a cell membrane, and in addition it is unstable in physiological conditions. Accordingly, a biocompatible nanocarrier able to deliver siRNA into cells is needed. In this work, we synthesized biocompatible positively charged nanoparticles (NPs) following a two-step process that involves ring opening polymerization (ROP) and emulsion free radical polymerization (EFRP). Firstly, we proved the possibility of fine tuning the NPs’ characteristics (e.g. size and surface charge) by changing the synthetic process parameters. Then the capability in loading and delivering undamaged siRNA into a cancer cell cytoplasm has been shown. This latter process occurs through the biodegradation of the polymer constituting the NPs, whose kinetics can be tuned by adjusting the polymer’s MW. Finally, the ability of NPs to carry siRNA inside the cells in order to inhibit their target gene has been demonstrated using green flourescent protein positive cells.

  12. Pulmonary administration of small interfering RNA: The route to go?

    Science.gov (United States)

    Ruigrok, M J R; Frijlink, H W; Hinrichs, W L J

    2016-08-10

    Ever since the discovery of RNA interference (RNAi), which is a post-transcriptional gene silencing mechanism, researchers have been studying the therapeutic potential of using small interfering RNA (siRNA) to treat diseases that are characterized by excessive gene expression. Excessive gene expression can be particularly harmful if it occurs in a vulnerable organ such as the lungs as they are essential for physiological respiration. Consequently, RNAi could offer an approach to treat such lung diseases. Parenteral administration of siRNA has been shown to be difficult due to degradation by nucleases in the systemic circulation and excretion by the kidneys. To avoid these issues and to achieve local delivery and local effects, pulmonary administration has been proposed as an alternative administration route. Regarding this application, various animal studies have been conducted over the past few years. Therefore, this review presents a critical analysis of publications where pulmonary administration of siRNA in animals has been reported. Such an analysis is necessary to determine the feasibility of this administration route and to define directions for future research. First, we provide background information on lungs, pulmonary administration, and delivery vectors. Thereafter, we present and discuss relevant animal studies. Though nearly all publications reported positive outcomes, several reoccurring challenges were identified. They relate to 1) the necessity, efficacy, and safety of delivery vectors, 2) the biodistribution of siRNA in tissues other than the lungs, 3) the poor correlation between in vitro and in vivo models, and 4) the long-term effects upon (repeated) administration of siRNA. Finally, we present recommendations for future research to define the route to go: towards safer and more effective pulmonary administration of siRNA. PMID:27235976

  13. Computational prediction of miRNA genes from small RNA sequencing data

    Directory of Open Access Journals (Sweden)

    Wenjing eKang

    2015-01-01

    Full Text Available Next-generation sequencing now for the first time allows researchers to gauge the depth and variation of entire transcriptomes. However, now as rare transcripts can be detected that are present in cells at single copies, more advanced computational tools are needed to accurately annotate and profile them. miRNAs are 22 nucleotide small RNAs (sRNAs that post-transcriptionally reduce the output of protein coding genes. They have established roles in numerous biological processes, including cancers and other diseases. During miRNA biogenesis, the sRNAs are sequentially cleaved from precursor molecules that have a characteristic hairpin RNA structure. The vast majority of new miRNA genes that are discovered are mined from small RNA sequencing (sRNA-seq, which can detect more than a billion RNAs in a single run. However, given that many of the detected RNAs are degradation products from all types of transcripts, the accurate identification of miRNAs remain a non-trivial computational problem. Here we review the tools available to predict animal miRNAs from sRNA sequencing data. We present tools for generalist and specialist use cases, including prediction from massively pooled data or in species without reference genome. We also present wet-lab methods used to validate predicted miRNAs, and approaches to computationally benchmark prediction accuracy. For each tool, we reference validation experiments and benchmarking efforts. Last, we discuss the future of the field.

  14. Noncoding RNA small nucleolar RNA host gene 1 promote cell proliferation in nonsmall cell lung cancer

    Directory of Open Access Journals (Sweden)

    J You

    2014-01-01

    Full Text Available Background: Nonsmall cell lung cancer (NSCLC is the major cause of cancer death worldwide. Increasing evidence shows that noncoding RNAs (ncRNAs are widely involved in the development and progression of NSCLC. ncRNA small nucleolar RNA host gene 1 (SNHG1 has not been studied in cancer, especially its role in lung cancer remains unknown. Our studies were designed to investigate the expression and biological significance of SNHG1 in lung cancer. SNHG1 may be a novel ncRNA in early diagnosis in lung cancer. Methods: Noncoding RNA SNHG1 expression in 7 lung cancer cell lines was measured by quantitative real-time polymerase chain reaction. RNA interference approaches were used to find the biological functions of SNHG1. The effect of SNHG1 on proliferation was evaluated by cell count and crystal violet stains. Results: Noncoding RNA SNHG1 expression was significantly upregulated in lung cancer cells when compared with normal bronchial epithelial cells. In addition, in vitro assays our results indicated that knockdown of SNHG1 inhibited cell proliferation. Conclusions: Our data indicated that ncRNA SNHG1 is significantly upregulated in NSCLC cell lines and may represent a new biomarker and a potential therapeutic target for NSCLC intervention.

  15. Silent no more: Endogenous small RNA pathways promote gene expression.

    Science.gov (United States)

    Wedeles, Christopher J; Wu, Monica Z; Claycomb, Julie M

    2014-01-01

    Endogenous small RNA pathways related to RNA interference (RNAi) play a well-documented role in protecting host genomes from the invasion of foreign nucleic acids. In C. elegans, the PIWI type Argonaute, PRG-1, through an association with 21U-RNAs, mediates a genome surveillance process by constantly scanning the genome for potentially deleterious invading elements. Upon recognition of foreign nucleic acids, PRG-1 initiates a cascade of cytoplasmic and nuclear events that results in heritable epigenetic silencing of these transcripts and their coding genomic loci. If the PRG-1/21U-RNA genome surveillance pathway has the capacity to target most of the C. elegans transcriptome, what mechanisms exist to protect endogenous transcripts from being silenced by this pathway? In this commentary, we discuss three recent publications that implicate the CSR-1 small RNA pathway in the heritable activation of germline transcripts, propose a model as to why not all epialleles behave similarly, and touch on the practical implications of these findings.

  16. Small RNA expression and strain specificity in the rat

    Directory of Open Access Journals (Sweden)

    de Bruijn Ewart

    2010-04-01

    Full Text Available Abstract Background Digital gene expression (DGE profiling has become an established tool to study RNA expression. Here, we provide an in-depth analysis of small RNA DGE profiles from two different rat strains (BN-Lx and SHR from six different rat tissues (spleen, liver, brain, testis, heart, kidney. We describe the expression patterns of known and novel micro (miRNAs and piwi-interacting (piRNAs. Results We confirmed the expression of 588 known miRNAs (54 in antisense orientation and identified 56 miRNAs homologous to known human or mouse miRNAs, as well as 45 new rat miRNAs. Furthermore, we confirmed specific A to I editing in brain for mir-376a/b/c and identified mir-377 as a novel editing target. In accordance with earlier findings, we observed a highly tissue-specific expression pattern for all tissues analyzed. The brain was found to express the highest number of tissue-specific miRNAs, followed by testis. Notably, our experiments also revealed robust strain-specific differential miRNA expression in the liver that is caused by genetic variation between the strains. Finally, we identified two types of germline-specific piRNAs in testis, mapping either to transposons or in strand-specific clusters. Conclusions Taken together, the small RNA compendium described here advances the annotation of small RNAs in the rat genome. Strain and tissue-specific expression patterns furthermore provide a strong basis for studying the role of small RNAs in regulatory networks as well as biological process like physiology and neurobiology that are extensively studied in this model system.

  17. Repertoire of bovine miRNA and miRNA-like small regulatory RNAs expressed upon viral infection.

    Directory of Open Access Journals (Sweden)

    Evgeny A Glazov

    Full Text Available MicroRNA (miRNA and other types of small regulatory RNAs play a crucial role in the regulation of gene expression in eukaryotes. Several distinct classes of small regulatory RNAs have been discovered in recent years. To extend the repertoire of small RNAs characterized in mammals and to examine relationship between host miRNA expression and viral infection we used Illumina's ultrahigh throughput sequencing approach. We sequenced three small RNA libraries prepared from cell line derived from the adult bovine kidney under normal conditions and upon infection of the cell line with Bovine herpesvirus 1. We used a bioinformatics approach to distinguish authentic mature miRNA sequences from other classes of small RNAs and short RNA fragments represented in the sequencing data. Using this approach we detected 219 out of 356 known bovine miRNAs and 115 respective miRNA* sequences. In addition we identified five new bovine orthologs of known mammalian miRNAs and discovered 268 new cow miRNAs many of which are not identifiable in other mammalian genomes and thus might be specific to the ruminant lineage. In addition we found seven new bovine mirtron candidates. We also discovered 10 small nucleolar RNA (snoRNA loci that give rise to small RNA with possible miRNA-like function. Results presented in this study extend our knowledge of the biology and evolution of small regulatory RNAs in mammals and illuminate mechanisms of small RNA biogenesis and function. New miRNA sequences and the original sequencing data have been submitted to miRNA repository (miRBase and NCBI GEO archive respectively. We envisage that these resources will facilitate functional annotation of the bovine genome and promote further functional and comparative genomics studies of small regulatory RNA in mammals.

  18. Adenylylation of small RNA sequencing adapters using the TS2126 RNA ligase I.

    Science.gov (United States)

    Lama, Lodoe; Ryan, Kevin

    2016-01-01

    Many high-throughput small RNA next-generation sequencing protocols use 5' preadenylylated DNA oligonucleotide adapters during cDNA library preparation. Preadenylylation of the DNA adapter's 5' end frees from ATP-dependence the ligation of the adapter to RNA collections, thereby avoiding ATP-dependent side reactions. However, preadenylylation of the DNA adapters can be costly and difficult. The currently available method for chemical adenylylation of DNA adapters is inefficient and uses techniques not typically practiced in laboratories profiling cellular RNA expression. An alternative enzymatic method using a commercial RNA ligase was recently introduced, but this enzyme works best as a stoichiometric adenylylating reagent rather than a catalyst and can therefore prove costly when several variant adapters are needed or during scale-up or high-throughput adenylylation procedures. Here, we describe a simple, scalable, and highly efficient method for the 5' adenylylation of DNA oligonucleotides using the thermostable RNA ligase 1 from bacteriophage TS2126. Adapters with 3' blocking groups are adenylylated at >95% yield at catalytic enzyme-to-adapter ratios and need not be gel purified before ligation to RNA acceptors. Experimental conditions are also reported that enable DNA adapters with free 3' ends to be 5' adenylylated at >90% efficiency. PMID:26567315

  19. Nicotiana small RNA sequences support a host genome origin of cucumber mosaic virus satellite RNA.

    Directory of Open Access Journals (Sweden)

    Kiran Zahid

    2015-01-01

    Full Text Available Satellite RNAs (satRNAs are small noncoding subviral RNA pathogens in plants that depend on helper viruses for replication and spread. Despite many decades of research, the origin of satRNAs remains unknown. In this study we show that a β-glucuronidase (GUS transgene fused with a Cucumber mosaic virus (CMV Y satellite RNA (Y-Sat sequence (35S-GUS:Sat was transcriptionally repressed in N. tabacum in comparison to a 35S-GUS transgene that did not contain the Y-Sat sequence. This repression was not due to DNA methylation at the 35S promoter, but was associated with specific DNA methylation at the Y-Sat sequence. Both northern blot hybridization and small RNA deep sequencing detected 24-nt siRNAs in wild-type Nicotiana plants with sequence homology to Y-Sat, suggesting that the N. tabacum genome contains Y-Sat-like sequences that give rise to 24-nt sRNAs capable of guiding RNA-directed DNA methylation (RdDM to the Y-Sat sequence in the 35S-GUS:Sat transgene. Consistent with this, Southern blot hybridization detected multiple DNA bands in Nicotiana plants that had sequence homology to Y-Sat, suggesting that Y-Sat-like sequences exist in the Nicotiana genome as repetitive DNA, a DNA feature associated with 24-nt sRNAs. Our results point to a host genome origin for CMV satRNAs, and suggest novel approach of using small RNA sequences for finding the origin of other satRNAs.

  20. Small RNA-directed epigenetic natural variation in Arabidopsis thaliana.

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    Jixian Zhai

    2008-04-01

    Full Text Available Progress in epigenetics has revealed mechanisms that can heritably regulate gene function independent of genetic alterations. Nevertheless, little is known about the role of epigenetics in evolution. This is due in part to scant data on epigenetic variation among natural populations. In plants, small interfering RNA (siRNA is involved in both the initiation and maintenance of gene silencing by directing DNA methylation and/or histone methylation. Here, we report that, in the model plant Arabidopsis thaliana, a cluster of approximately 24 nt siRNAs found at high levels in the ecotype Landsberg erecta (Ler could direct DNA methylation and heterochromatinization at a hAT element adjacent to the promoter of FLOWERING LOCUS C (FLC, a major repressor of flowering, whereas the same hAT element in ecotype Columbia (Col with almost identical DNA sequence, generates a set of low abundance siRNAs that do not direct these activities. We have called this hAT element MPF for Methylated region near Promoter of FLC, although de novo methylation triggered by an inverted repeat transgene at this region in Col does not alter its FLC expression. DNA methylation of the Ler allele MPF is dependent on genes in known silencing pathways, and such methylation is transmissible to Col by genetic crosses, although with varying degrees of penetrance. A genome-wide comparison of Ler and Col small RNAs identified at least 68 loci matched by a significant level of approximately 24 nt siRNAs present specifically in Ler but not Col, where nearly half of the loci are related to repeat or TE sequences. Methylation analysis revealed that 88% of the examined loci (37 out of 42 were specifically methylated in Ler but not Col, suggesting that small RNA can direct epigenetic differences between two closely related Arabidopsis ecotypes.

  1. Advances in targeted delivery of small interfering RNA using simple bioconjugates

    DEFF Research Database (Denmark)

    Nielsen, Christoffer; Kjems, Jørgen; Sørensen, Kristine Rothaus;

    2014-01-01

    INTRODUCTION: Development of drugs based on RNA interference by small interfering RNA (siRNA) has been progressing slowly due to a number of challenges associated with the in vivo behavior of siRNA. A central problem is controlling siRNA delivery to specific cell types. Here, we review existing l...

  2. Analysis of microRNA transcriptome by deep sequencing of small RNA libraries of peripheral blood

    Directory of Open Access Journals (Sweden)

    Kumar Lalit

    2010-05-01

    Full Text Available Abstract Background MicroRNAs are a class of small non-coding RNAs that regulate mRNA expression at the post - transcriptional level and thereby many fundamental biological processes. A number of methods, such as multiplex polymerase chain reaction, microarrays have been developed for profiling levels of known miRNAs. These methods lack the ability to identify novel miRNAs and accurately determine expression at a range of concentrations. Deep or massively parallel sequencing methods are providing suitable platforms for genome wide transcriptome analysis and have the ability to identify novel transcripts. Results The results of analysis of small RNA sequences obtained by Solexa technology of normal peripheral blood mononuclear cells, tumor cell lines K562 and HL60 are presented. In general K562 cells displayed overall low level of miRNA population and also low levels of DICER. Some of the highly expressed miRNAs in the leukocytes include several members of the let-7 family, miR-21, 103, 185, 191 and 320a. Comparison of the miRNA profiles of normal versus K562 or HL60 cells revealed a specific set of differentially expressed molecules. Correlation of the miRNA with that of mRNA expression profiles, obtained by microarray, revealed a set of target genes showing inverse correlation with miRNA levels. Relative expression levels of individual miRNAs belonging to a cluster were found to be highly variable. Our computational pipeline also predicted a number of novel miRNAs. Some of the predictions were validated by Real-time RT-PCR and or RNase protection assay. Organization of some of the novel miRNAs in human genome suggests that these may also be part of existing clusters or form new clusters. Conclusions We conclude that about 904 miRNAs are expressed in human leukocytes. Out of these 370 are novel miRNAs. We have identified miRNAs that are differentially regulated in normal PBMC with respect to cancer cells, K562 and HL60. Our results suggest that

  3. Quantitative characteristics of gene regulation by small RNA.

    Directory of Open Access Journals (Sweden)

    Erel Levine

    2007-09-01

    Full Text Available An increasing number of small RNAs (sRNAs have been shown to regulate critical pathways in prokaryotes and eukaryotes. In bacteria, regulation by trans-encoded sRNAs is predominantly found in the coordination of intricate stress responses. The mechanisms by which sRNAs modulate expression of its targets are diverse. In common to most is the possibility that interference with the translation of mRNA targets may also alter the abundance of functional sRNAs. Aiming to understand the unique role played by sRNAs in gene regulation, we studied examples from two distinct classes of bacterial sRNAs in Escherichia coli using a quantitative approach combining experiment and theory. Our results demonstrate that sRNA provides a novel mode of gene regulation, with characteristics distinct from those of protein-mediated gene regulation. These include a threshold-linear response with a tunable threshold, a robust noise resistance characteristic, and a built-in capability for hierarchical cross-talk. Knowledge of these special features of sRNA-mediated regulation may be crucial toward understanding the subtle functions that sRNAs can play in coordinating various stress-relief pathways. Our results may also help guide the design of synthetic genetic circuits that have properties difficult to attain with protein regulators alone.

  4. MicroRNA: Small RNA mediators of the brains genomic response to environmental stress.

    Science.gov (United States)

    Hollins, Sharon L; Cairns, Murray J

    2016-08-01

    The developmental processes that establish the synaptic architecture of the brain while retaining capacity for activity-dependent remodeling, are complex and involve a combination of genetic and epigenetic influences. Dysregulation of these processes can lead to problems with neural circuitry which manifest in humans as a range of neurodevelopmental syndromes, such as schizophrenia, bipolar disorder and fragile X mental retardation. Recent studies suggest that prenatal, postnatal and intergenerational environmental factors play an important role in the aetiology of stress-related psychopathology. A number of these disorders have been shown to display epigenetic changes in the postmortem brain that reflect early life experience. These changes affect the regulation of gene expression though chromatin remodeling (transcriptional) and post-transcriptional influences, especially small noncoding microRNA (miRNA). These dynamic and influential molecules appear to play an important function in both brain development and its adaption to stress. In this review, we examine the role of miRNA in mediating the brain's response to both prenatal and postnatal environmental perturbations and explore how stress- induced alterations in miRNA expression can regulate the stress response via modulation of the immune system. Given the close relationship between environmental stress, miRNA, and brain development/function, we assert that miRNA hold a significant position at the molecular crossroads between neural development and adaptations to environmental stress. A greater understanding of the dynamics that mediate an individual's predisposition to stress-induced neuropathology has major human health benefits and is an important area of research. PMID:27317386

  5. Global Annotation of Small RNA and MicroRNA Mature Sequences from Developing Ovules of Gossypium hirsutum L.

    Institute of Scientific and Technical Information of China (English)

    ABDURAKHMONOV Ibrokhim Y; DEVOR Eric J; HUANG Ling-yan; BURIEV Zabardast T; MAKAMOV Abdusalom; SHERMATOV Shukhrat E; ABDUKARIMOV Abdusattor

    2008-01-01

    @@ The involvement of small RNAs in cotton fiber development is under explored.The objective of this work was to directly clone,annotate,and analyze small RNAs of developing ovules to reveal the candidate small interfering RNA/microRNAs (siRNAs/miRNAs) involved in cotton ovule and fiber development.We cloned small RNA sequences from 0~10 days post anthesis (DPA) developing ovules of Gossypium hirsutum var.

  6. In Silico Reconstruction of Viral Genomes from Small RNAs Improves Virus-Derived Small Interfering RNA Profiling

    OpenAIRE

    Vodovar, Nicolas; Goic, Bertsy; Blanc, Hervé; Saleh, Maria-Carla

    2011-01-01

    RNA interference (RNAi) is the essential component of antiviral immunity in invertebrates and plants. One of the landmarks of the antiviral RNAi response is the production of virus-derived small interfering RNA (vsiRNA) from viral double-stranded RNA (dsRNA). vsiRNAs constitute a fragmented image of the viral genome sequence that results from Dicer cleavage. vsiRNA sequence profiling is used extensively as a surrogate to study the antiviral RNAi response by determining the nature of the viral...

  7. miRge - A Multiplexed Method of Processing Small RNA-Seq Data to Determine MicroRNA Entropy.

    Directory of Open Access Journals (Sweden)

    Alexander S Baras

    Full Text Available Small RNA RNA-seq for microRNAs (miRNAs is a rapidly developing field where opportunities still exist to create better bioinformatics tools to process these large datasets and generate new, useful analyses. We built miRge to be a fast, smart small RNA-seq solution to process samples in a highly multiplexed fashion. miRge employs a Bayesian alignment approach, whereby reads are sequentially aligned against customized mature miRNA, hairpin miRNA, noncoding RNA and mRNA sequence libraries. miRNAs are summarized at the level of raw reads in addition to reads per million (RPM. Reads for all other RNA species (tRNA, rRNA, snoRNA, mRNA are provided, which is useful for identifying potential contaminants and optimizing small RNA purification strategies. miRge was designed to optimally identify miRNA isomiRs and employs an entropy based statistical measurement to identify differential production of isomiRs. This allowed us to identify decreasing entropy in isomiRs as stem cells mature into retinal pigment epithelial cells. Conversely, we show that pancreatic tumor miRNAs have similar entropy to matched normal pancreatic tissues. In a head-to-head comparison with other miRNA analysis tools (miRExpress 2.0, sRNAbench, omiRAs, miRDeep2, Chimira, UEA small RNA Workbench, miRge was faster (4 to 32-fold and was among the top-two methods in maximally aligning miRNAs reads per sample. Moreover, miRge has no inherent limits to its multiplexing. miRge was capable of simultaneously analyzing 100 small RNA-Seq samples in 52 minutes, providing an integrated analysis of miRNA expression across all samples. As miRge was designed for analysis of single as well as multiple samples, miRge is an ideal tool for high and low-throughput users. miRge is freely available at http://atlas.pathology.jhu.edu/baras/miRge.html.

  8. New perspectives on the diversification of the RNA interference system: insights from comparative genomics and small RNA sequencing.

    Science.gov (United States)

    Burroughs, Alexander Maxwell; Ando, Yoshinari; Aravind, L

    2014-01-01

    Our understanding of the pervasive involvement of small RNAs in regulating diverse biological processes has been greatly augmented by recent application of deep-sequencing technologies to small RNA across diverse eukaryotes. We review the currently known small RNA classes and place them in context of the reconstructed evolutionary history of the RNA interference (RNAi) protein machinery. This synthesis indicates that the earliest versions of eukaryotic RNAi systems likely utilized small RNA processed from three types of precursors: (1) sense-antisense transcriptional products, (2) genome-encoded, imperfectly complementary hairpin sequences, and (3) larger noncoding RNA precursor sequences. Structural dissection of PIWI proteins along with recent discovery of novel families (including Med13 of the Mediator complex) suggest that emergence of a distinct architecture with the N-terminal domains (also occurring separately fused to endoDNases in prokaryotes) formed via duplication of an ancestral unit was key to their recruitment as primary RNAi effectors and use of small RNAs of certain preferred lengths. Prokaryotic PIWI proteins are typically components of several RNA-directed DNA restriction or CRISPR/Cas systems. However, eukaryotic versions appear to have emerged from a subset that evolved RNA-directed RNAi. They were recruited alongside RNaseIII domains and RNA-dependent RNA polymerase (RdRP) domains, also from prokaryotic systems, to form the core eukaryotic RNAi system. Like certain regulatory systems, RNAi diversified into two distinct but linked arms concomitant with eukaryotic nucleocytoplasmic compartmentalization. Subsequent elaboration of RNAi proceeded via diversification of the core protein machinery through lineage-specific expansions and recruitment of new components from prokaryotes (nucleases and small RNA-modifying enzymes), allowing for diversification of associating small RNAs. PMID:24311560

  9. Small RNA transcriptome investigation based on next-generation sequencing technology

    Institute of Scientific and Technical Information of China (English)

    Linglin Zhou; Xueying Li; Qi Liu; Fangqing Zhao; Jinyu Wu

    2011-01-01

    Over the past decade,there has been a growing realization that studying the small RNA transcriptome is essential for understanding the complexity of transcriptional regulation.With an increased throughput and a reduced cost,next-generation sequencing technology has provided an unprecedented opportunity to measure the extent and complexity of small RNA transcriptome.Meanwhile,the large amount of obtained data and varied technology platforms have also posed multiple challenges for effective data analysis and mining.To provide some insight into the small RNA transcriptome investigation,this review describes the major small RNA classes,experimental methods to identify small RNAs,and available bioinformatics tools and databases.

  10. From early lessons to new frontiers: the worm as a treasure trove of small RNA biology

    OpenAIRE

    YOUNGMAN, ELAINE M.; Claycomb, Julie M

    2014-01-01

    In the past 20 years, the tiny soil nematode Caenorhabditis elegans has provided critical insights into our understanding of the breadth of small RNA-mediated gene regulatory activities. The first microRNA was identified in C. elegans in 1993, and the understanding that dsRNA was the driving force behind RNA-mediated gene silencing came from experiments performed in C. elegans in 1998. Likewise, early genetic screens in C. elegans for factors involved in RNA interference pointed to conserved ...

  11. Broad-Spectrum Antiviral Activity of Small Interfering RNA Targeting the Conserved RNA Termini of Lassa Virus▿

    OpenAIRE

    Müller, Stefanie; Günther, Stephan

    2007-01-01

    Small interfering RNAs targeting the conserved RNA termini upstream of NP and L gene were found to reduce reporter gene expression from Lassa virus replicon and Lassa virus mRNA expression construct and to inhibit replication of different Lassa virus strains, lymphocytic choriomeningitis virus, and Mopeia virus in cell culture.

  12. From early lessons to new frontiers: the worm as a treasure trove of small RNA biology.

    Science.gov (United States)

    Youngman, Elaine M; Claycomb, Julie M

    2014-01-01

    In the past 20 years, the tiny soil nematode Caenorhabditis elegans has provided critical insights into our understanding of the breadth of small RNA-mediated gene regulatory activities. The first microRNA was identified in C. elegans in 1993, and the understanding that dsRNA was the driving force behind RNA-mediated gene silencing came from experiments performed in C. elegans in 1998. Likewise, early genetic screens in C. elegans for factors involved in RNA interference pointed to conserved mechanisms for small RNA-mediated gene silencing pathways, placing the worm squarely among the founding fathers of a now extensive field of molecular biology. Today, the worm continues to be at the forefront of ground-breaking insight into small RNA-mediated biology. Recent studies have revealed with increasing mechanistic clarity that C. elegans possesses an extensive nuclear small RNA regulatory network that encompasses not only gene silencing but also gene activating roles. Further, a portrait is emerging whereby small RNA pathways play key roles in integrating responses to environmental stimuli and transmitting epigenetic information about such responses from one generation to the next. Here we discuss endogenous small RNA pathways in C. elegans and the insight worm biology has provided into the mechanisms employed by these pathways. We touch on the increasingly spectacular diversity of small RNA biogenesis and function, and discuss the relevance of lessons learned in the worm for human biology. PMID:25505902

  13. High-Level Accumulation of Exogenous Small RNAs Not Affecting Endogenous Small RNA Biogenesis and Function in Plants

    Institute of Scientific and Technical Information of China (English)

    SHEN Wan-xia; Neil A Smith; ZHOU Chang-yong; WANG Ming-bo

    2014-01-01

    RNA silencing is a fundamental plant defence and gene control mechanism in plants that are directed by 20-24 nucleotide (nt) small interfering RNA (siRNA) and microRNA (miRNA). Infection of plants with viral pathogens or transformation of plants with RNA interference (RNAi) constructs is usually associated with high levels of exogenous siRNAs, but it is unclear if these siRNAs interfere with endogenous small RNA pathways and hence affect plant development. Here we provide evidence that viral satellite RNA (satRNA) infection does not affect siRNA and miRNA biogenesis or plant growth despite the extremely high level of satRNA-derived siRNAs. We generated transgenic Nicotiana benthamiana plants that no longer develop the speciifc yellowing symptoms generally associated with infection by Cucumber mosaic virus (CMV) Y-satellite RNA (Y-Sat). We then used these plants to show that CMV Y-Sat infection did not cause any visible phenotypic changes in comparison to uninfected plants, despite the presence of high-level Y-Sat siRNAs. Furthermore, we showed that the accumulation of hairpin RNA (hpRNA)-derived siRNAs or miRNAs, and the level of siRNA-directed transgene silencing, are not signiifcantly affected by CMV Y-Sat infection. Taken together, our results suggest that the high levels of exogenous siRNAs associated with viral infection or RNAi-inducing transgenes do not saturate the endogenous RNA silencing machineries and have no signiifcant impact on normal plant development.

  14. Transcription of the major neurospora crassa microRNA-like small RNAs relies on RNA polymerase III.

    Directory of Open Access Journals (Sweden)

    Qiuying Yang

    Full Text Available Most plant and animal microRNAs (miRNAs are transcribed by RNA polymerase II. We previously discovered miRNA-like small RNAs (milRNAs in the filamentous fungus Neurospora crassa and uncovered at least four different pathways for milRNA production. To understand the evolutionary origin of milRNAs, we determined the roles of polymerases II and III (Pol II and Pol III in milRNA transcription. Our results show that Pol III is responsible for the transcription of the major milRNAs produced in this organism. The inhibition of Pol III activity by an inhibitor or by gene silencing abolishes the production of most abundant milRNAs and pri-milRNAs. In addition, Pol III associates with these milRNA producing loci. Even though silencing of Pol II does not affect the synthesis of the most abundant milRNAs, Pol II or both Pol II and Pol III are associated with some milRNA-producing loci, suggesting a regulatory interaction between the two polymerases for some milRNA transcription. Furthermore, we show that one of the Pol III-transcribed milRNAs is derived from a tRNA precursor, and its biogenesis requires RNase Z, which cleaves the tRNA moiety to generate pre-milRNA. Our study identifies the transcriptional machinery responsible for the synthesis of fungal milRNAs and sheds light on the evolutionary origin of eukaryotic small RNAs.

  15. Diverse Evolutionary Trajectories for Small RNA Biogenesis Genes in the Oomycete Genus Phytophthora

    OpenAIRE

    Stephanie R Bollmann; Fang, Yufeng; Press, Caroline M.; Tyler, Brett M.; Niklaus J Grünwald

    2016-01-01

    Gene regulation by small RNA pathways is ubiquitous among eukaryotes, but little is known about small RNA pathways in the Stramenopile kingdom. Phytophthora, a genus of filamentous oomycetes, contains many devastating plant pathogens, causing multibillion-dollar damage to crops, ornamental plants, and natural environments. The genomes of several oomycetes including Phytophthora species such as the soybean pathogen P. sojae, have been sequenced, allowing evolutionary analysis of small RNA-proc...

  16. Myelin basic protein synthesis is regulated by small non-coding RNA 715.

    Science.gov (United States)

    Bauer, Nina M; Moos, Christina; van Horssen, Jack; Witte, Maarten; van der Valk, Paul; Altenhein, Benjamin; Luhmann, Heiko J; White, Robin

    2012-09-01

    Oligodendroglial Myelin Basic Protein (MBP) synthesis is essential for myelin formation in the central nervous system. During oligodendrocyte differentiation, MBP mRNA is kept in a translationally silenced state while intracellularly transported, until neuron-derived signals initiate localized MBP translation. Here we identify the small non-coding RNA 715 (sncRNA715) as an inhibitor of MBP translation. SncRNA715 localizes to cytoplasmic granular structures and associates with MBP mRNA transport granule components. We also detect increased levels of sncRNA715 in demyelinated chronic human multiple sclerosis lesions, which contain MBP mRNA but lack MBP protein.

  17. Intrinsic noise in post-transcriptional gene regulation by small non-coding RNA.

    Science.gov (United States)

    Jia, Ya; Liu, Wangheng; Li, Anbang; Yang, Lijian; Zhan, Xuan

    2009-07-01

    Small non-coding RNA (sRNA) plays very important role in the post transcriptional regulation in various organisms. In complex regulatory networks, highly significant relative fluctuations in RNAs copy numbers can not be neglected due to very small copy number of individual RNA molecules. Here we consider two simple regulation schemes, where one is single target gene regulated by a sRNA and the other is two target mRNAs (mRNA(R) and mRNA(T)) regulated by one sRNA. The Fano factor (a measure of the relative size of the internal fluctuations) formulae of RNA molecules in the post transcriptional regulation are theoretically derived by using of the Langevin theory. For single target gene regulated by a sRNA, it is shown that the intrinsic noise of both mRNA and sRNA approaches the bare Poissonian limit in the regimen of both target RNA silencing and surviving. However, the strong anti-correlation between the fluctuations of two components result in a large intrinsic fluctuations in the level of RNA molecules in the regimen of crossover. For two target mRNAs regulated by one sRNA, in the regimen of crossover, it is found that, with the increasing of transcription rate of target mRNA(T), the maximal intrinsic fluctuation of RNA molecules is shifted from sRNA to target mRNA(R), and then to target mRNA(T). The intrinsic noise intensity of target mRNA(R) is determined by both the transcriptional rate of itself and that of sRNA, and independent of the transcriptional rate of the other target mRNA(T). PMID:19403234

  18. Spliceosomal small nuclear RNAs of Tetrahymena thermophila and some possible snRNA-snRNA base-pairing interactions

    DEFF Research Database (Denmark)

    Orum, H; Nielsen, Henrik; Engberg, J

    1991-01-01

    We have identified and characterized the full set of spliceosomal small nuclear RNAs (snRNAs; U1, U2, U4, U5 and U6) from the ciliated protozoan Tetrahymena thermophila. With the exception of U4 snRNA, the sizes of the T. thermophila snRNAs are closely similar to their metazoan homologues. The T...... organisms. Furthermore, secondary structures closely similar to phylogenetically proven models can be inferred from the T. thermophila data. Analysis of the snRNA sequences identifies three potential snRNA-snRNA base-pairing interactions, all of which are consistent with available phylogenetic data. Two...

  19. From early lessons to new frontiers: The worm as a treasure trove of small RNA biology

    Directory of Open Access Journals (Sweden)

    Elaine M. Youngman

    2014-11-01

    Full Text Available In the past twenty years, the tiny soil nematode C. elegans has provided critical insights into our understanding of the breadth of small RNA-mediated gene regulatory activities. The first microRNA was identified in C. elegans in 1993, and the understanding that dsRNA was the driving force behind RNA-mediated gene silencing came from experiments performed in C. elegans in 1998. Likewise, early genetic screens in C. elegans for factors involved in RNAi pointed to conserved mechanisms for small RNA-mediated gene silencing pathways, placing the worm squarely among the founding fathers of a now extensive field of molecular biology. Today, the worm continues to be at the forefront of ground-breaking insight into small RNA-mediated biology. Recent studies have revealed with increasing mechanistic clarity that C. elegans possesses an extensive nuclear small RNA regulatory network that encompasses not only gene silencing but also gene activating roles. Further, a portrait is emerging whereby small RNA pathways play key roles in integrating responses to environmental stimuli and transmitting epigenetic information about such responses from one generation to the next. Here we discuss endogenous small RNA pathways in C. elegans and the insight worm biology has provided into the mechanisms employed by these pathways. We touch on the increasingly spectacular diversity of small RNA biogenesis and function, and discuss the relevance of lessons learned in the worm for human biology.

  20. Empirical insights into the stochasticity of small RNA sequencing

    Science.gov (United States)

    Qin, Li-Xuan; Tuschl, Thomas; Singer, Samuel

    2016-04-01

    The choice of stochasticity distribution for modeling the noise distribution is a fundamental assumption for the analysis of sequencing data and consequently is critical for the accurate assessment of biological heterogeneity and differential expression. The stochasticity of RNA sequencing has been assumed to follow Poisson distributions. We collected microRNA sequencing data and observed that its stochasticity is better approximated by gamma distributions, likely because of the stochastic nature of exponential PCR amplification. We validated our findings with two independent datasets, one for microRNA sequencing and another for RNA sequencing. Motivated by the gamma distributed stochasticity, we provided a simple method for the analysis of RNA sequencing data and showed its superiority to three existing methods for differential expression analysis using three data examples of technical replicate data and biological replicate data.

  1. Systemic delivery of siRNA in pumpkin by a plant PHLOEM SMALL RNA-BINDING PROTEIN 1-ribonucleoprotein complex.

    Science.gov (United States)

    Ham, Byung-Kook; Li, Gang; Jia, Weitao; Leary, Julie A; Lucas, William J

    2014-11-01

    In plants, the vascular system, specifically the phloem, functions in delivery of small RNA (sRNA) to exert epigenetic control over developmental and defense-related processes. Although the importance of systemic sRNA delivery has been established, information is currently lacking concerning the nature of the protein machinery involved in this process. Here, we show that a PHLOEM SMALL-RNA BINDING PROTEIN 1 (PSRP1) serves as the basis for formation of an sRNA ribonucleoprotein complex (sRNPC) that delivers sRNA (primarily 24 nt) to sink organs. Assembly of this complex is facilitated through PSRP1 phosphorylation by a phloem-localized protein kinase, PSRPK1. During long-distance transport, PSRP1-sRNPC is stable against phloem phosphatase activity. Within target tissues, phosphatase activity results in disassembly of PSRP1-sRNPC, a process that is probably required for unloading cargo sRNA into surrounding cells. These findings provide an insight into the mechanism involved in delivery of sRNA associated with systemic gene silencing in plants. PMID:25227635

  2. Diverse evolutionary trajectories for small RNA biogenesis genes in the oomycete genus Phytophthora

    Science.gov (United States)

    Gene regulation by small RNA pathways is ubiquitous among eukaryotes, but little is known about small RNA pathways in the Stramenopile kingdom. Phytophthora, a genus of filamentous oomycetes, contains many devastating plant pathogens, causing multibillion-dollar damage to crops, ornamental plants, ...

  3. An assessment of bacterial small RNA target prediction programs

    OpenAIRE

    Pain, Adrien; Ott, Alban; Amine, Hamza; Rochat, Tatiana; Bouloc, Philippe; Gautheret, Daniel

    2015-01-01

    Most bacterial regulatory RNAs exert their function through base-pairing with target RNAs. Computational prediction of targets is a busy research field that offers biologists a variety of web sites and software. However, it is difficult for a non-expert to evaluate how reliable those programs are. Here, we provide a simple benchmark for bacterial sRNA target prediction based on trusted E. coli sRNA/target pairs. We use this benchmark to assess the most recent RNA target predictors as well as ...

  4. Global small RNA chaperone Hfq and regulatory small RNAs are important virulence regulators in Erwinia amylovora.

    Science.gov (United States)

    Zeng, Quan; McNally, R Ryan; Sundin, George W

    2013-04-01

    Hfq is a global small RNA (sRNA) chaperone that interacts with Hfq-regulated sRNAs and functions in the posttranscriptional regulation of gene expression. In this work, we identified Hfq to be a virulence regulator in the Gram-negative fire blight pathogen Erwinia amylovora. Deletion of hfq in E. amylovora Ea1189 significantly reduced bacterial virulence in both immature pear fruits and apple shoots. Analysis of virulence determinants in strain Ea1189Δhfq showed that Hfq exerts pleiotropic regulation of amylovoran exopolysaccharide production, biofilm formation, motility, and the type III secretion system (T3SS). Further characterization of biofilm regulation by Hfq demonstrated that Hfq limits bacterial attachment to solid surfaces while promoting biofilm maturation. Characterization of T3SS regulation by Hfq revealed that Hfq positively regulates the translocation and secretion of the major type III effector DspE and negatively controls the secretion of the putative translocator HrpK and the type III effector Eop1. Lastly, 10 Hfq-regulated sRNAs were identified using a computational method, and two of these sRNAs, RprA and RyhA, were found to be required for the full virulence of E. amylovora.

  5. Transfer RNA-derived small RNAs in the cancer transcriptome

    OpenAIRE

    Green, Darrell; Fraser, William; Dalmay, Tamas

    2016-01-01

    The cellular lifetime includes stages such as differentiation, proliferation, division, senescence and apoptosis.These stages are driven by a strictly ordered process of transcription dynamics. Molecular disruption to RNA polymerase assembly, chromatin remodelling and transcription factor binding through to RNA editing, splicing, post-transcriptional regulation and ribosome scanning can result in significant costs arising from genome instability. Cancer development is one example of when such...

  6. Intersection of small RNA pathways in Arabidopsis thaliana sub-nuclear domains.

    Directory of Open Access Journals (Sweden)

    Olga Pontes

    Full Text Available In Arabidopsis thaliana, functionally diverse small RNA (smRNA pathways bring about decreased RNA accumulation of target genes via several different mechanisms. Cytological experiments have suggested that the processing of microRNAs (miRNAs and heterochromatic small interfering RNAs (hc-siRNAs occurs within a specific nuclear domain that can present Cajal Body (CB characteristics. It is unclear whether single or multiple smRNA-related domains are found within the same CB and how specialization of the smRNA pathways is determined within this specific sub-compartment. To ascertain whether nuclear smRNA centers are spatially related, we localized key proteins required for siRNA or miRNA biogenesis by immunofluorescence analysis. The intranuclear distribution of the proteins revealed that hc-siRNA, miRNA and trans-acting siRNA (ta-siRNA pathway proteins accumulate and colocalize within a sub-nuclear structure in the nucleolar periphery. Furthermore, colocalization of miRNA- and siRNA-pathway members with CB markers, and reduced wild-type localization patterns in CB mutants indicates that proper nuclear localization of these proteins requires CB integrity. We hypothesize that these nuclear domains could be important for RNA silencing and may partially explain the functional redundancies and interactions among components of the same protein family. The CB may be the place in the nucleus where Dicer-generated smRNA precursors are processed and assigned to a specific pathway, and where storage, recycling or assembly of RNA interference components takes place.

  7. The VP3 factor from viruses of Birnaviridae family suppresses RNA silencing by binding both long and small RNA duplexes.

    Directory of Open Access Journals (Sweden)

    Adrian Valli

    Full Text Available RNA silencing is directly involved in antiviral defense in a wide variety of eukaryotic organisms, including plants, fungi, invertebrates, and presumably vertebrate animals. The study of RNA silencing-mediated antiviral defences in vertebrates is hampered by the overlap with other antiviral mechanisms; thus, heterologous systems are often used to study the interplay between RNA silencing and vertebrate-infecting viruses. In this report we show that the VP3 protein of the avian birnavirus Infectious bursal disease virus (IBDV displays, in addition to its capacity to bind long double-stranded RNA, the ability to interact with double-stranded small RNA molecules. We also demonstrate that IBDV VP3 prevents the silencing mediated degradation of a reporter mRNA, and that this silencing suppression activity depends on its RNA binding ability. Furthermore, we find that the anti-silencing activity of IBDV VP3 is shared with the homologous proteins expressed by both insect- and fish-infecting birnaviruses. Finally, we show that IBDV VP3 can functionally replace the well-characterized HCPro silencing suppressor of Plum pox virus, a potyvirus that is unable to infect plants in the absence of an active silencing suppressor. Altogether, our results support the idea that VP3 protects the viral genome from host sentinels, including those of the RNA silencing machinery.

  8. Subcellular distribution of small interfering RNA: directed delivery through RNA polymerase III expression cassettes and localization by in situ hybridization.

    Science.gov (United States)

    Paul, Cynthia P

    2005-01-01

    Reduction in the expression of specific genes through small interfering RNAs (siRNAs) is dependent on the colocalization of siRNAs with other components of the RNA interference (RNAi) pathways within the cell. The expression of siRNAs within cells from cassettes that are derived from genes transcribed by RNA polymerase III (pol III) and provide for selective subcellular distribution of their products can be used to direct siRNAs to the cellular pathways. Expression from the human U6 promoter, resulting in siRNA accumulation in the nucleus, is effective in reducing gene expression, whereas cytoplasmic and nucleolar localization of the siRNA when expressed from the 5S or 7 SL promoters is not effective. The distribution of siRNA within the cell is determined by fluorescence in situ hybridization. Although the long uninterrupted duplex of siRNA makes it difficult to detect with DNA oligonucleotide probes, labeled oligonucleotide probes with 2'-O-methyl RNA backbones provide the stability needed for a strong signal. These methods contribute to studies of the interconnected cellular RNAi pathways and are useful in adapting RNAi as a tool to determine gene function and develop RNA-based therapeutics. PMID:15644179

  9. Structural features of microRNA (miRNA) precursors and their relevance to miRNA biogenesis and small interfering RNA/short hairpin RNA design.

    Science.gov (United States)

    Krol, Jacek; Sobczak, Krzysztof; Wilczynska, Urszula; Drath, Maria; Jasinska, Anna; Kaczynska, Danuta; Krzyzosiak, Wlodzimierz J

    2004-10-01

    We have established the structures of 10 human microRNA (miRNA) precursors using biochemical methods. Eight of these structures turned out to be different from those that were computer-predicted. The differences localized in the terminal loop region and at the opposite side of the precursor hairpin stem. We have analyzed the features of these structures from the perspectives of miRNA biogenesis and active strand selection. We demonstrated the different thermodynamic stability profiles for pre-miRNA hairpins harboring miRNAs at their 5'- and 3'-sides and discussed their functional implications. Our results showed that miRNA prediction based on predicted precursor structures may give ambiguous results, and the success rate is significantly higher for the experimentally determined structures. On the other hand, the differences between the predicted and experimentally determined structures did not affect the stability of termini produced through "conceptual dicing." This result confirms the value of thermodynamic analysis based on mfold as a predictor of strand section by RNAi-induced silencing complex (RISC). PMID:15292246

  10. Small interfering RNA-based molecular therapy of cancers

    Institute of Scientific and Technical Information of China (English)

    Wei Guo; Wangbing Chen; Wendan Yu; Wenlin Huang; Wuguo Deng

    2013-01-01

    RNA interference (RNAi) has become a gold standard for validating gene function in basic life science research and provides a promising therapeutic modality for cancer and other diseases. This mini-review focuses on the potential of smal interfering RNAs (siRNAs) in anticancer treatment, including the establishment and screening of cancer-associated siRNA libraries and their applications in anticancer drug target discovery and cancer therapy. This article also describes the current delivery approaches of siRNAs using lipids, polymers, and, in particular, gold nanoparticles to induce significant gene silencing and tumor growth regression.

  11. Small interfering RNA delivery by polyethylenimine-functionalised porous silicon nanoparticles.

    Science.gov (United States)

    Hasanzadeh Kafshgari, M; Alnakhli, M; Delalat, B; Apostolou, S; Harding, F J; Mäkilä, E; Salonen, J J; Kuss, B J; Voelcker, N H

    2015-12-01

    In this study, thermally hydrocarbonised porous silicon nanoparticles (THCpSiNPs) capped with polyethylenimine (PEI) were fabricated, and their potential for small interfering RNA (siRNA) delivery was investigated in an in vitro glioblastoma model. PEI coating following siRNA loading enhanced the sustained release of siRNA, and suppressed burst release effects. The positively-charged surface improved the internalisation of the nanoparticles across the cell membrane. THCpSiNP-mediated siRNA delivery reduced mRNA expression of the MRP1 gene, linked to the resistence of glioblastoma to chemotherapy, by 63% and reduced MRP1-protein levels by 70%. MRP1 siRNA loaded nanoparticles did not induce cytotoxicity in glioblastoma cells, but markedly reduced cell proliferation. In summary, the results demonstrated that non-cytotoxic cationic THCpSiNPs are promising vehicles for therapeutic siRNA delivery.

  12. Initiation of RNA Polymerization and Polymerase Encapsidation by a Small dsRNA Virus.

    Directory of Open Access Journals (Sweden)

    Aaron M Collier

    2016-04-01

    Full Text Available During the replication cycle of double-stranded (ds RNA viruses, the viral RNA-dependent RNA polymerase (RdRP replicates and transcribes the viral genome from within the viral capsid. How the RdRP molecules are packaged within the virion and how they function within the confines of an intact capsid are intriguing questions with answers that most likely vary across the different dsRNA virus families. In this study, we have determined a 2.4 Å resolution structure of an RdRP from the human picobirnavirus (hPBV. In addition to the conserved polymerase fold, the hPBV RdRP possesses a highly flexible 24 amino acid loop structure located near the C-terminus of the protein that is inserted into its active site. In vitro RNA polymerization assays and site-directed mutagenesis showed that: (1 the hPBV RdRP is fully active using both ssRNA and dsRNA templates; (2 the insertion loop likely functions as an assembly platform for the priming nucleotide to allow de novo initiation; (3 RNA transcription by the hPBV RdRP proceeds in a semi-conservative manner; and (4 the preference of virus-specific RNA during transcription is dictated by the lower melting temperature associated with the terminal sequences. Co-expression of the hPBV RdRP and the capsid protein (CP indicated that, under the conditions used, the RdRP could not be incorporated into the recombinant capsids in the absence of the viral genome. Additionally, the hPBV RdRP exhibited higher affinity towards the conserved 5'-terminal sequence of the viral RNA, suggesting that the RdRP molecules may be encapsidated through their specific binding to the viral RNAs during assembly.

  13. Small noncoding RNA modulates japanese encephalitis virus replication and translation in trans

    Directory of Open Access Journals (Sweden)

    Fan Yi-Hsin

    2011-11-01

    Full Text Available Abstract Background Sequence and structural elements in the 3'-untranslated region (UTR of Japanese encephalitis virus (JEV are known to regulate translation and replication. We previously reported an abundant accumulation of small subgenomic flaviviral RNA (sfRNA which is collinear with the highly conserved regions of the 3'-UTR in JEV-infected cells. However, function of the sfRNA in JEV life cycle remains unknown. Results Northern blot and real-time RT-PCR analyses indicated that the sfRNA becomes apparent at the time point at which minus-strand RNA (antigenome reaches a plateau suggesting a role for sfRNA in the regulation of antigenome synthesis. Transfection of minus-sense sfRNA into JEV-infected cells, in order to counter the effects of plus-sense sfRNA, resulted in higher levels of antigenome suggesting that the presence of the sfRNA inhibits antigenome synthesis. Trans-acting effect of sfRNA on JEV translation was studied using a reporter mRNA containing the luciferase gene fused to partial coding regions of JEV and flanked by the respective JEV UTRs. In vivo and in vitro translation revealed that sfRNA inhibited JEV translation. Conclusions Our results indicate that sfRNA modulates viral translation and replication in trans.

  14. Intravesical administration of small interfering RNA targeting PLK-1 successfully prevents the growth of bladder cancer

    OpenAIRE

    Nogawa, Masaki; Yuasa, Takeshi; Kimura, Shinya; Tanaka, Motoyoshi; Kuroda, Junya; SATO, Kiyoshi; Yokota, Asumi; Segawa, Hidekazu; Toda, Yoshinobu; Kageyama, Susumu; YOSHIKI, Tatsuhiro; Okada, Yusaku; Maekawa, Taira

    2005-01-01

    The mainstay in the management of invasive bladder cancer continues to be radical cystectomy. With regard to improvement of quality of life, however, therapies that preserve the bladder are desirable. We investigated the use of intravesical PLK-1 small interfering RNA (siRNA) against bladder cancer. Patients with bladder cancers expressing high levels of PLK-1 have a poor prognosis compared with patients with low expression. Using siRNA/cationic liposomes, the expression of endogenous PLK-1 c...

  15. Fluorescent tag is not a reliable marker for small RNA transfection in the presence of serum

    Indian Academy of Sciences (India)

    Jing Han; Qi-Wei Wang; Shi-Qiang Wang

    2013-09-01

    Chemically synthetic siRNA and miRNA have become powerful tools to study gene function in the past decade. Fluorescent dyes covalently attached to the 5′ or 3′ ends of synthetic small RNAs are widely used for fluorescently imaging and detection of these RNAs. However, the reliability of fluorescent tags as small RNA markers in different conditions has not attracted enough attention. We used Cy3-labelled small RNAs to explore the reliability of fluorescent tags as small RNA markers in cell cultures involving serum. A strong Cy3-fluorescence signal was observed in the cytoplasm of the cells transfected with Cy3-miR24 in the culture medium containing fetal bovine serum (FBS), but qRT-PCR results showed that little miR24 were detected in these cells. Further study demonstrated that small RNAs were degraded in the presence of FBS, suggesting that it was Cy3-RNA fragments, rather than the original Cy3-miR24, diffused into cells. These phenomena disappeared when FBS was replaced by boiled-FBS, further supporting that the Cy3-fluorescence we observed in cells in the presence of FBS could not represent the presence of intact small RNAs. These findings addressed that fluorescent tags are not reliable for small RNA transfection in the presence of serum in culture.

  16. Inhibiting severe acute respiratory syndrome-associated coronavirus by small interfering RNA

    Institute of Scientific and Technical Information of China (English)

    张仁礼; 郭中敏; 陆家海; 孟锦绣; 周灿权; 詹希美; 黄冰; 余新炳; 黄民; 潘兴华; 凌文华; 陈系古; 万卓越; 郑焕英; 鄢心革; 王一飞; 冉延超; 刘新健; 马俊鑫; 王承宇; 张必良

    2003-01-01

    Objective To evaluate the effectiveness of small interfering RNA (siRNA) on inhibiting severe acute respiratory syndrome (SARS)-associated coronavirus replication, and to lay bases for the future clinical application of siRNA for the treatment of viral infectious diseases.Methods Vero-E6 cells was transfected with siRNA before SARS virus infection, and the effectiveness of siRNA interference was evaluated by observing the cytopathic effect (CPE) on Vero-E6 cells.Results Five pairs of siRNA showed ability to reduce CPE dose dependently, and two of them had the best effect. Conclusion siRNA may be effective in inhibiting SARS-associated coronavirus replication.

  17. Phytophthora have distinct endogenous small RNA populations that include short interfering and microRNAs

    Science.gov (United States)

    In eukaryotes, RNA silencing pathways utilize 20–30-nucleotide small RNAs to regulate gene expression, specify and maintain chromatin structure, and repress viruses and mobile genetic elements. RNA silencing was likely present in the common ancestor of modern eukaryotes, but most research has focuse...

  18. Nucleic acids encoding phloem small RNA-binding proteins and transgenic plants comprising them

    Science.gov (United States)

    Lucas, William J.; Yoo, Byung-Chun; Lough, Tony J.; Varkonyi-Gasic, Erika

    2007-03-13

    The present invention provides a polynucleotide sequence encoding a component of the protein machinery involved in small RNA trafficking, Cucurbita maxima phloem small RNA-binding protein (CmPSRB 1), and the corresponding polypeptide sequence. The invention also provides genetic constructs and transgenic plants comprising the polynucleotide sequence encoding a phloem small RNA-binding protein to alter (e.g., prevent, reduce or elevate) non-cell autonomous signaling events in the plants involving small RNA metabolism. These signaling events are involved in a broad spectrum of plant physiological and biochemical processes, including, for example, systemic resistance to pathogens, responses to environmental stresses, e.g., heat, drought, salinity, and systemic gene silencing (e.g., viral infections).

  19. Radiosensitization by small interfering RNAs (siRNA) targeting ATM

    International Nuclear Information System (INIS)

    Previous work by us (Guha, C., et al, Gene Therapy 7, 2000 and Fan, Z., et al, Human Gene Therapy 7, 2000), using antisense ATM approaches demonstrated radiosensitization of prostate and glioblastoma cell lines. In an attempt to further develop radiosensitizing gene therapy strategies for attenuation of ATM protein expression, we screened a series of siRNAs against ATM in human transformed kidney and cervical carcinoma cells. siRNAs were constructed as double-stranded ATM siRNA or the siRNA Hairpin cloned into pSilencer 1.0 -U6 expression vectors and transfected into HeLa and 293 cells. All transfected cell-lines were clonally expanded and isolated for Western blot analysis. Clonogenic survival assay (0 - 10Gy single dose or 2Gy x 2q 4hr separation) for selected transfectant lines was perfomed. Cell cycle progression and S-phase fraction were determined by FACScan analysis. Significant down-regulation of ATM expression occurred as early as 48hrs in both oligonucleotide and plasmid-transfected cells. Protein down-regulation was dependent on target sequences selected and independent of format, whether in oligonucleotide only or as hairpin-plasmid. These siRNAs also demonstrated cytotoxicity as assessed by reduction of plating efficiencies in clonogenic assay. ATM siRNA-transfected cells exhibited enhanced radiosensitivity, compared to cells transfected with control vectors. These data suggest that attenuation of ATM by transfection of siRNAs against ATM could be useful tools for studying the role of ATM in radiosensitivty of tumors. Adenoviral vectors expressing these siRNAs are being developed for potential use in radiosensitizing gene therapy

  20. Small regulatory RNA-induced growth rate heterogeneity of Bacillus subtilis.

    OpenAIRE

    Mars, Ruben A. T.; Pierre Nicolas; Mariano Ciccolini; Ewoud Reilman; Alexander Reder; Marc Schaffer; Ulrike Mäder; Uwe Völker; Jan Maarten van Dijl; Denham, Emma L.

    2015-01-01

    Isogenic bacterial populations can consist of cells displaying heterogeneous physiological traits. Small regulatory RNAs (sRNAs) could affect this heterogeneity since they act by fine-tuning mRNA or protein levels to coordinate the appropriate cellular behavior. Here we show that the sRNA RnaC/S1022 from the Gram-positive bacterium Bacillus subtilis can suppress exponential growth by modulation of the transcriptional regulator AbrB. Specifically, the post-transcriptional abrB-RnaC/S1022 inter...

  1. Final report for ER65039, The Role of Small RNA in Biomass Deposition

    Energy Technology Data Exchange (ETDEWEB)

    Hudson, Matthew E. [Univ. of Illinois, Urbana, IL (United States)

    2015-03-12

    Our objective in this project was to discover the role of sRNA in regulating both biomass biosynthesis and perenniality in the Andropogoneae feedstock grasses. Our central hypothesis was that there is a time-and space specific sRNA network playing a crucial role in regulating processes associated with cell wall biosynthesis, flowering time control, overwintering/juvenility, and nutrient sequestration in the feedstock grasses. To address this, we performed a large scale biological project consisting of the growth of material, generation of Illumina libraries, sequencing and analysis for small RNA, mRNA and Degradome / cmRNA. Our subsidiary objectives included analysis of the biology of small RNAs and the cell wall composition of Miscanthus. These objectives have all been completed, one publication is in print, one is submitted and several more are in progress.

  2. PSRna: Prediction of small RNA secondary structures based on reverse complementary folding method.

    Science.gov (United States)

    Li, Jin; Xu, Chengzhen; Wang, Lei; Liang, Hong; Feng, Weixing; Cai, Zhongxi; Wang, Ying; Cong, Wang; Liu, Yunlong

    2016-08-01

    Prediction of RNA secondary structures is an important problem in computational biology and bioinformatics, since RNA secondary structures are fundamental for functional analysis of RNA molecules. However, small RNA secondary structures are scarce and few algorithms have been specifically designed for predicting the secondary structures of small RNAs. Here we propose an algorithm named "PSRna" for predicting small-RNA secondary structures using reverse complementary folding and characteristic hairpin loops of small RNAs. Unlike traditional algorithms that usually generate multi-branch loops and 5[Formula: see text] end self-folding, PSRna first estimated the maximum number of base pairs of RNA secondary structures based on the dynamic programming algorithm and a path matrix is constructed at the same time. Second, the backtracking paths are extracted from the path matrix based on backtracking algorithm, and each backtracking path represents a secondary structure. To improve accuracy, the predicted RNA secondary structures are filtered based on their free energy, where only the secondary structure with the minimum free energy was identified as the candidate secondary structure. Our experiments on real data show that the proposed algorithm is superior to two popular methods, RNAfold and RNAstructure, in terms of sensitivity, specificity and Matthews correlation coefficient (MCC). PMID:27045556

  3. Bias in ligation-based small RNA sequencing library construction is determined by adaptor and RNA structure.

    Directory of Open Access Journals (Sweden)

    Ryan T Fuchs

    Full Text Available High-throughput sequencing (HTS has become a powerful tool for the detection of and sequence characterization of microRNAs (miRNA and other small RNAs (sRNA. Unfortunately, the use of HTS data to determine the relative quantity of different miRNAs in a sample has been shown to be inconsistent with quantitative PCR and Northern Blot results. Several recent studies have concluded that the major contributor to this inconsistency is bias introduced during the construction of sRNA libraries for HTS and that the bias is primarily derived from the adaptor ligation steps, specifically where single stranded adaptors are sequentially ligated to the 3' and 5'-end of sRNAs using T4 RNA ligases. In this study we investigated the effects of ligation bias by using a pool of randomized ligation substrates, defined mixtures of miRNA sequences and several combinations of adaptors in HTS library construction. We show that like the 3' adaptor ligation step, the 5' adaptor ligation is also biased, not because of primary sequence, but instead due to secondary structures of the two ligation substrates. We find that multiple secondary structural factors influence final representation in HTS results. Our results provide insight about the nature of ligation bias and allowed us to design adaptors that reduce ligation bias and produce HTS results that more accurately reflect the actual concentrations of miRNAs in the defined starting material.

  4. Comparative genomics of small RNA regulatory pathway components in vector mosquitoes

    Directory of Open Access Journals (Sweden)

    Foy Brian D

    2008-09-01

    Full Text Available Abstract Background Small RNA regulatory pathways (SRRPs control key aspects of development and anti-viral defense in metazoans. Members of the Argonaute family of catalytic enzymes degrade target RNAs in each of these pathways. SRRPs include the microRNA, small interfering RNA (siRNA and PIWI-type gene silencing pathways. Mosquitoes generate viral siRNAs when infected with RNA arboviruses. However, in some mosquitoes, arboviruses survive antiviral RNA interference (RNAi and are transmitted via mosquito bite to a subsequent host. Increased knowledge of these pathways and functional components should increase understanding of the limitations of anti-viral defense in vector mosquitoes. To do this, we compared the genomic structure of SRRP components across three mosquito species and three major small RNA pathways. Results The Ae. aegypti, An. gambiae and Cx. pipiens genomes encode putative orthologs for all major components of the miRNA, siRNA, and piRNA pathways. Ae. aegypti and Cx. pipiens have undergone expansion of Argonaute and PIWI subfamily genes. Phylogenetic analyses were performed for these protein families. In addition, sequence pattern recognition algorithms MEME, MDScan and Weeder were used to identify upstream regulatory motifs for all SRRP components. Statistical analyses confirmed enrichment of species-specific and pathway-specific cis-elements over the rest of the genome. Conclusion Analysis of Argonaute and PIWI subfamily genes suggests that the small regulatory RNA pathways of the major arbovirus vectors, Ae. aegypti and Cx. pipiens, are evolving faster than those of the malaria vector An. gambiae and D. melanogaster. Further, protein and genomic features suggest functional differences between subclasses of PIWI proteins and provide a basis for future analyses. Common UCR elements among SRRP components indicate that 1 key components from the miRNA, siRNA, and piRNA pathways contain NF-kappaB-related and Broad complex

  5. In silico reconstruction of viral genomes from small RNAs improves virus-derived small interfering RNA profiling.

    Science.gov (United States)

    Vodovar, Nicolas; Goic, Bertsy; Blanc, Hervé; Saleh, Maria-Carla

    2011-11-01

    RNA interference (RNAi) is the essential component of antiviral immunity in invertebrates and plants. One of the landmarks of the antiviral RNAi response is the production of virus-derived small interfering RNA (vsiRNA) from viral double-stranded RNA (dsRNA). vsiRNAs constitute a fragmented image of the viral genome sequence that results from Dicer cleavage. vsiRNA sequence profiling is used extensively as a surrogate to study the antiviral RNAi response by determining the nature of the viral dsRNA molecules exposed to and processed by the RNAi machinery. The accuracy of these profiles depends on the actual viral genome sequence used as a reference to align vsiRNA reads, and the interpretation of inaccurate profiles can be misleading. Using Flock house virus and Drosophila melanogaster as a model RNAi-competent organism, we show accurate reconstruction of full-length virus reference sequence from vsiRNAs and prediction of the structure of defective interfering particles (DIs). We developed a Perl script, named Paparazzi, that reconstitutes viral genomes through an iterative alignment/consensus call procedure using a related reference sequence as scaffold. As prevalent DI-derived reads introduce artifacts during reconstruction, Paparazzi eliminates DI-specific reads to improve the quality of the reconstructed genome. Paparazzi constitutes a promising alternative to Sanger sequencing in this context and an effective tool to study antiviral RNAi mechanisms by accurately quantifying vsiRNA along the replicating viral genome. We further discuss Paparazzi as a companion tool for virus discovery as it provides full-length genome sequences and corrects for potential artifacts of assembly. PMID:21880776

  6. In Silico Reconstruction of Viral Genomes from Small RNAs Improves Virus-Derived Small Interfering RNA Profiling ▿ † ‡

    Science.gov (United States)

    Vodovar, Nicolas; Goic, Bertsy; Blanc, Hervé; Saleh, Maria-Carla

    2011-01-01

    RNA interference (RNAi) is the essential component of antiviral immunity in invertebrates and plants. One of the landmarks of the antiviral RNAi response is the production of virus-derived small interfering RNA (vsiRNA) from viral double-stranded RNA (dsRNA). vsiRNAs constitute a fragmented image of the viral genome sequence that results from Dicer cleavage. vsiRNA sequence profiling is used extensively as a surrogate to study the antiviral RNAi response by determining the nature of the viral dsRNA molecules exposed to and processed by the RNAi machinery. The accuracy of these profiles depends on the actual viral genome sequence used as a reference to align vsiRNA reads, and the interpretation of inaccurate profiles can be misleading. Using Flock house virus and Drosophila melanogaster as a model RNAi-competent organism, we show accurate reconstruction of full-length virus reference sequence from vsiRNAs and prediction of the structure of defective interfering particles (DIs). We developed a Perl script, named Paparazzi, that reconstitutes viral genomes through an iterative alignment/consensus call procedure using a related reference sequence as scaffold. As prevalent DI-derived reads introduce artifacts during reconstruction, Paparazzi eliminates DI-specific reads to improve the quality of the reconstructed genome. Paparazzi constitutes a promising alternative to Sanger sequencing in this context and an effective tool to study antiviral RNAi mechanisms by accurately quantifying vsiRNA along the replicating viral genome. We further discuss Paparazzi as a companion tool for virus discovery as it provides full-length genome sequences and corrects for potential artifacts of assembly. PMID:21880776

  7. Silencing-associated and meiosis-specific small RNA pathways in Paramecium tetraurelia

    Science.gov (United States)

    Lepère, Gersende; Nowacki, Mariusz; Serrano, Vincent; Gout, Jean-François; Guglielmi, Gérard; Duharcourt, Sandra; Meyer, Eric

    2009-01-01

    Distinct small RNA pathways are involved in the two types of homology-dependent effects described in Paramecium tetraurelia, as shown by a functional analysis of Dicer and Dicer-like genes and by the sequencing of small RNAs. The siRNAs that mediate post-transcriptional gene silencing when cells are fed with double-stranded RNA (dsRNA) were found to comprise two subclasses. DCR1-dependent cleavage of the inducing dsRNA generates ∼23-nt primary siRNAs from both strands, while a different subclass of ∼24-nt RNAs, characterized by a short untemplated poly-A tail, is strictly antisense to the targeted mRNA, suggestive of secondary siRNAs that depend on an RNA-dependent RNA polymerase. An entirely distinct pathway is responsible for homology-dependent regulation of developmental genome rearrangements after sexual reproduction. During early meiosis, the DCL2 and DCL3 genes are required for the production of a highly complex population of ∼25-nt scnRNAs from all types of germline sequences, including both strands of exons, introns, intergenic regions, transposons and Internal Eliminated Sequences. A prominent 5′-UNG signature, and a minor fraction showing the complementary signature at positions 21–23, indicate that scnRNAs are cleaved from dsRNA precursors as duplexes with 2-nt 3′ overhangs at both ends, followed by preferential stabilization of the 5′-UNG strand. PMID:19103667

  8. Inhibition of hepatitis B virus surface antigen expression by small hairpin RNA in vitro

    Institute of Scientific and Technical Information of China (English)

    Zheng-Gang Yang; Zhi Chen; Qin Ni; Ning Xu; Jun-Bin Shao; Hang-Ping Yao

    2005-01-01

    AIM: To explore the anti-hepatitis B virus effect of RNA interference (RNAi) using small hairpin RNA (shRNA)expression vector.METHODS: Hepatitis B virus surface antigen green fluorescent protein (HBs-GFP) fusion vector and shRNA expression vectors were constructed and cotransfected transiently into HepG2 cells. mRNAs extracted from HepG2 cells were detected by real-time PCR. Fluorescence of HBs-GFP protein was detected by fluorescence-activated cell sorting (FACS). The effective shRNA expression vector was transfected into HepG2.2.15 cells. HBsAg and HBeAg in HepG2.2.15 cells were analyzed by radioimmunoassay (RIA) method.RESULTS: FACS revealed that shRNA targeting at HBsAg reduced the GFP signal by 56% compared to the control.Real-time PCR showed that HBs-GFP mRNA extracted from HepG2 cells cotransfected with pAVU6+27 and HBs-GFP expression plasmids decreased by 90% compared to the empty vector control. The expressions of HBsAg and HBeAg were also inhibited by 43% and 64%, respectively.CONCLUSION: RNAi using shRNA expression vector can inhibit the expression of HBsAg, providing a fresh approach to screening the efficient small interfering RNAs (siRNAs).

  9. Gene silencing in tick cell lines using small interfering or long double-stranded RNA.

    Science.gov (United States)

    Barry, Gerald; Alberdi, Pilar; Schnettler, Esther; Weisheit, Sabine; Kohl, Alain; Fazakerley, John K; Bell-Sakyi, Lesley

    2013-03-01

    Gene silencing by RNA interference (RNAi) is an important research tool in many areas of biology. To effectively harness the power of this technique in order to explore tick functional genomics and tick-microorganism interactions, optimised parameters for RNAi-mediated gene silencing in tick cells need to be established. Ten cell lines from four economically important ixodid tick genera (Amblyomma, Hyalomma, Ixodes and Rhipicephalus including the sub-species Boophilus) were used to examine key parameters including small interfering RNA (siRNA), double stranded RNA (dsRNA), transfection reagent and incubation time for silencing virus reporter and endogenous tick genes. Transfection reagents were essential for the uptake of siRNA whereas long dsRNA alone was taken up by most tick cell lines. Significant virus reporter protein knockdown was achieved using either siRNA or dsRNA in all the cell lines tested. Optimum conditions varied according to the cell line. Consistency between replicates and duration of incubation with dsRNA were addressed for two Ixodes scapularis cell lines; IDE8 supported more consistent and effective silencing of the endogenous gene subolesin than ISE6, and highly significant knockdown of the endogenous gene 2I1F6 in IDE8 cells was achieved within 48 h incubation with dsRNA. In summary, this study shows that gene silencing by RNAi in tick cell lines is generally more efficient with dsRNA than with siRNA but results vary between cell lines and optimal parameters need to be determined for each experimental system.

  10. RNA-Seq of the nucleolus reveals abundant SNORD44-derived small RNAs.

    Directory of Open Access Journals (Sweden)

    Baoyan Bai

    Full Text Available Small non-coding RNAs represent RNA species that are not translated to proteins, but which have diverse and broad functional activities in physiological and pathophysiological states. The knowledge of these small RNAs is rapidly expanding in part through the use of massive parallel (deep sequencing efforts. We present here the first deep sequencing of small RNomes in subcellular compartments with particular emphasis on small RNAs (sRNA associated with the nucleolus. The vast majority of the cellular, cytoplasmic and nuclear sRNAs were identified as miRNAs. In contrast, the nucleolar sRNAs had a unique size distribution consisting of 19-20 and 25 nt RNAs, which were predominantly composed of small snoRNA-derived box C/D RNAs (termed as sdRNA. Sequences from 47 sdRNAs were identified, which mapped to both 5' and 3' ends of the snoRNAs, and retained conserved box C or D motifs. SdRNA reads mapping to SNORD44 comprised 74% of all nucleolar sdRNAs, and were confirmed by Northern blotting as comprising both 20 and 25 nt RNAs. A novel 120 nt SNORD44 form was also identified. The expression of the SNORD44 sdRNA and 120 nt form was independent of Dicer/Drosha-mediated processing pathways but was dependent on the box C/D snoRNP proteins/sno-ribonucleoproteins fibrillarin and NOP58. The 120 nt SNORD44-derived RNA bound to fibrillarin suggesting that C/D sno-ribonucleoproteins are involved in regulating the stability or processing of SNORD44. This study reveals sRNA cell-compartment specific expression and the distinctive unique composition of the nucleolar sRNAs.

  11. RNA-Seq of the nucleolus reveals abundant SNORD44-derived small RNAs.

    Science.gov (United States)

    Bai, Baoyan; Yegnasubramanian, Srinivasan; Wheelan, Sarah J; Laiho, Marikki

    2014-01-01

    Small non-coding RNAs represent RNA species that are not translated to proteins, but which have diverse and broad functional activities in physiological and pathophysiological states. The knowledge of these small RNAs is rapidly expanding in part through the use of massive parallel (deep) sequencing efforts. We present here the first deep sequencing of small RNomes in subcellular compartments with particular emphasis on small RNAs (sRNA) associated with the nucleolus. The vast majority of the cellular, cytoplasmic and nuclear sRNAs were identified as miRNAs. In contrast, the nucleolar sRNAs had a unique size distribution consisting of 19-20 and 25 nt RNAs, which were predominantly composed of small snoRNA-derived box C/D RNAs (termed as sdRNA). Sequences from 47 sdRNAs were identified, which mapped to both 5' and 3' ends of the snoRNAs, and retained conserved box C or D motifs. SdRNA reads mapping to SNORD44 comprised 74% of all nucleolar sdRNAs, and were confirmed by Northern blotting as comprising both 20 and 25 nt RNAs. A novel 120 nt SNORD44 form was also identified. The expression of the SNORD44 sdRNA and 120 nt form was independent of Dicer/Drosha-mediated processing pathways but was dependent on the box C/D snoRNP proteins/sno-ribonucleoproteins fibrillarin and NOP58. The 120 nt SNORD44-derived RNA bound to fibrillarin suggesting that C/D sno-ribonucleoproteins are involved in regulating the stability or processing of SNORD44. This study reveals sRNA cell-compartment specific expression and the distinctive unique composition of the nucleolar sRNAs.

  12. Potent host-directed small-molecule inhibitors of myxovirus RNA-dependent RNA-polymerases.

    Directory of Open Access Journals (Sweden)

    Stefanie A Krumm

    Full Text Available Therapeutic targeting of host cell factors required for virus replication rather than of pathogen components opens new perspectives to counteract virus infections. Anticipated advantages of this approach include a heightened barrier against the development of viral resistance and a broadened pathogen target spectrum. Myxoviruses are predominantly associated with acute disease and thus are particularly attractive for this approach since treatment time can be kept limited. To identify inhibitor candidates, we have analyzed hit compounds that emerged from a large-scale high-throughput screen for their ability to block replication of members of both the orthomyxovirus and paramyxovirus families. This has returned a compound class with broad anti-viral activity including potent inhibition of different influenza virus and paramyxovirus strains. After hit-to-lead chemistry, inhibitory concentrations are in the nanomolar range in the context of immortalized cell lines and human PBMCs. The compound shows high metabolic stability when exposed to human S-9 hepatocyte subcellular fractions. Antiviral activity is host-cell species specific and most pronounced in cells of higher mammalian origin, supporting a host-cell target. While the compound induces a temporary cell cycle arrest, host mRNA and protein biosynthesis are largely unaffected and treated cells maintain full metabolic activity. Viral replication is blocked at a post-entry step and resembles the inhibition profile of a known inhibitor of viral RNA-dependent RNA-polymerase (RdRp activity. Direct assessment of RdRp activity in the presence of the reagent reveals strong inhibition both in the context of viral infection and in reporter-based minireplicon assays. In toto, we have identified a compound class with broad viral target range that blocks host factors required for viral RdRp activity. Viral adaptation attempts did not induce resistance after prolonged exposure, in contrast to rapid

  13. Characterization of Argonaute-related small RNA pathways in Caenorhabditis elegans

    OpenAIRE

    Batista, Pedro Jorge de Oliveira Rodrigues

    2010-01-01

    Tese de doutoramento, Biologia (Genética), Universidade de Lisboa, Faculdade de Ciências, 2011 In Small-RNA-mediated pathways, small RNAs engage a protein of the Argonaute family and utilize base-pairing interactions to identify and regulate complementary genetic information. My research has focused on understanding how diverse classes of small RNAs in the model organism Caenorhabditis elegans interact with specific members of the Argonaute protein family to carry out unique bi...

  14. The small 6C RNA of Corynebacterium glutamicum is involved in the SOS response.

    Science.gov (United States)

    Pahlke, Jennifer; Dostálová, Hana; Holátko, Jiří; Degner, Ursula; Bott, Michael; Pátek, Miroslav; Polen, Tino

    2016-09-01

    The 6C RNA family is a class of small RNAs highly conserved in Actinobacteria, including the genera Mycobacterium, Streptomyces and Corynebacterium whose physiological function has not yet been elucidated. We found that strong transcription of the cgb_03605 gene, which encodes 6C RNA in C. glutamicum, was driven by the SigA- and SigB-dependent promoter Pcgb_03605. 6C RNA was detected at high level during exponential growth phase (180 to 240 molcules per cell) which even increased at the entry of the stationary phase. 6C RNA level did not decrease within 240 min after transcription had been stopped with rifampicin, which suggests high 6C RNA stability. The expression of cgb_03605 further increased approximately twofold in the presence of DNA-damaging mitomycin C (MMC) and nearly threefold in the absence of LexA. Deletion of the 6C RNA gene cgb_03605 resulted in a higher sensitivity of C. glutamicum toward MMC and UV radiation. These results indicate that 6C RNA is involved in the DNA damage response. Both 6C RNA level-dependent pausing of cell growth and branched cell morphology in response to MMC suggest that 6C RNA may also be involved in a control of cell division.

  15. Biogenesis of RNA Polymerases II and III Requires the Conserved GPN Small GTPases in Saccharomyces cerevisiae

    Science.gov (United States)

    Minaker, Sean W.; Filiatrault, Megan C.; Ben-Aroya, Shay; Hieter, Philip; Stirling, Peter C.

    2013-01-01

    The GPN proteins are a poorly characterized and deeply evolutionarily conserved family of three paralogous small GTPases, Gpn1, 2, and 3. The founding member, GPN1/NPA3/XAB1, is proposed to function in nuclear import of RNA polymerase II along with a recently described protein called Iwr1. Here we show that the previously uncharacterized protein Gpn2 binds both Gpn3 and Npa3/Gpn1 and that temperature-sensitive alleles of Saccharomyces cerevisiae GPN2 and GPN3 exhibit genetic interactions with RNA polymerase II mutants, hypersensitivity to transcription inhibition, and defects in RNA polymerase II nuclear localization. Importantly, we identify previously unrecognized RNA polymerase III localization defects in GPN2, GPN3, and IWR1 mutant backgrounds but find no localization defects of unrelated nuclear proteins or of RNA polymerase I. Previously, it was unclear whether the GPN proteins and Iwr1 had overlapping function in RNA polymerase II assembly or import. In this study, we show that the nuclear import defect of iwr1Δ, but not the GPN2 or GPN3 mutant defects, is partially suppressed by fusion of a nuclear localization signal to the RNA polymerase II subunit Rpb3. These data, combined with strong genetic interactions between GPN2 and IWR1, suggest that the GPN proteins function upstream of Iwr1 in RNA polymerase II and III biogenesis. We propose that the three GPN proteins execute a common, and likely essential, function in RNA polymerase assembly and transport. PMID:23267056

  16. The small 6C RNA of Corynebacterium glutamicum is involved in the SOS response.

    Science.gov (United States)

    Pahlke, Jennifer; Dostálová, Hana; Holátko, Jiří; Degner, Ursula; Bott, Michael; Pátek, Miroslav; Polen, Tino

    2016-09-01

    The 6C RNA family is a class of small RNAs highly conserved in Actinobacteria, including the genera Mycobacterium, Streptomyces and Corynebacterium whose physiological function has not yet been elucidated. We found that strong transcription of the cgb_03605 gene, which encodes 6C RNA in C. glutamicum, was driven by the SigA- and SigB-dependent promoter Pcgb_03605. 6C RNA was detected at high level during exponential growth phase (180 to 240 molcules per cell) which even increased at the entry of the stationary phase. 6C RNA level did not decrease within 240 min after transcription had been stopped with rifampicin, which suggests high 6C RNA stability. The expression of cgb_03605 further increased approximately twofold in the presence of DNA-damaging mitomycin C (MMC) and nearly threefold in the absence of LexA. Deletion of the 6C RNA gene cgb_03605 resulted in a higher sensitivity of C. glutamicum toward MMC and UV radiation. These results indicate that 6C RNA is involved in the DNA damage response. Both 6C RNA level-dependent pausing of cell growth and branched cell morphology in response to MMC suggest that 6C RNA may also be involved in a control of cell division. PMID:27362471

  17. The influenza A virus NS1 protein binds small interfering RNAs and suppresses RNA silencing in plants

    NARCIS (Netherlands)

    Bucher, E.C.; Hemmes, J.C.; Haan, de P.; Goldbach, R.W.; Prins, M.W.

    2004-01-01

    RNA silencing comprises a set of sequence-specific RNA degradation pathways that occur in a wide range of eukaryotes, including animals, fungi and plants. A hallmark of RNA silencing is the presence of small interfering RNA molecules (siRNAs). The siRNAs are generated by cleavage of larger double-st

  18. Detailed characterization of the posttranscriptional gene-silencing-related small RNA in a GUS gene-silenced tobacco

    NARCIS (Netherlands)

    Hutvágner, G.; Mlynarova, L.; Nap, J.P.H.

    2000-01-01

    Posttranscriptional gene-silencing phenomena such as cosuppression and RNA interference are associated with the occurrence of small, about 21-23 nt short RNA species homologous to the silenced gene. We here show that the small RNA present in silenced transgenic plants can easily be detected in total

  19. A cost-effective method for Illumina small RNA-Seq library preparation using T4 RNA ligase 1 adenylated adapters

    Directory of Open Access Journals (Sweden)

    Chen Yun-Ru

    2012-09-01

    Full Text Available Abstract Background Deep sequencing is a powerful tool for novel small RNA discovery. Illumina small RNA sequencing library preparation requires a pre-adenylated 3’ end adapter containing a 5’,5’-adenyl pyrophosphoryl moiety. In the absence of ATP, this adapter can be ligated to the 3’ hydroxyl group of small RNA, while RNA self-ligation and concatenation are repressed. Pre-adenylated adapters are one of the most essential and costly components required for library preparation, and few are commercially available. Results We demonstrate that DNA oligo with 5’ phosphate and 3’ amine groups can be enzymatically adenylated by T4 RNA ligase 1 to generate customized pre-adenylated adapters. We have constructed and sequenced a small RNA library for tomato (Solanum lycopersicum using the T4 RNA ligase 1 adenylated adapter. Conclusion We provide an efficient and low-cost method for small RNA sequencing library preparation, which takes two days to complete and costs around $20 per library. This protocol has been tested in several plant species for small RNA sequencing including sweet potato, pepper, watermelon, and cowpea, and could be readily applied to any RNA samples.

  20. Comparative analysis of the small RNA transcriptomes of Pinus contorta and Oryza sativa.

    Science.gov (United States)

    Morin, Ryan D; Aksay, Gozde; Dolgosheina, Elena; Ebhardt, H Alexander; Magrini, Vincent; Mardis, Elaine R; Sahinalp, S Cenk; Unrau, Peter J

    2008-04-01

    The diversity of microRNAs and small-interfering RNAs has been extensively explored within angiosperms by focusing on a few key organisms such as Oryza sativa and Arabidopsis thaliana. A deeper division of the plants is defined by the radiation of the angiosperms and gymnosperms, with the latter comprising the commercially important conifers. The conifers are expected to provide important information regarding the evolution of highly conserved small regulatory RNAs. Deep sequencing provides the means to characterize and quantitatively profile small RNAs in understudied organisms such as these. Pyrosequencing of small RNAs from O. sativa revealed, as expected, approximately 21- and approximately 24-nt RNAs. The former contained known microRNAs, and the latter largely comprised intergenic-derived sequences likely representing heterochromatin siRNAs. In contrast, sequences from Pinus contorta were dominated by 21-nt small RNAs. Using a novel sequence-based clustering algorithm, we identified sequences belonging to 18 highly conserved microRNA families in P. contorta as well as numerous clusters of conserved small RNAs of unknown function. Using multiple methods, including expressed sequence folding and machine learning algorithms, we found a further 53 candidate novel microRNA families, 51 appearing specific to the P. contorta library. In addition, alignment of small RNA sequences to the O. sativa genome revealed six perfectly conserved classes of small RNA that included chloroplast transcripts and specific types of genomic repeats. The conservation of microRNAs and other small RNAs between the conifers and the angiosperms indicates that important RNA silencing processes were highly developed in the earliest spermatophytes. Genomic mapping of all sequences to the O. sativa genome can be viewed at http://microrna.bcgsc.ca/cgi-bin/gbrowse/rice_build_3/.

  1. Temporal small RNA transcriptome profiling unraveled partitioned miRNA expression in developing maize endosperms between reciprocal crosses.

    Science.gov (United States)

    Xin, Mingming; Yang, Guanghui; Yao, Yingyin; Peng, Huiru; Hu, Zhaorong; Sun, Qixin; Wang, Xiangfeng; Ni, Zhongfu

    2015-01-01

    In angiosperms, the endosperm nurtures the embryo and provides nutrients for seed germination. To identify the expression pattern of small interfering RNA in the developing maize endosperm, we have performed high-throughput small RNA transcriptome sequencing of kernels at 0, 3, and 5 days after pollination (DAP) and endosperms at 7, 10, and 15 DAP using B73 and Mo17 reciprocal crosses in previous study. Here, we further explored these small RNA-seq data to investigate the potential roles of miRNAs in regulating the gene expression process. In total, 57 conserved miRNAs and 18 novel miRNAs were observed highly expressed in maize endosperm. Temporal expression profiling indicated that these miRNAs exhibited dynamic and partitioned expression patterns at different developmental stages between maize reciprocal crosses, and quantitative RT-PCR results further confirmed our observation. In addition, we found a subset of distinct tandem miRNAs are generated from a single stem-loop structure in maize that might be conserved in monocots. Furthermore, a SNP variation of Zma-miR408-5p at 11th base position was characterized between B73 and Mo17 which might lead to completely different functions in repressing targets. More interestingly, Zma-miR408-5p exhibited B73-biased expression pattern in the B73 and Mo17 reciprocal hybrid endosperms at 7, 10, and 15 DAP according to the reads abundance with SNPs and CAPS experiment. Together, this study suggests that miRNA plays a crucial role in regulating endosperm development, and exhibited distinct expression patterns in developing endosperm between maize reciprocal crosses. PMID:26442057

  2. Small Regulatory RNA-Induced Growth Rate Heterogeneity of Bacillus subtilis

    OpenAIRE

    Mars, Ruben A. T.; Nicolas, Pierre; Ciccolini, Mariano; Reilman, Ewoud; Reder, Alexander; Schaffer, Marc; Maeder, Ulrike; Voelker, Uwe; van Dijl, Jan Maarten; Denham, Emma L.

    2015-01-01

    Author Summary Bacterial cells that share the same genetic information can display very different phenotypes, even if they grow under identical conditions. Despite the relevance of this population heterogeneity for processes like drug resistance and development, the molecular players that induce heterogenic phenotypes are often not known. Here we report that in the Gram-positive model bacterium Bacillus subtilis a small regulatory RNA (sRNA) can induce heterogeneity in growth rates by increas...

  3. MicroRNA-based Cancer Therapeutics: Big Hope from Small RNAs

    OpenAIRE

    Bhardwaj, Arun; Singh, Seema; Ajay P Singh

    2010-01-01

    Tremendous progress has been made during the last few years in identification of novel tumor-associated microRNAs and experimental validation of their cancer relevant gene targets. Indeed, these small non-coding RNAs are now known to modulate many biological pathways related to cancer progression, metastasis and therapy-resistance. Therefore, modulating miRNA functions may provide novel therapeutic opportunities for cancer treatment. This article reviews recent literature on the role of miRNA...

  4. Anomalous uptake and circulatory characteristics of the plant-based small RNA MIR2911

    Science.gov (United States)

    Yang, Jian; Hotz, Tremearne; Broadnax, LaCassidy; Yarmarkovich, Mark; Elbaz-Younes, Ismail; Hirschi, Kendal D.

    2016-01-01

    Inconsistent detection of plant-based dietary small RNAs in circulation has thwarted the use of dietary RNA therapeutics. Here we demonstrate mice consuming diets rich in vegetables displayed enhanced serum levels of the plant specific small RNA MIR2911. Differential centrifugation, size-exclusion chromatography, and proteinase K treatment of plant extracts suggest this RNA resides within a proteinase K-sensitive complex. Plant derived MIR2911 was more bioavailable than the synthetic RNA. Furthermore, MIR2911 exhibited unusual digestive stability compared with other synthetic plant microRNAs. The characteristics of circulating MIR2911 were also unusual as it was not associated with exosomes and fractionated as a soluble complex that was insensitive to proteinase K treatment, consistent with MIR2911 being stabilized by modifications conferred by the host. These results indicate that intrinsic stability and plant-based modifications orchestrate consumer uptake of this anomalous plant based small RNA and invite revisiting plant-based microRNA therapeutic approaches. PMID:27251858

  5. Genome-scale mRNA and small RNA transcriptomic insights into initiation of citrus apomixis

    Science.gov (United States)

    Long, Jian-Mei; Liu, Zheng; Wu, Xiao-Meng; Fang, Yan-Ni; Jia, Hui-Hui; Xie, Zong-Zhou; Deng, Xiu-Xin; Guo, Wen-Wu

    2016-01-01

    Nucellar embryony (NE) is an adventitious form of apomixis common in citrus, wherein asexual embryos initiate directly from nucellar cells surrounding the embryo sac. NE enables the fixation of desirable agronomic traits and the production of clonal offspring of virus-free rootstock, but impedes progress in hybrid breeding. In spite of the great importance of NE in citrus breeding and commercial production, little is understood about the underlying molecular mechanisms. In this study, the stages of nucellar embryo initiation (NEI) were determined for two polyembryonic citrus cultivars via histological observation. To explore the genes and regulatory pathways involved in NEI, we performed mRNA-seq and sRNA-seq analyses of ovules immediately prior to and at stages during NEI in the two pairs of cultivars. A total of 305 differentially expressed genes (DEGs) were identified between the poly- and monoembryonic ovules. Gene ontology (GO) analysis revealed that several processes are significantly enriched based on DEGs. In particular, response to stress, and especially response to oxidative stress, was over-represented in polyembryonic ovules. Nearly 150 miRNAs, comprising ~90 conserved and ~60 novel miRNAs, were identified in the ovules of either cultivar pair. Only two differentially expressed miRNAs (DEMs) were identified, of which the novel miRN23-5p was repressed whereas the targets accumulated in the polyembryonic ovules. This integrated study on the transcriptional and post-transcriptional regulatory profiles between poly- and monoembryonic citrus ovules provides new insights into the mechanism of NE, which should contribute to revealing the regulatory mechanisms of plant apomixis. PMID:27619233

  6. Characterization of the small RNA component of leaves and fruits from four different cucurbit species

    Directory of Open Access Journals (Sweden)

    Jagadeeswaran Guru

    2012-07-01

    Full Text Available Abstract Background MicroRNAs (miRNAs are a class of non-coding small RNAs involved in post-transcriptional regulation of gene expression critical for plant growth and development, stress responses and other diverse biological processes in plants. The Cucurbitaceae or cucurbit family represents some of economically important species, particularly those with edible and medicinal fruits. Genomic tools for the molecular analysis of members of this family are just emerging. Partial draft genome sequence became available recently for cucumber and watermelon facilitating investigation of the small RNA component of the transcriptomes in cucurbits. Results We generated four small RNA libraries from bottle gourd (Lagenaria siceraria, Cucurbita moschata, Cucurbita pepo, and, watermelon (Citrullus lanatus var. lanatus in order to identify conserved and novel lineage specific miRNAs in these cucurbits. Deep sequencing of small RNA libraries from these species resulted in 1,597,263, 532,948, 601,388, and 493,384 unique sRNA reads from bottle gourd, moschata, pepo and watermelon, respectively. Sequence analysis of these four libraries resulted in identification of 21 miRNA families that are highly conserved and 8 miRNA families that are moderately conserved in diverse dicots. We also identified 4 putative novel miRNAs in these plant species. Furthermore, the tasiRNAs were identified and their biogenesis was determined in these cucurbits. Small RNA blot analysis or q-PCR analyses of leaf and fruit tissues of these cucurbits showed differential expression of several conserved miRNAs. Interestingly, the abundance of several miRNAs in leaves and fruits of closely related C. moschata and C. pepo was also distinctly different. Target genes for the most conserved miRNAs are also predicted. Conclusion High-throughput sequencing of small RNA libraries from four cucurbit species has provided a glimpse of small RNA component in their transcriptomes. The analysis also

  7. 7SK small nuclear RNA inhibits cancer cell proliferation through apoptosis induction.

    Science.gov (United States)

    Keramati, Farid; Seyedjafari, Ehsan; Fallah, Parviz; Soleimani, Masoud; Ghanbarian, Hossein

    2015-04-01

    7SK small nuclear RNA (snRNA) is a 331-333-bp non-coding RNA, which recruits HEXIM 1/2 protein to inhibit positive elongation factor b (P-TEFb) activity. P-TEFb is an essential factor in alleviating promoter-proximal paused RNA polymerase II (Pol II) and initiating the productive elongation phase of gene transcription. Without this protein, Pol II will remain in its hypophosphorylated state, and no transcription occurs. In this study, we inhibited P-TEFb activity by over-expressing 7SK snRNA in human embryonic kidney (HEK) 293T cancer cell line. This inhibition led to a significant decrease in cell viability, which can be due to the transcription inhibition. Moreover, 7SK snRNA over-expression promoted apoptosis in cancerous cells. Our results suggest 7SK snRNA as a potential endogenous anti-cancer agent, and to the best of our knowledge, this is the first study that uses a long non-coding RNA's over-expression against cancer cell growth and proliferation.

  8. Alpha-synuclein suppression by targeted small interfering RNA in the primate substantia nigra.

    Directory of Open Access Journals (Sweden)

    Alison L McCormack

    Full Text Available The protein alpha-synuclein is involved in the pathogenesis of Parkinson's disease and other neurodegenerative disorders. Its toxic potential appears to be enhanced by increased protein expression, providing a compelling rationale for therapeutic strategies aimed at reducing neuronal alpha-synuclein burden. Here, feasibility and safety of alpha-synuclein suppression were evaluated by treating monkeys with small interfering RNA (siRNA directed against alpha-synuclein. The siRNA molecule was chemically modified to prevent degradation by exo- and endonucleases and directly infused into the left substantia nigra. Results compared levels of alpha-synuclein mRNA and protein in the infused (left vs. untreated (right hemisphere and revealed a significant 40-50% suppression of alpha-synuclein expression. These findings could not be attributable to non-specific effects of siRNA infusion since treatment of a separate set of animals with luciferase-targeting siRNA produced no changes in alpha-synuclein. Infusion with alpha-synuclein siRNA, while lowering alpha-synuclein expression, had no overt adverse consequences. In particular, it did not cause tissue inflammation and did not change (i the number and phenotype of nigral dopaminergic neurons, and (ii the concentrations of striatal dopamine and its metabolites. The data represent the first evidence of successful anti-alpha-synuclein intervention in the primate substantia nigra and support further development of RNA interference-based therapeutics.

  9. Optimization of transfection efficiency of small interfering RNA in purified human prolactinoma cells

    Institute of Scientific and Technical Information of China (English)

    MENG Qing-hu; SONG Yong-mei; ZHAO Jiang; YU Chun-jiang; ZHAN Qi-min

    2011-01-01

    Background Control of hypersecretion of certain hormones is one of the key targets in the treatment of pituitary adenomas. RNA interference has been shown to inhibit protein expression, and thus it may represent a promising method for the treatment of pituitary adenomas. In the present study, transfection efficiency of small interfering RNA (siRNA) was optimized in human prolactinoma cells.Methods First, a method was optimized to extract highly purified human prolactinoma cells in vitro. The extracted cells were verified to retain the physiological features of prolactin (PRL) secretion. Second, three conditions for siRNA transfection were tested by the evaluation of transfection efficiency and cell viability. The proper transfection condition was verified for human prolactinoma cells. Third, the siRNA for prolactin was transfected into the human prolactinoma cells, and the suppression of PRL mRNA was evaluated by quantitative real-time reverse transcription-PCR.Conclusion It is possible to inhibit hormone hypersecretion by RNA interference, that may eventually enable therapeutic siRNA drugs developed.

  10. 微孢子虫核糖体小亚单位RNA(ssUrRNA)基因%Small Subunit Ribosomal RNA Genes of Microsporidia

    Institute of Scientific and Technical Information of China (English)

    王见杨; 黄可威; 毛西成; 赵 昀; 陆长德

    2001-01-01

    微孢子虫是广泛分布于自然界的细胞内原虫类寄生物。它们可寄生于整个生物界。微孢子虫是真核生物,但其核糖体及核糖体RNA(rRNA)为原核生物型。为探讨9种家蚕病原性微孢子虫的种属地位及亲缘关系,对已广泛用于生物进化分类的核糖体小亚单位RNA(asurRNA)基因进行了研究。由微孢子虫ssurRNA基因序列同源性分析所构建的系统进化发育树及Southam杂交分析表明,这9种微孢子虫同为Nosema属,为同属不同种。%Microsporidia are ubiquitous intracellular parasitic protozoa infecting all types of animals. Their ribosomes and rRNAs are of prokaryotic size. In order to better understand their phylogenetic relationship and identify the uncertain species, the sequences of the small subunit ribosomal RNA (ssurRNA, 16 S rRNA) genesof nine microsporidia infectious to the silkworm, Bombyx mori, were determined. The results of phylogenetic trees and Southern blotting suggest all the nine strains of icrosporidia are various species of the genus Nosema.

  11. A deep analysis of the small non-coding RNA population in Schistosoma japonicum eggs.

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    Pengfei Cai

    Full Text Available BACKGROUND: Schistosoma japonicum is a parasitic flatworm that causes zoonotic schistosomiasis. The typical outcome of schistosomiasis is hepatic granuloma and fibrosis, which is primarily induced by soluble egg-derived antigens. Although schistosomal eggs represent an important pathogenic stage to the host, the biology of this critical stage is largely unknown. We previously investigated the expression profiles of sncRNAs during different developmental stages of this parasite. However, using small RNA extracted from egg-deposited liver tissues generated limited information about sncRNAs in eggs. Here, we characterized the complete small RNAome in this stage of the parasite after optimization of RNA purification. METHODOLOGY AND PRINCIPAL FINDINGS: A library, SjE, was constructed with the small RNA extracted from S. japonicum eggs and subjected to high-throughput sequencing. The data were depicted by comprehensive bioinformatic analysis to explore the expression features of sncRNAs in the egg stage. MicroRNAs accounted for about one quarter of the total small RNA population in this stage, with a strongly biased expression pattern of certain miRNA family members. Sja-miR-71, sja-miR-71-5p, and sja-miR-36-3p were suggested to play important roles in embryo development. A panel of transfer RNA fragments (tRFs precisely processed from the 5' end of mature tRNAs was identified for the first time, which represented a strong egg stage-biased expression. The tRNA-Ala derived small RNAs were the most highly expressed Sj-tRFs in eggs. Further, the expression of siRNAs from 29 types of well-defined transposable elements (TEs was observed to be relatively stable among different developmental stages. CONCLUSIONS AND SIGNIFICANCE: In this study, we characterized the sncRNA profile in the egg stage of S. japonicum. Featured expression of sncRNAs, especially the tRNA-derived small RNAs, was identified, which was further compared with that of other developmental

  12. Asymmetric purine-pyrimidine distribution in cellular small RNA population of papaya

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    Aryal Rishi

    2012-12-01

    Full Text Available Abstract Background The small RNAs (sRNA are a regulatory class of RNA mainly represented by the 21 and 24-nucleotide size classes. The cellular sRNAs are processed by RNase III family enzyme dicer (Dicer like in plant from a self-complementary hairpin loop or other type of RNA duplexes. The papaya genome has been sequenced, but its microRNAs and other regulatory RNAs are yet to be analyzed. Results We analyzed the genomic features of the papaya sRNA population from three sRNA deep sequencing libraries made from leaves, flowers, and leaves infected with Papaya Ringspot Virus (PRSV. We also used the deep sequencing data to annotate the micro RNA (miRNA in papaya. We identified 60 miRNAs, 24 of which were conserved in other species, and 36 of which were novel miRNAs specific to papaya. In contrast to the Chargaff’s purine-pyrimidine equilibrium, cellular sRNA was significantly biased towards a purine rich population. Of the two purine bases, higher frequency of adenine was present in 23nt or longer sRNAs, while 22nt or shorter sRNAs were over represented by guanine bases. However, this bias was not observed in the annotated miRNAs in plants. The 21nt species were expressed from fewer loci but expressed at higher levels relative to the 24nt species. The highly expressed 21nt species were clustered in a few isolated locations of the genome. The PRSV infected leaves showed higher accumulation of 21 and 22nt sRNA compared to uninfected leaves. We observed higher accumulation of miRNA* of seven annotated miRNAs in virus-infected tissue, indicating the potential function of miRNA* under stressed conditions. Conclusions We have identified 60 miRNAs in papaya. Our study revealed the asymmetric purine-pyrimidine distribution in cellular sRNA population. The 21nt species of sRNAs have higher expression levels than 24nt sRNA. The miRNA* of some miRNAs shows higher accumulation in PRSV infected tissues, suggesting that these strands are not totally

  13. Small RNA pathways and diversity in model legumes: lessons from genomics.

    Science.gov (United States)

    Bustos-Sanmamed, Pilar; Bazin, Jérémie; Hartmann, Caroline; Crespi, Martin; Lelandais-Brière, Christine

    2013-01-01

    Small non-coding RNAs (smRNA) participate in the regulation of development, cell differentiation, adaptation to environmental constraints and defense responses in plants. They negatively regulate gene expression by degrading specific mRNA targets, repressing their translation or modifying chromatin conformation through homologous interaction with target loci. MicroRNAs (miRNA) and short-interfering RNAs (siRNA) are generated from long double stranded RNA (dsRNA) that are cleaved into 20-24-nucleotide dsRNAs by RNase III proteins called DICERs (DCL). One strand of the duplex is then loaded onto effective complexes containing different ARGONAUTE (AGO) proteins. In this review, we explored smRNA diversity in model legumes and compiled available data from miRBAse, the miRNA database, and from 22 reports of smRNA deep sequencing or miRNA identification genome-wide in three legumes: Medicago truncatula, soybean (Glycine max) and Lotus japonicus. In addition to conserved miRNAs present in other plant species, 229, 179, and 35 novel miRNA families were identified respectively in these 3 legumes, among which several seems legume-specific. New potential functions of several miRNAs in the legume-specific nodulation process are discussed. Furthermore, a new category of siRNA, the phased siRNAs, which seems to mainly regulate disease-resistance genes, was recently discovered in legumes. Despite that the genome sequence of model legumes are not yet fully completed, further analysis was performed by database mining of gene families and protein characteristics of DCLs and AGOs in these genomes. Although most components of the smRNA pathways are conserved, identifiable homologs of key smRNA players from non-legumes, like AGO10 or DCL4, could not yet be detected in M. truncatula available genomic and expressed sequence (EST) databases. In contrast to Arabidopsis, an important gene diversification was observed in the three legume models (for DCL2, AGO4, AGO2, and AGO10) or

  14. Small RNA pathways and diversity in model legumes: lessons from genomics.

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    Pilar eBustos-Sanmamed

    2013-07-01

    Full Text Available Small non coding RNAs (smRNA participate in the regulation of development, cell differentiation, adaptation to environmental constraints and defense responses in plants. They negatively regulate gene expression by degrading specific mRNA targets, repressing their translation or modifying chromatin conformation through homologous interaction with target loci. MicroRNAs (miRNA and short-interfering RNAs (siRNA are generated from long double stranded RNA (dsRNA that are cleaved into 20- to 24-nucleotide dsRNAs by RNase III proteins called DICERs (DCL. One strand of the duplex is then loaded onto effective complexes containing different ARGONAUTE (AGO proteins. In this review, we explored smRNA diversity in model legumes and compiled available data from miRBAse, the miRNA database, and from 22 reports of smRNA deep sequencing or miRNA identification genome-wide in Medicago truncatula, Glycine max and Lotus japonicus. In addition to conserved miRNAs present in other plant species, 229, 179 and 35 novel miRNA families were identified respectively in these 3 legumes, among which several seems legume-specific. New potential functions of several miRNAs in the legume-specific nodulation process are discussed. Furthermore, a new category of siRNA, the phased siRNAs, which seems to mainly regulate disease-resistance genes, was recently discovered in legumes. Despite that the genome sequence of model legumes are not yet fully completed, further analysis was performed by database mining of gene families and protein characteristics of DCLs and AGOs in these genomes. Although most components of the smRNA pathways are conserved, identifiable homologs of key smRNA players from non-legumes could not yet be detected in M. truncatula available genomic and expressed sequence databases. In addition, an important gene diversification was observed in the three legumes. Functional significance of these variant isoforms may reflect peculiarities of smRNA biogenesis in

  15. Undesired small RNAs originate from an artificial microRNA precursor in transgenic petunia (Petunia hybrida.

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    Yulong Guo

    Full Text Available Although artificial microRNA (amiRNA technology has been used frequently in gene silencing in plants, little research has been devoted to investigating the accuracy of amiRNA precursor processing. In this work, amiRNAchs1 (amiRchs1, based on the Arabidopsis miR319a precursor, was expressed in order to suppress the expression of CHS genes in petunia. The transgenic plants showed the CHS gene-silencing phenotype. A modified 5' RACE technique was used to map small-RNA-directed cleavage sites and to detect processing intermediates of the amiRchs1 precursor. The results showed that the target CHS mRNAs were cut at the expected sites and that the amiRchs1 precursor was processed from loop to base. The accumulation of small RNAs in amiRchs1 transgenic petunia petals was analyzed using the deep-sequencing technique. The results showed that, alongside the accumulation of the desired artificial microRNAs, additional small RNAs that originated from other regions of the amiRNA precursor were also accumulated at high frequency. Some of these had previously been found to be accumulated at low frequency in the products of ath-miR319a precursor processing and some of them were accompanied by 3'-tailing variant. Potential targets of the undesired small RNAs were discovered in petunia and other Solanaceae plants. The findings draw attention to the potential occurrence of undesired target silencing induced by such additional small RNAs when amiRNA technology is used. No appreciable production of secondary small RNAs occurred, despite the fact that amiRchs1 was designed to have perfect complementarity to its CHS-J target. This confirmed that perfect pairing between an amiRNA and its targets is not the trigger for secondary small RNA production. In conjunction with the observation that amiRNAs with perfect complementarity to their target genes show high efficiency and specificity in gene silencing, this finding has an important bearing on future applications of ami

  16. Heat shock increases lifetime of a small RNA and induces its accumulation in cells.

    Science.gov (United States)

    Tatosyan, Karina A; Kramerov, Dmitri A

    2016-08-01

    4.5SH and 4.5SI RNA are two abundant small non-coding RNAs specific for several related rodent families including Muridae. These RNAs have a number of common characteristics such as the short length (about 100nt), transcription by RNA polymerase III, and origin from Short Interspersed Elements (SINEs). However, their stabilities in cells substantially differ: the half-life of 4.5SH RNA is about 20min, while that of 4.5SI RNA is 22h. Here we studied the influence of cell stress such as heat shock or viral infection on these two RNAs. We found that the level of 4.5SI RNA did not change in stressed cells; whereas heat shock increased the abundance of 4.5SH RNA 3.2-10.5 times in different cell lines; and viral infection, 5 times. Due to the significant difference in the turnover rates of these two RNAs, a similar activation of their transcription by heat shock increases the level of the short-lived 4.5SH RNA and has minor effect on the level of the long-lived 4.5SI RNA. In addition, the accumulation of 4.5SH RNA results not only from the induction of its transcription but also from a substantial retardation of its decay. To our knowledge, it is the first example of a short-lived non-coding RNA whose elongated lifetime contributes significantly to its accumulation in stressed cells.

  17. Systematic coarse-grained modeling of complexation between small interfering RNA and polycations

    Energy Technology Data Exchange (ETDEWEB)

    Wei, Zonghui [Graduate Program in Applied Physics, Northwestern University, Evanston, Illinois 60208 (United States); Luijten, Erik, E-mail: luijten@northwestern.edu [Graduate Program in Applied Physics, Northwestern University, Evanston, Illinois 60208 (United States); Department of Materials Science and Engineering, Northwestern University, Evanston, Illinois 60208 (United States); Department of Engineering Sciences and Applied Mathematics, Northwestern University, Evanston, Illinois 60208 (United States); Department of Physics and Astronomy, Northwestern University, Evanston, Illinois 60208 (United States)

    2015-12-28

    All-atom molecular dynamics simulations can provide insight into the properties of polymeric gene-delivery carriers by elucidating their interactions and detailed binding patterns with nucleic acids. However, to explore nanoparticle formation through complexation of these polymers and nucleic acids and study their behavior at experimentally relevant time and length scales, a reliable coarse-grained model is needed. Here, we systematically develop such a model for the complexation of small interfering RNA (siRNA) and grafted polyethyleneimine copolymers, a promising candidate for siRNA delivery. We compare the predictions of this model with all-atom simulations and demonstrate that it is capable of reproducing detailed binding patterns, charge characteristics, and water release kinetics. Since the coarse-grained model accelerates the simulations by one to two orders of magnitude, it will make it possible to quantitatively investigate nanoparticle formation involving multiple siRNA molecules and cationic copolymers.

  18. Functional specialization of the small interfering RNA pathway in response to virus infection.

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    Joao Trindade Marques

    Full Text Available In Drosophila, post-transcriptional gene silencing occurs when exogenous or endogenous double stranded RNA (dsRNA is processed into small interfering RNAs (siRNAs by Dicer-2 (Dcr-2 in association with a dsRNA-binding protein (dsRBP cofactor called Loquacious (Loqs-PD. siRNAs are then loaded onto Argonaute-2 (Ago2 by the action of Dcr-2 with another dsRBP cofactor called R2D2. Loaded Ago2 executes the destruction of target RNAs that have sequence complementarity to siRNAs. Although Dcr-2, R2D2, and Ago2 are essential for innate antiviral defense, the mechanism of virus-derived siRNA (vsiRNA biogenesis and viral target inhibition remains unclear. Here, we characterize the response mechanism mediated by siRNAs against two different RNA viruses that infect Drosophila. In both cases, we show that vsiRNAs are generated by Dcr-2 processing of dsRNA formed during viral genome replication and, to a lesser extent, viral transcription. These vsiRNAs seem to preferentially target viral polyadenylated RNA to inhibit viral replication. Loqs-PD is completely dispensable for silencing of the viruses, in contrast to its role in silencing endogenous targets. Biogenesis of vsiRNAs is independent of both Loqs-PD and R2D2. R2D2, however, is required for sorting and loading of vsiRNAs onto Ago2 and inhibition of viral RNA expression. Direct injection of viral RNA into Drosophila results in replication that is also independent of Loqs-PD. This suggests that triggering of the antiviral pathway is not related to viral mode of entry but recognition of intrinsic features of virus RNA. Our results indicate the existence of a vsiRNA pathway that is separate from the endogenous siRNA pathway and is specifically triggered by virus RNA. We speculate that this unique framework might be necessary for a prompt and efficient antiviral response.

  19. Targeted Delivery of Small Interfering RNA Using Reconstituted High-Density Lipoprotein Nanoparticles

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    Mian M.K. Shahzad

    2011-04-01

    Full Text Available RNA interference holds tremendous potential as a therapeutic approach, especially in the treatment of malignant tumors. However, efficient and biocompatible delivery methods are needed for systemic delivery of small interfering RNA (siRNA. To maintain a high level of growth, tumor cells scavenge high-density lipoprotein (HDL particles by overexpressing its receptor: scavenger receptor type B1 (SR-B1. In this study, we exploited this cellular characteristic to achieve efficient siRNA delivery and established a novel formulation of siRNA by incorporating it into reconstituted HDL (rHDL nanoparticles. Here, we demonstrate that rHDL nanoparticles facilitate highly efficient systemic delivery of siRNA in vivo, mediated by the SR-B1. Moreover, in therapeutic proof-of-concept studies, these nanoparticles were effective in silencing the expression of two proteins that are key to cancer growth and metastasis (signal transducer and activator of transcription 3 and focal adhesion kinase in orthotopic mouse models of ovarian and colorectal cancer. These data indicate that an rHDL nanoparticle is a novel and highly efficient siRNA carrier, and therefore, this novel technology could serve as the foundation for new cancer therapeutic approaches.

  20. Small regulatory RNA-induced growth rate heterogeneity of Bacillus subtilis.

    Science.gov (United States)

    Mars, Ruben A T; Nicolas, Pierre; Ciccolini, Mariano; Reilman, Ewoud; Reder, Alexander; Schaffer, Marc; Mäder, Ulrike; Völker, Uwe; van Dijl, Jan Maarten; Denham, Emma L

    2015-03-01

    Isogenic bacterial populations can consist of cells displaying heterogeneous physiological traits. Small regulatory RNAs (sRNAs) could affect this heterogeneity since they act by fine-tuning mRNA or protein levels to coordinate the appropriate cellular behavior. Here we show that the sRNA RnaC/S1022 from the Gram-positive bacterium Bacillus subtilis can suppress exponential growth by modulation of the transcriptional regulator AbrB. Specifically, the post-transcriptional abrB-RnaC/S1022 interaction allows B. subtilis to increase the cell-to-cell variation in AbrB protein levels, despite strong negative autoregulation of the abrB promoter. This behavior is consistent with existing mathematical models of sRNA action, thus suggesting that induction of protein expression noise could be a new general aspect of sRNA regulation. Importantly, we show that the sRNA-induced diversity in AbrB levels generates heterogeneity in growth rates during the exponential growth phase. Based on these findings, we hypothesize that the resulting subpopulations of fast- and slow-growing B. subtilis cells reflect a bet-hedging strategy for enhanced survival of unfavorable conditions. PMID:25790031

  1. Small regulatory RNA-induced growth rate heterogeneity of Bacillus subtilis.

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    Ruben A T Mars

    2015-03-01

    Full Text Available Isogenic bacterial populations can consist of cells displaying heterogeneous physiological traits. Small regulatory RNAs (sRNAs could affect this heterogeneity since they act by fine-tuning mRNA or protein levels to coordinate the appropriate cellular behavior. Here we show that the sRNA RnaC/S1022 from the Gram-positive bacterium Bacillus subtilis can suppress exponential growth by modulation of the transcriptional regulator AbrB. Specifically, the post-transcriptional abrB-RnaC/S1022 interaction allows B. subtilis to increase the cell-to-cell variation in AbrB protein levels, despite strong negative autoregulation of the abrB promoter. This behavior is consistent with existing mathematical models of sRNA action, thus suggesting that induction of protein expression noise could be a new general aspect of sRNA regulation. Importantly, we show that the sRNA-induced diversity in AbrB levels generates heterogeneity in growth rates during the exponential growth phase. Based on these findings, we hypothesize that the resulting subpopulations of fast- and slow-growing B. subtilis cells reflect a bet-hedging strategy for enhanced survival of unfavorable conditions.

  2. Micro RNA: Small but full of mystery and use (review article

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    M.R. Noori Daloii

    2006-07-01

    Full Text Available MicroRNAs form a class of small non-coding RNA molecules. With only 21-23 nucleutide in length, they have an important role in gene expression. These molecules bind to their target mRNA molecules and repress the protein expression via mRNA degradation or blocking the translation machine of the cell. From the advent of molecular biology microRNA molecules were out of focus, however huge amount of studies in the past few years revealed a lot of facts about their nature. Nowadays around 1600 different microRNA are discovered in human, animals, plant and even viruses. In this review article the most recent data in the history, genes, expression and process of these molecules are introduced. Furthermore, the findings about diverse roles of these molecules in normal and abnormal conditions, cancer in particular, are shown. Finally, the differences to siRNA molecules and the prospect of microRNA have been explained.

  3. Divergent patterns of endogenous small RNA populations from seed and vegetative tissues of Glycine max

    Science.gov (United States)

    Background: Small non-coding RNAs (smRNAs) are known to have major roles in gene regulation in eukaryotes. In plants, knowledge of the biogenesis and mechanisms of action of smRNA classes including microRNAs (miRNAs), short interfering RNAs (siRNAs), and trans-acting siRNAs (tasiRNAs) has been gaine...

  4. Regulation of bacterial photosynthesis genes by the small noncoding RNA PcrZ.

    Science.gov (United States)

    Mank, Nils N; Berghoff, Bork A; Hermanns, Yannick N; Klug, Gabriele

    2012-10-01

    The small RNA PcrZ (photosynthesis control RNA Z) of the facultative phototrophic bacterium Rhodobacter sphaeroides is induced upon a drop of oxygen tension with similar kinetics to those of genes for components of photosynthetic complexes. High expression of PcrZ depends on PrrA, the response regulator of the PrrB/PrrA two-component system with a central role in redox regulation in R. sphaeroides. In addition the FnrL protein, an activator of some photosynthesis genes at low oxygen tension, is involved in redox-dependent expression of this small (s)RNA. Overexpression of full-length PcrZ in R. sphaeroides affects expression of a small subset of genes, most of them with a function in photosynthesis. Some mRNAs from the photosynthetic gene cluster were predicted to be putative PcrZ targets and results from an in vivo reporter system support these predictions. Our data reveal a negative effect of PcrZ on expression of its target mRNAs. Thus, PcrZ counteracts the redox-dependent induction of photosynthesis genes, which is mediated by protein regulators. Because PrrA directly activates photosynthesis genes and at the same time PcrZ, which negatively affects photosynthesis gene expression, this is one of the rare cases of an incoherent feed-forward loop including an sRNA. Our data identified PcrZ as a trans acting sRNA with a direct regulatory function in formation of photosynthetic complexes and provide a model for the control of photosynthesis gene expression by a regulatory network consisting of proteins and a small noncoding RNA.

  5. Temporal small RNA transcriptome profiling unraveled partitioned miRNA expression in developing maize endosperms between reciprocal crosses

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    Mingming eXin

    2015-09-01

    Full Text Available In angiosperms, the endosperm nurtures embryo and provides nutrients for seed germination. We have performed high-throughput small RNA transcriptome sequencing of kernels at 0, 3, and 5 days after pollination (DAP and endosperms at 7, 10, and 15 DAP by using B73 and Mo17 reciprocal crosses in previous study. Here, we further explored these small RNA-seq data to investigate the potential roles of miRNAs in regulating gene expression process. In total, 57 conserved miRNAs and 18 novel miRNAs were observed highly expressed in maize endosperm. Temporal expression profiling indicates these miRNAs exhibited dynamic and partitioned expression patterns at different developmental stages between maize reciprocal crosses, and qRT-PCR results further confirmed our observation. In addition, we found a subset of distinct tandem miRNAs are generated from a single stem-loop structure in maize which might be conserved in monocots. Furthermore, a SNP variation of Zma-miR408-5p at 11th base position was characterized between B73 and Mo17 which leads to completely different functions in repressing targets. More interestingly, Zma-miR408-5p exhibited B73-biased expression pattern in the B73 and Mo17 reciprocal hybrid endosperms at 7, 10 and 15 DAP according to the reads abundance with SNPs and CAPS experiment.

  6. Silencing invariant chains of dendritic cells enhances anti-tumor immunity using small-interfering RNA

    Institute of Scientific and Technical Information of China (English)

    KE Shan; CHEN Xue-hua; ZHU Zheng-gang; LI Jian-fang; YU Bei-qin; GU Qin-long; LIU Bing-ya

    2010-01-01

    Background Genetic modification of dendritic cells (DCs) has been used as an effective approach to enhance anti-tumor immunity. RNA interference (RNAi), which can cause the degradation of any RNA in a sequence-specific manner, is a post-transcriptional gene silencing mechanism. In this study, small-interfering RNA (siRNA) specific for the Ii gene was transfected into DCs, and the anti-tumor immunity of Ii-silenced DCs was assessed.Methods The silencing effect of siRNA was evaluated by Western blotting and real-time PCR analyses. In vitro cytotoxic activity of T cells was evaluated using a Cytotox 96(R) non-radioactive cytotoxicity assay kit. The time to tumor onset and the tumor volumes were used as reliable indices to assess the anti-tumor immunity in vivo. To further examine the mechanisms underlying the anti-tumor immunity, flow cytometry analysis was used.Results The Ii expression of DCs was significantly reduced after Ii siRNA transfection. Significant in vitro anti-tumor ability was exhibited when DCs were co-transfected with Ii siRNA plus endogenous tumor antigen (P <0.05). Furthermore,tumor growth was greatly inhibited when mice were immunized with DCs transfected with Ii siRNA plus tumor antigen prior to or subsequent to tumor implantation. Flow cytometry analysis in vitro and in vivo indicated that both CD4+ and CD8+ T cells were significantly activated in the Ii siRNA group (P <0.05).Conclusion Silencing of the Ii gene of DCs may offer a potential approach to enhance DC-based anti-tumor immunity.

  7. Rapid delivery of small interfering RNA by biosurfactant MEL-A-containing liposomes.

    Science.gov (United States)

    Inoh, Yoshikazu; Furuno, Tadahide; Hirashima, Naohide; Kitamoto, Dai; Nakanishi, Mamoru

    2011-10-28

    The downregulation of gene expression by RNA interference holds great potential for genetic analysis and gene therapy. However, a more efficient delivery system for small interfering RNA (siRNA) into the target cells is required for wide fields such as cell biology, physiology, and clinical application. Non-viral vectors are stronger candidates than viral vectors because they are safer and easier to prepare. We have previously used a new method for gene transfection by combining cationic liposomes with the biosurfactant mannosylerythritol lipid-A (MEL-A). The novel MEL-A-containing cationic liposomes rapidly delivered DNA (plasmids and oligonucleotides) into the cytosol and nucleus through membrane fusion between liposomes and the plasma membrane, and consequently, enhanced the gene transfection efficiency. In this study, we determined the efficiency of MEL-A-containing cationic liposomes for siRNA delivery. We observed that exogenous and endogenous protein expression was suppressed by approximately 60% at 24h after brief (30 min) incubation of target cells with MEL-A-containing cationic liposome/siRNA complexes. Confocal microscopic analysis showed that suppression of protein expression was caused by rapid siRNA delivery into the cytosol. We found that the MEL-A-containing cationic liposomes directly delivered siRNA into the cytoplasm by the membrane fusion in addition to endocytotic pathway whereas Lipofectamine RNAiMax delivered siRNA only by the endocytotic pathway. It seems that the ability to rapidly and directly deliver siRNA into the cytosol using MEL-A-containing cationic liposomes is able to reduce immune responses, cytotoxicity, and other side effects caused by viral vectors in clinical applications. PMID:22001930

  8. Small interference RNA targeting tissue factor inhibits human lung adenocarcinoma growth in vitro and in vivo

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    Wang Jianing

    2011-05-01

    Full Text Available Abstract Background The human coagulation trigger tissue factor (TF is overexpressed in several types of cancer and involved in tumor growth, vascularization, and metastasis. To explore the role of TF in biological processes of lung adenocarcinoma, we used RNA interference (RNAi technology to silence TF in a lung adenocarcinoma cell line A549 with high-level expression of TF and evaluate its antitumor effects in vitro and in vivo. Methods The specific small interfering RNA (siRNA designed for targeting human TF was transfected into A549 cells. The expression of TF was detected by reverse transcription-PCR and Western blot. Cell proliferation was measured by MTT and clonogenic assays. Cell apoptosis was assessed by flow cytometry. The metastatic potential of A549 cells was determined by wound healing, the mobility and Matrigel invasion assays. Expressions of PI3K/Akt, Erk1/2, VEGF and MMP-2/-9 in transfected cells were detected by Western blot. In vivo, the effect of TF-siRNA on the growth of A549 lung adenocarcinoma xenografts in nude mice was investigated. Results TF -siRNA significantly reduced the expression of TF in the mRNA and protein levels. The down-regulation of TF in A549 cells resulted in the suppression of cell proliferation, invasion and metastasis and induced cell apoptosis in dose-dependent manner. Erk MAPK, PI3K/Akt pathways as well as VEGF and MMP-2/-9 expressions were inhibited in TF-siRNA transfected cells. Moreover, intratumoral injection of siRNA targeting TF suppressed the tumor growth of A549 cells in vivo model of lung adenocarcinoma. Conclusions Down-regulation of TF using siRNA could provide a potential approach for gene therapy against lung adenocarcinoma, and the antitumor effects may be associated with inhibition of Erk MAPK, PI3K/Akt pathways.

  9. Unraveling the conformational determinants of LARP7 and 7SK small nuclear RNA by theoretical approaches.

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    Xu, Lei; Kong, Ren; Zhu, Jingyu; Sun, Huiyong; Chang, Shan

    2016-07-19

    LARP7, a member of the La-related proteins (LARPs), shares a conserved La module comprising the La-motif (LAM) and the RNA-recognition motif (RRM1), binding exclusively to the non-coding RNA 7SK. LARP7 is a component of the small nuclear ribonucleoprotein (7SKsnRNP) required for the stability and function of the RNA, and implicated in the transcription termination and regulation of translation. In the current work, molecular dynamics simulations were employed to investigate the recently determined crystal structures of the La module of LARP7 in complexs with a stretch of uridines at the 3'-end of 7SK in the presence and absence of RNA and two different mutants. The structural stabilities of the four systems provided by the simulations are consistent with the experimental data. Principal component analysis (PCA) and free energy landscape (FEL) were used to explore the dominant motions and the functional dynamics between the two ends of the superhelical structures in both RNA-bound and RNA-free systems. The final values of the intramolecular angle formed by the Cα atoms of Arg30, Lys53 and Pro189 are ∼96° and 125° for the RNA-bound and RNA-free systems, highlighting the importance of the binding of the 3'-end of RNA 7SK for system stability. The dynamic cross-correlation maps (DCCM) were utilized to evaluate the conformational changes in different mutants, and small values were found around the residues 29-50 and 100-120 in the F168A system, whereas large values were found around the residues 120-160 and 170-189 in the E130A system. The time evolutions of the hydrogen-bond distances of the terminal uridine U-1 and Asp54 and that of the penultimate residue U-2 and Gln41 were monitored to compare their conformational changes, and the results suggest that the E130A mutant may have an important effect on the RNA binding, which is consistent with site-directed mutagenesis. This study provides some new insights into the understanding of the recognition mechanism

  10. Barcoded cDNA library preparation for small RNA profiling by next-generation sequencing

    OpenAIRE

    Hafner, Markus; Renwick, Neil; Farazi, Thalia A.; Mihailovi, Aleksandra; Pena, John T.G.; Tuschl, Thomas

    2012-01-01

    The characterization of post-transcriptional gene regulation by small regulatory (20–30 nt) RNAs, particularly miRNAs and piRNAs, has become a major focus of research in recent years. A prerequisite for characterizing small RNAs is their identification and quantification across different developmental stages, and in normal and disease tissues, as well as model cell lines. Here we present a step-by-step protocol for generating barcoded small RNA cDNA libraries compatible with Illumina HiSeq se...

  11. Small RNA Sequencing Based Identification of MiRNAs in Daphnia magna.

    Directory of Open Access Journals (Sweden)

    Ercan Selçuk Ünlü

    Full Text Available Small RNA molecules are short, non-coding RNAs identified for their crucial role in post-transcriptional regulation. A well-studied example includes miRNAs (microRNAs which have been identified in several model organisms including the freshwater flea and planktonic crustacean Daphnia. A model for epigenetic-based studies with an available genome database, the identification of miRNAs and their potential role in regulating Daphnia gene expression has only recently garnered interest. Computational-based work using Daphnia pulex, has indicated the existence of 45 miRNAs, 14 of which have been experimentally verified. To extend this study, we took a sequencing approach towards identifying miRNAs present in a small RNA library isolated from Daphnia magna. Using Perl codes designed for comparative genomic analysis, 815,699 reads were obtained from 4 million raw reads and run against a database file of known miRNA sequences. Using this approach, we have identified 205 putative mature miRNA sequences belonging to 188 distinct miRNA families. Data from this study provides critical information necessary to begin an investigation into a role for these transcripts in the epigenetic regulation of Daphnia magna.

  12. Identification of microRNA-like small RNAs from fungal parasite Nosema ceranae.

    Science.gov (United States)

    Huang, Qiang; Evans, Jay D

    2016-01-01

    We previously found transcripts encoding Dicer and Argonaute which are involved in the production of microRNAs, in the honey bee parasite Nosema ceranae. In order to identify microRNAs in N. ceranae, we sequenced small RNAs from midgut tissues of infected honey bees at 24 h intervals for 6 days post infection, covering the complete reproduction cycle for this intracellular parasite. We predicted six microRNA-like small RNAs, all of which were confirmed via RT-qPCR assays. This is the first evidence for microRNA-like small RNAs generated by a microsporidian species, providing new insights into host-parasite interactions involving this widespread taxonomic group. PMID:26678507

  13. A survey of small RNA population during FR-induced apical hook opening

    Directory of Open Access Journals (Sweden)

    Ying eLi

    2014-04-01

    Full Text Available Photomorphogenesis is a mechanism employed by plants to regulate their architecture and developmental program in response to light conditions. As they emerge into light for the first time, dark-grown seedlings employ a rapid and finely-controlled photomorphogenic signaling network. Small RNAs have increasingly been revealed to play an important role in regulating multiple aspects of plant development, by modulating the stability of mRNAs. The rapid alteration of the mRNA transcriptome is a known hallmark of the de-etiolation response, thus we investigated the small RNA transcriptome during this process in specific seedling tissues. Here we describe a survey of the small RNA expression profile in four tissues of etiolated soybean seedlings, the cotyledons, hypocotyl and the convex and concave sides of the apical hook. We also investigate how this profile responds to a one-hour far-red light treatment. Our data suggests that miRNAs show a different global profile between these tissues and treatments, suggesting a possible role for tissue- and treatment-specific expression in the differential morphology of the seedling on de-etiolation. Further evidence for the role of miRNA in light-regulated development is given by the de-etiolation responses of a hypomorphic ago1 mutant, which displays reduced and delayed photomorphogenic responses in apical hook and cotyledon angle to far-red light.

  14. Soybean ribulose bisphosphate carboxylase small subunit: Mechanisms and determinants of RNA turnover. Annual progress report

    Energy Technology Data Exchange (ETDEWEB)

    Meagher, R.B. [Georgia Univ., Athens, GA (United States). Dept. of Genetics

    1993-12-31

    An in vitro degradation system has been developed from petunia and soybean polysomes in order to investigate the mechanisms and determinants controlling RNA turnover in higher plants. This system faithfully degrades soybean ribulose-1,5-bisphosphate carboxylase small subunit (rbcS) mRNA into the same products observed in total RNA preparations. In previous years it was shown that the most stable products represent a nested constellation of fragments, which are shortened from their 3{prime} ends, and have intact 5{prime} ends. Exogenous rbcS RNA tagged with novel 5{prime} sequence 15 or 56 bp long were synthesized in vitro as Sp6 and T7 runoff transcripts, respectively. When added to the system they were degraded faithfully into constellation of products which were 15 or 56 bp longer than the endogenous products, respectively. Detailed kinetics on the appearance of these exogenous products confirmed degradation proceeds in an overall 3{prime} to 5{prime} direction but suggested that there are multiple pathways through which the RNA may be degraded. To further demonstrate a precursor product relationships, in vitro synthesized transcripts truncated at their 3{prime} ends were shown to degrade into the expected smaller fragments previously mapped in the 5{prime} portion of the rbcS RNA.

  15. Rapid delivery of small interfering RNA by biosurfactant MEL-A-containing liposomes

    Energy Technology Data Exchange (ETDEWEB)

    Inoh, Yoshikazu; Furuno, Tadahide [School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya 464-8650 (Japan); Hirashima, Naohide [Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603 (Japan); Kitamoto, Dai [National Institute of Advanced Industrial Science and Technology (AIST), Central 5, 1-1 Higashi, Tsukuba 305-8565 (Japan); Nakanishi, Mamoru, E-mail: mamoru@dpc.agu.ac.jp [School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya 464-8650 (Japan)

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer We use MEL-A-containing cationic liposomes for siRNA delivery. Black-Right-Pointing-Pointer MEL-A-containing cationic liposomes can efficiently and rapidly deliver siRNA into the cytoplasm. Black-Right-Pointing-Pointer Rapid delivery of siRNA is due to the membrane fusion between liposomes and plasma membrane. -- Abstract: The downregulation of gene expression by RNA interference holds great potential for genetic analysis and gene therapy. However, a more efficient delivery system for small interfering RNA (siRNA) into the target cells is required for wide fields such as cell biology, physiology, and clinical application. Non-viral vectors are stronger candidates than viral vectors because they are safer and easier to prepare. We have previously used a new method for gene transfection by combining cationic liposomes with the biosurfactant mannosylerythritol lipid-A (MEL-A). The novel MEL-A-containing cationic liposomes rapidly delivered DNA (plasmids and oligonucleotides) into the cytosol and nucleus through membrane fusion between liposomes and the plasma membrane, and consequently, enhanced the gene transfection efficiency. In this study, we determined the efficiency of MEL-A-containing cationic liposomes for siRNA delivery. We observed that exogenous and endogenous protein expression was suppressed by approximately 60% at 24 h after brief (30 min) incubation of target cells with MEL-A-containing cationic liposome/siRNA complexes. Confocal microscopic analysis showed that suppression of protein expression was caused by rapid siRNA delivery into the cytosol. We found that the MEL-A-containing cationic liposomes directly delivered siRNA into the cytoplasm by the membrane fusion in addition to endocytotic pathway whereas Lipofectamine Trade-Mark-Sign RNAiMax delivered siRNA only by the endocytotic pathway. It seems that the ability to rapidly and directly deliver siRNA into the cytosol using MEL-A-containing cationic

  16. Diagnostic and prognostic signatures from the small non-coding RNA transcriptome in prostate cancer

    DEFF Research Database (Denmark)

    Martens-Uzunova, E S; Jalava, S E; Dits, N F;

    2011-01-01

    Prostate cancer (PCa) is the most frequent male malignancy and the second most common cause of cancer-related death in Western countries. Current clinical and pathological methods are limited in the prediction of postoperative outcome. It is becoming increasingly evident that small non-coding RNA...... RNAs (snoRNAs) and transfer RNAs (tRNAs). From microarray analysis, we derived a miRNA diagnostic classifier that accurately distinguishes normal from cancer samples. Furthermore, we were able to construct a PCa prognostic predictor that independently forecasts postoperative outcome. Importantly...

  17. Identification of novel small RNAs and characterization of the 6S RNA of Coxiella burnetii.

    Directory of Open Access Journals (Sweden)

    Indu Warrier

    Full Text Available Coxiella burnetii, an obligate intracellular bacterial pathogen that causes Q fever, undergoes a biphasic developmental cycle that alternates between a metabolically-active large cell variant (LCV and a dormant small cell variant (SCV. As such, the bacterium undoubtedly employs complex modes of regulating its lifecycle, metabolism and pathogenesis. Small RNAs (sRNAs have been shown to play important regulatory roles in controlling metabolism and virulence in several pathogenic bacteria. We hypothesize that sRNAs are involved in regulating growth and development of C. burnetii and its infection of host cells. To address the hypothesis and identify potential sRNAs, we subjected total RNA isolated from Coxiella cultured axenically and in Vero host cells to deep-sequencing. Using this approach, we identified fifteen novel C. burnetii sRNAs (CbSRs. Fourteen CbSRs were validated by Northern blotting. Most CbSRs showed differential expression, with increased levels in LCVs. Eight CbSRs were upregulated (≥2-fold during intracellular growth as compared to growth in axenic medium. Along with the fifteen sRNAs, we also identified three sRNAs that have been previously described from other bacteria, including RNase P RNA, tmRNA and 6S RNA. The 6S regulatory sRNA of C. burnetii was found to accumulate over log phase-growth with a maximum level attained in the SCV stage. The 6S RNA-encoding gene (ssrS was mapped to the 5' UTR of ygfA; a highly conserved linkage in eubacteria. The predicted secondary structure of the 6S RNA possesses three highly conserved domains found in 6S RNAs of other eubacteria. We also demonstrate that Coxiella's 6S RNA interacts with RNA polymerase (RNAP in a specific manner. Finally, transcript levels of 6S RNA were found to be at much higher levels when Coxiella was grown in host cells relative to axenic culture, indicating a potential role in regulating the bacterium's intracellular stress response by interacting with RNAP during

  18. Effect of small interfering RNA targeting survivin gene on biological behaviour of bladder cancer

    Institute of Scientific and Technical Information of China (English)

    HOU Jian-quan; HE Jun; WANG Xiao-lin; WEN Duan-gai; CHEN Zi-xing

    2006-01-01

    Background Bladder cancer is the most common type of urinary system tumours. It is frequently associated with genetic mutations that deregulate the cell cycle and render these tumours resistant to apoptosis. Survivin, a newly discovered member inhibitor of apoptosis protein (IAP) family in several human cancers, by inducing cell proliferation and inhibiting apoptosis is frequently activated in bladder cancer. We studied the influence of small interfering RNA (siRNA) targeting survivin on the biological behaviour of bladder cancer cells.Methods A double strand survivin target sequence specific siRNA was designed and synthesized. After transfection of bladder cancer cell line T24 by siRNA/liposome complex with increasing concentrations(50-200 nmol/L), the transfectant cells were intratumourally injected at different doses (5 μg or 50μg). The effects were measured in vitro and in vivo.Results The selected siRNA efficiently down-regulated survivin mRNA expression in a dose and time dependent manner. The maximal effect was achieved at the concentration of 100 nmol/L, at which survivin expression level was down-regulated by 75.91%. The inhibition rate of cell growth was 55.29% (P<0.01) and the markedly increased apoptotic rate was 45.70% (P<0.01). In vivo intratumoural injection of 50 μg siRNA-survivin could notably prevent the growth of bladder cancer (P<0.01) in xenografted animals.Conclusion The application of siRNA-survivin could markedly inhibit survivin expression in bladder cancer cell line by inducing apoptosis and inhibiting the growth of the tumour. It may become a new gene therapy tool for bladder cancer.

  19. CCR5 small interfering RNA ameliorated joint inflammation in rats with adjuvant-induced arthritis.

    Science.gov (United States)

    Duan, Hongmei; Yang, Pingting; Fang, Fang; Ding, Shuang; Xiao, Weiguo

    2014-12-01

    Rheumatoid arthritis (RA) is a systemic inflammatory disease. C-C chemokine receptor type 5 (CCR5) is found in inflamed synovium of RA patients and is necessary for formation of RA. We aimed to check whether delivery of CCR5-specific small interfering RNA (siRNA) via electroporation suppresses local inflammation in arthritis rats. Vectors encoding siRNA that target CCR5 or negative control siRNA were constructed for gene silencing and the silencing effects of suppressing CCR5 expression in synovium examined by western blot. The vector with strongest effect was delivered into the knee joint of adjuvant-induced arthritis (AIA) rats by the in vivo electroporation method 7, 10, 13, and 16 days after immunization with Complete Freund's adjuvant. During an observation of 28 days, behavior, paw swelling, arthritis and histopathologic scoring were estimated. The expression level of CCR5 in synovium was evaluated by western blot and real-time PCR. Anti-CCR5 D1 siRNA was effectively inhibited CCR5 expression in vitro. Moreover, delivery of the siRNA into inflammatory joint also suppressed the expression of CCR5 in vivo and markedly suppressed paw swelling and inflammation. Local electroporation of anti-CCR5 siRNA into the left inflamed joints could achieve the silencing of CCR5 gene and alleviate local inflammation just in the knee joint injected with siRNA other than the opposite joint. Inhibition of CCR5 expression may provide a potential for treatment of RA.

  20. Gene Suppression of Mouse Testis In Vivo Using Small Interfering RNA Derived from Plasmid Vectors

    International Nuclear Information System (INIS)

    We evaluated whether inhibiting gene expression by small interfering RNA (siRNA) can be used for an in vivo model using a germ cell-specific gene (Tex101) as a model target in mouse testis. We generated plasmid-based expression vectors of siRNA targeting the Tex101 gene and transfected them into postnatal day 10 mouse testes by in vivo electroporation. After optimizing the electroporation conditions using a vector transfected into the mouse testis, a combination of high- and low-voltage pulses showed excellent transfection efficiency for the vectors with minimal tissue damage, but gene suppression was transient. Gene suppression by in vivo electroporation may be helpful as an alternative approach when designing experiments to unravel the basic role of testicular molecules

  1. Elucidation of the Mechanism of Gene Silencing using Small Interferin RNA: DNA Hybrid Molecules

    Energy Technology Data Exchange (ETDEWEB)

    Dugan, L

    2006-02-08

    The recent discovery that short hybrid RNA:DNA molecules (siHybrids) induce long-term silencing of gene expression in mammalian cells conflicts with the currently hypothesized mechanisms explaining the action of small, interfering RNA (siRNA). As a first step to elucidating the mechanism for this effect, we set out to quantify the delivery of siHybrids and determine their cellular localization in mammalian cells. We then tracked the segregation of the siHybrids into daughter cells after cell division. Markers for siHybrid delivery were shown to enter cells with and without the use of a transfection agent. Furthermore, delivery without transfection agent only occurred after a delay of 2-4 hours, suggesting a degradation process occurring in the cell culture media. Therefore, we studied the effects of nucleases and backbone modifications on the stability of siHybrids under cell culture conditions.

  2. Deep sequencing analysis of small noncoding RNA and mRNA targets of the global post-transcriptional regulator, Hfq

    DEFF Research Database (Denmark)

    Sittka, A; Lucchini, S; Papenfort, K;

    2008-01-01

    Recent advances in high-throughput pyrosequencing (HTPS) technology now allow a thorough analysis of RNA bound to cellular proteins, and, therefore, of post-transcriptional regulons. We used HTPS to discover the Salmonella RNAs that are targeted by the common bacterial Sm-like protein, Hfq. Initial...... transcriptomic analysis revealed that Hfq controls the expression of almost a fifth of all Salmonella genes, including several horizontally acquired pathogenicity islands (SPI-1, -2, -4, -5), two sigma factor regulons, and the flagellar gene cascade. Subsequent HTPS analysis of 350,000 cDNAs, derived from RNA co......-immunoprecipitation (coIP) with epitope-tagged Hfq or control coIP, identified 727 mRNAs that are Hfq-bound in vivo. The cDNA analysis discovered new, small noncoding RNAs (sRNAs) and more than doubled the number of sRNAs known to be expressed in Salmonella to 64; about half of these are associated with Hfq. Our analysis...

  3. tRF2Cancer: A web server to detect tRNA-derived small RNA fragments (tRFs) and their expression in multiple cancers.

    Science.gov (United States)

    Zheng, Ling-Ling; Xu, Wei-Lin; Liu, Shun; Sun, Wen-Ju; Li, Jun-Hao; Wu, Jie; Yang, Jian-Hua; Qu, Liang-Hu

    2016-07-01

    tRNA-derived small RNA fragments (tRFs) are one class of small non-coding RNAs derived from transfer RNAs (tRNAs). tRFs play important roles in cellular processes and are involved in multiple cancers. High-throughput small RNA (sRNA) sequencing experiments can detect all the cellular expressed sRNAs, including tRFs. However, distinguishing genuine tRFs from RNA fragments generated by random degradation remains a major challenge. In this study, we developed an integrated web-based computing system, tRF2Cancer, to accurately identify tRFs from sRNA deep-sequencing data and evaluate their expression in multiple cancers. The binomial test was introduced to evaluate whether reads from a small RNA-seq data set represent tRFs or degraded fragments. A classification method was then used to annotate the types of tRFs based on their sites of origin in pre-tRNA or mature tRNA. We applied the pipeline to analyze 10 991 data sets from 32 types of cancers and identified thousands of expressed tRFs. A tool called 'tRFinCancer' was developed to facilitate the users to inspect the expression of tRFs across different types of cancers. Another tool called 'tRFBrowser' shows both the sites of origin and the distribution of chemical modification sites in tRFs on their source tRNA. The tRF2Cancer web server is available at http://rna.sysu.edu.cn/tRFfinder/. PMID:27179031

  4. tRF2Cancer: A web server to detect tRNA-derived small RNA fragments (tRFs) and their expression in multiple cancers.

    Science.gov (United States)

    Zheng, Ling-Ling; Xu, Wei-Lin; Liu, Shun; Sun, Wen-Ju; Li, Jun-Hao; Wu, Jie; Yang, Jian-Hua; Qu, Liang-Hu

    2016-07-01

    tRNA-derived small RNA fragments (tRFs) are one class of small non-coding RNAs derived from transfer RNAs (tRNAs). tRFs play important roles in cellular processes and are involved in multiple cancers. High-throughput small RNA (sRNA) sequencing experiments can detect all the cellular expressed sRNAs, including tRFs. However, distinguishing genuine tRFs from RNA fragments generated by random degradation remains a major challenge. In this study, we developed an integrated web-based computing system, tRF2Cancer, to accurately identify tRFs from sRNA deep-sequencing data and evaluate their expression in multiple cancers. The binomial test was introduced to evaluate whether reads from a small RNA-seq data set represent tRFs or degraded fragments. A classification method was then used to annotate the types of tRFs based on their sites of origin in pre-tRNA or mature tRNA. We applied the pipeline to analyze 10 991 data sets from 32 types of cancers and identified thousands of expressed tRFs. A tool called 'tRFinCancer' was developed to facilitate the users to inspect the expression of tRFs across different types of cancers. Another tool called 'tRFBrowser' shows both the sites of origin and the distribution of chemical modification sites in tRFs on their source tRNA. The tRF2Cancer web server is available at http://rna.sysu.edu.cn/tRFfinder/.

  5. Complex intra-operonic dynamics mediated by a small RNA in Streptomyces coelicolor.

    Directory of Open Access Journals (Sweden)

    Hindra

    Full Text Available Streptomyces are predominantly soil-dwelling bacteria that are best known for their multicellular life cycle and their prodigious metabolic capabilities. They are also renowned for their regulatory capacity and flexibility, with each species encoding >60 sigma factors, a multitude of transcription factors, and an increasing number of small regulatory RNAs. Here, we describe our characterization of a conserved small RNA (sRNA, scr4677. In the model species Streptomyces coelicolor, this sRNA is located in the intergenic region separating SCO4677 (an anti-sigma factor-encoding gene and SCO4676 (a putative regulatory protein-encoding gene, close to the SCO4676 translation start site in an antisense orientation. There appears to be considerable genetic interplay between these different gene products, with wild type expression of scr4677 requiring function of the anti-sigma factor SCO4677, and scr4677 in turn influencing the abundance of SCO4676-associated transcripts. The scr4677-mediated effects were independent of RNase III (a double stranded RNA-specific nuclease, with RNase III having an unexpectedly positive influence on the level of SCO4676-associated transcripts. We have shown that both SCO4676 and SCO4677 affect the production of the blue-pigmented antibiotic actinorhodin under specific growth conditions, and that this activity appears to be independent of scr4677.

  6. A group-I intron in the mitochondrial small subunit ribosomal RNA gene of Sclerotinia sclerotiorum.

    Science.gov (United States)

    Carbone, I; Anderson, J B; Kohn, L M

    1995-01-01

    A 1,380-bp intervening sequence within the mitochondrial small subunit ribosomal RNA (mt SSU rRNA) gene of the fungus Sclerotinia sclerotiorum has been sequenced and identified as a group-I intron. This is the first report of an intron in the mt SSU rRNA gene. The intron shows close similarity in secondary structure to the subgroup-IC2 introns from Podospora (ND3i1, ND5i2, and COIi5) and Neurospora (ND5i1). The intron has an open reading frame (ORF) that encodes a putative protein of 420 amino acids which contains two copies of the LAGLI-DADG motif. The ORF belongs to a family of ORFs identified in Podospora (ND3i1, ND4Li1, ND4Li2, ND5i2, and COIi5) and Neurospora (ND5i1). The putative 420-aa polypeptide is also similar to a site-specific endonuclease in the chloroplast large subunit ribosomal RNA (LSU rRNA) gene of the green alga Chlamydomonas eugametos. In each clone of S. sclerotiorum examined, including several clones which were sampled over a 3-year period from geographically separated sites, all isolates either had the intron or lacked the intron within the mt SSU rRNA gene. Screening by means of Southern hybridization and PCR amplification detected the intron in the mt SSU rRNA genes of S. minor, S. trifoliorum and Sclerotium cepivorum, but not in other members of the Sclerotiniaceae, such as Botrytis anamorphs of Botryotinia spp., or in other ascomycetous and basidiomycetous fungi. PMID:7788720

  7. Blockage of receptor-interacting protein 2 expression by small interfering RNA in murine macrophages

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    This study aims to demonstrate that blocking the receptor-interacting protein2(Rip2)expression can decrease inflammatory cytokine production by macrophage and protect mice from endotoxin lethality.Murine Rip2 small interfering RNA(siRNA)plasmids were constructed and transfected into macrophage and Rip2 expression was detected with reverse transcription-polymerase chain reaction(RT-PCR)and western blot.Cell proliferation was assayed with MTT.TNF-α concentration was assayed with ELISA and high-mobility group box 1 protein(HMGB1)level with semi-quantitative western blot after lipopolysaccharide(LPS)stimulation.LPS challenge was given after the plasmids were injected into mice and the survival rate was calculated.Rip2 siRNA plasmid could block the mRNA and protein expression of Rip2 and promote cell proliferation.Blocking Rip2 could attenuate LPS-induced TNF-~ and HMGB1 production.The HMGB1 expression in the liver decreased to(40.21±11.03)pg/g,and serum TNF-α level decreased to(300.43±59.26)ng/L(P<0.05).The survival rate of mice from endotoxemia was also improved(P<0.05).The results demonstrate that Rip2 siRNA plasmid can block the expression of Rip2,decrease the production of TNF-α and HMGB1 and protect mice from fatal endotoxemia.

  8. Progress of Targeting Transforming Growth Factor-β1 Small Interfering RNA in Liver Fibrosis

    Institute of Scientific and Technical Information of China (English)

    Xuan Zhou; Xue-feng Yang

    2014-01-01

    Liver fibrosis is a common pathological consequence of a variety of chronic stimuli, including viral, autoimmune, drug-induced, cholestatic and metabolic diseases. Fibrosis is driven by a dynamic process involving increased synthesis of matrix components and a failure of physiological mechanisms of matrix turnover. Activation of hepatic stellate cells (HSCs) remains a central event in fibrosis. HSCs are the main source of extracellular matrix (ECM). Transforming growth factor-beta (TGF-β), which is the fibrogenic master cytokine, can induce the activation of HSCs to produce a large amount of ECM, and is capable of inducing apoptosis of liver cells. RNA interference (RNAi) is a novel gene disruption technology. Studies have shown that small interfering RNA (siRNA) targeting TGF-β1 may inhibit the activation and proliferation of HSCs, suppress ECM synthesis and block liver fibrosis. TGF-β1 siRNA-mediated gene silencing therapy provides a new avenue for liver fibrosis. This review summarizes recent progresses in research on HSCs, TGF-β1 and TGF-β1 siRNA in liver fibrosis.

  9. Delivery of Small Interfering RNA by Peptide-Targeted Mesoporous Silica Nanoparticle-Supported Lipid Bilayers

    OpenAIRE

    Ashley, Carlee E.; CARNES, ERIC C.; Epler, Katharine E.; Padilla, David P.; Phillips, Genevieve K.; Castillo, Robert E.; Wilkinson, Dan C.; Wilkinson, Brian S.; Burgard, Cameron A.; Sewell, Robin M.; Townson, Jason L.; Chackerian, Bryce; Willman, Cheryl L.; Peabody, David S; Wharton, Walker

    2012-01-01

    The therapeutic potential of small interfering RNAs (siRNAs) is severely limited by the availability of delivery platforms that protect siRNA from degradation, deliver it to the target cell with high specificity and efficiency, and promote its endosomal escape and cytosolic dispersion. Here we report that mesoporous silica nanoparticle-supported lipid bilayers (or ‘protocells’), exhibit multiple properties that overcome many of the limitations of existing delivery platforms. Protocells have a...

  10. Structure and Genome Organization of Cherry Virus A (Capillovirus, Betaflexiviridae) from China Using Small RNA Sequencing.

    Science.gov (United States)

    Wang, Jiawei; Zhai, Ying; Liu, Weizhen; Dhingra, Amit; Pappu, Hanu R; Liu, Qingzhong

    2016-01-01

    Cherry virus A (CVA) (Capillovirus, Betaflexiviridae) is widely present in cherry-growing areas. We obtained the complete genome of a CVA isolate (CVA-TA) using small RNA deep sequencing, followed by overlapping reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE). The newly identified 5'-untranslated region (5'-UTR) from CVA-TA may form additional hairpin and loop structures to stabilize the CVA genome. PMID:27174277

  11. Small interference RNA targeting vascular endothelial growth factor gene effectively attenuates retinal neovascularization in mice model

    Institute of Scientific and Technical Information of China (English)

    KONG Yi-chun; SUN Bei; ZHAO Kan-xing; HAN Mei; WANG Yu-chuan

    2013-01-01

    Background The mechanism of retinal neovascularization is not understood completely.Many growth factors are involved in the process of retinal neovascularization,such as vascular endothelial growth factor (VEGF) and pigment epithelium-deprived factor (PEDF),which are the representatives of angiogenic and antiangiogenic molecules respectively.Oxygen induced retinopathy (OIR) is a useful model to investigate retinal neovascularization.The present study was conducted to investigate the feasibility of small interference RNA (siRNA) targeting VEGF gene in attenuating oxygen induced retinopathy (OIR) by regulating VEGF to PEDF ratio (VEGF/PEDF).Methods In vitro,cultured EOMA cells were transfected with VEGF-siRNA (psi-HITM/EGFPNEGF siRNA) and LipofectamineTM 2000 for 24,48,and 72 hours,respectively.Expression of VEGF mRNA was evaluated by real time polymerase chain reaction (PCR) and the level of VEGF protein was analyzed by Western blotting.In vivo,OIR model mice were established,the mice (C57BL/6J) received an intra-vitreal injection of 1 μl of mixture of psi-HITM/EGFPNEGF siRNA and Lipofectamine 2000.Expressions of retinal VEGF and PEDF protein were measured by Western blotting,retinal neovascularization was observed by fluorescein angiography,and quantified.Results In vitro psi-HITM/EGFP/VEGF siRNA treatment significantly reduced VEGF mRNA and protein expression.In vivo,with decreased VEGF and VEGF-PEDF ratio,significant attenuation of neovascular tufts,avascular regions,tortuous,and dilated blood vessels were observed in the interfered animals.Conclusions VEGF plays an important role in OIR,and the transfection of VEGF-siRNA can effectively downregulate VEGF expression in vivo,accompanied by the downregulation of VEGF-PEDF ratio,and simultaneous attenuation of retinal neovascularization was also observed.These findings suggest that VEGF/PEDF may serve as a potential target in the treatment of retinal neovascularization and RNA interference targeting VEGF expression

  12. Overexpression of miRNA-21 promotes radiation-resistance of non-small cell lung cancer

    International Nuclear Information System (INIS)

    MiRNA-21 was previously reported to be up-regulated in many kinds of cancer. In the present study, we want to investigate the potential role of miRNA-21 in non-small cell lung cancer. Expression of miRNA-21 was detected in 60 non-small cell lung cancer (NSCLC) samples and adjacent histologically normal tissue using RT-qPCR, Correlation between miRNA-21 expression and clinicopathological features of NSCLC was analyzed using statistical software. The effect of miRNA-21 expression on the growth and apoptosis of A549 cells induced by irradiation was examined. miRNA-21 expression increased in non-small cell lung cancer. Expression of miRNA-21 was positively associated with lymph node metastasis, clinical stage and poor prognosis. Multivariate Cox regression analysis showed that miRNA-21 was an independent prognostic factor for patients. Down-regulation of miRNA-21 inhibited proliferation and cell cycle progress of A549 cells and sensitized cells to radiation. Decreased miRNA-21 expression promoted the apoptosis of A549 cells induced by irradiation. miRNA-21 may be considered as a potential novel target for future development of specific therapeutic interventions in NSCLC

  13. Improving fold activation of small transcription activating RNAs (STARs) with rational RNA engineering strategies.

    Science.gov (United States)

    Meyer, Sarai; Chappell, James; Sankar, Sitara; Chew, Rebecca; Lucks, Julius B

    2016-01-01

    Regulatory RNAs have become integral components of the synthetic biology and bioengineering toolbox for controlling gene expression. We recently expanded this toolbox by creating small transcription activating RNAs (STARs) that act by disrupting the formation of a target transcriptional terminator hairpin placed upstream of a gene. While STARs are a promising addition to the repertoire of RNA regulators, much work remains to be done to optimize the fold activation of these systems. Here we apply rational RNA engineering strategies to improve the fold activation of two STAR regulators. We demonstrate that a combination of promoter strength tuning and multiple RNA engineering strategies can improve fold activation from 5.4-fold to 13.4-fold for a STAR regulator derived from the pbuE riboswitch terminator. We then validate the generality of our approach and show that these same strategies improve fold activation from 2.1-fold to 14.6-fold for an unrelated STAR regulator, opening the door to creating a range of additional STARs to use in a broad array of biotechnologies. We also establish that the optimizations preserve the orthogonality of these STARs between themselves and a set of RNA transcriptional repressors, enabling these optimized STARs to be used in sophisticated circuits. PMID:26134708

  14. MicroRNA-like small RNAs prediction in the development of Antrodia cinnamomea.

    Directory of Open Access Journals (Sweden)

    Yan-Liang Lin

    Full Text Available Antrodia cinnamomea, a precious, host-specific brown-rot fungus that has been used as a folk medicine in Taiwan for centuries is known to have diverse bioactive compounds with potent pharmaceutical activity. In this study, different fermentation states of A. cinnamomea (wild-type fruiting bodies and liquid cultured mycelium were sequenced using the next-generation sequencing (NGS technique. A 45.58 Mb genome encoding 6,522 predicted genes was obtained. High quality reads were assembled into a total of 13,109 unigenes. Using a previously constructed pipeline to search for microRNAs (miRNAs, we then identified 4 predicted conserved miRNA and 63 novel predicted miRNA-like small RNA (milRNA candidates. Target prediction revealed several interesting proteins involved in tri-terpenoid synthesis, mating type recognition, chemical or physical sensory protein and transporters predicted to be regulated by the miRNAs and milRNAs.

  15. Improving fold activation of small transcription activating RNAs (STARs) with rational RNA engineering strategies.

    Science.gov (United States)

    Meyer, Sarai; Chappell, James; Sankar, Sitara; Chew, Rebecca; Lucks, Julius B

    2016-01-01

    Regulatory RNAs have become integral components of the synthetic biology and bioengineering toolbox for controlling gene expression. We recently expanded this toolbox by creating small transcription activating RNAs (STARs) that act by disrupting the formation of a target transcriptional terminator hairpin placed upstream of a gene. While STARs are a promising addition to the repertoire of RNA regulators, much work remains to be done to optimize the fold activation of these systems. Here we apply rational RNA engineering strategies to improve the fold activation of two STAR regulators. We demonstrate that a combination of promoter strength tuning and multiple RNA engineering strategies can improve fold activation from 5.4-fold to 13.4-fold for a STAR regulator derived from the pbuE riboswitch terminator. We then validate the generality of our approach and show that these same strategies improve fold activation from 2.1-fold to 14.6-fold for an unrelated STAR regulator, opening the door to creating a range of additional STARs to use in a broad array of biotechnologies. We also establish that the optimizations preserve the orthogonality of these STARs between themselves and a set of RNA transcriptional repressors, enabling these optimized STARs to be used in sophisticated circuits.

  16. One step engineering of the small-subunit ribosomal RNA using CRISPR/Cas9.

    Science.gov (United States)

    Kannan, Krishna; Tsvetanova, Billyana; Chuang, Ray-Yuan; Noskov, Vladimir N; Assad-Garcia, Nacyra; Ma, Li; Hutchison Iii, Clyde A; Smith, Hamilton O; Glass, John I; Merryman, Chuck; Venter, J Craig; Gibson, Daniel G

    2016-01-01

    Bacteria are indispensable for the study of fundamental molecular biology processes due to their relatively simple gene and genome architecture. The ability to engineer bacterial chromosomes is quintessential for understanding gene functions. Here we demonstrate the engineering of the small-ribosomal subunit (16S) RNA of Mycoplasma mycoides, by combining the CRISPR/Cas9 system and the yeast recombination machinery. We cloned the entire genome of M. mycoides in yeast and used constitutively expressed Cas9 together with in vitro transcribed guide-RNAs to introduce engineered 16S rRNA genes. By testing the function of the engineered 16S rRNA genes through genome transplantation, we observed surprising resilience of this gene to addition of genetic elements or helix substitutions with phylogenetically-distant bacteria. While this system could be further used to study the function of the 16S rRNA, one could envision the "simple" M. mycoides genome being used in this setting to study other genetic structures and functions to answer fundamental questions of life. PMID:27489041

  17. One step engineering of the small-subunit ribosomal RNA using CRISPR/Cas9

    Science.gov (United States)

    Kannan, Krishna; Tsvetanova, Billyana; Chuang, Ray-Yuan; Noskov, Vladimir N.; Assad-Garcia, Nacyra; Ma, Li; Hutchison III, Clyde A.; Smith, Hamilton O.; Glass, John I.; Merryman, Chuck; Venter, J. Craig; Gibson, Daniel G.

    2016-01-01

    Bacteria are indispensable for the study of fundamental molecular biology processes due to their relatively simple gene and genome architecture. The ability to engineer bacterial chromosomes is quintessential for understanding gene functions. Here we demonstrate the engineering of the small-ribosomal subunit (16S) RNA of Mycoplasma mycoides, by combining the CRISPR/Cas9 system and the yeast recombination machinery. We cloned the entire genome of M. mycoides in yeast and used constitutively expressed Cas9 together with in vitro transcribed guide-RNAs to introduce engineered 16S rRNA genes. By testing the function of the engineered 16S rRNA genes through genome transplantation, we observed surprising resilience of this gene to addition of genetic elements or helix substitutions with phylogenetically-distant bacteria. While this system could be further used to study the function of the 16S rRNA, one could envision the “simple” M. mycoides genome being used in this setting to study other genetic structures and functions to answer fundamental questions of life. PMID:27489041

  18. Small RNA Control of Cell-to-Cell Communication in Vibrio Harveyi and Vibrio Cholerae

    Science.gov (United States)

    Svenningsen, Sine Lo

    Quorum sensing is a process of cell-to-cell communication, by which bacteria coordinate gene expression and behavior on a population-wide scale. Quorum sensing is accomplished through production, secretion, and subsequent detection of chemical signaling molecules termed autoinducers. The human pathogen Vibrio cholerae and the marine bioluminescent bacterium Vibrio harveyi incorporate information from multiple autoinducers, and also environmental signals and metabolic cues into their quorum-sensing pathways. At the core of these pathways lie several homologous small regulatory RNA molecules, the Quorum Regulatory RNAs. Small noncoding RNAs have emerged throughout the bacterial and eukaryotic kingdoms as key regulators of behavioral and developmental processes. Here, I review our present understanding of the role of the Qrr small RNAs in integrating quorum-sensing signals and in regulating the individual cells response to this information.

  19. Small RNA Regulators of Plant-Hemipteran Interactions: Micromanagers with Versatile Roles.

    Science.gov (United States)

    Sattar, Sampurna; Thompson, Gary A

    2016-01-01

    Non-coding small RNAs (sRNAs) in plants have important roles in regulating biological processes, including development, reproduction, and stress responses. Recent research indicates significant roles for sRNA-mediated gene silencing during plant-hemipteran interactions that involve all three of these biological processes. Plant responses to hemipteran feeding are determined by changes in the host transcriptome that appear to be fine-tuned by sRNAs. The role of sRNA in plant defense responses is complex. Different forms of sRNAs, with specific modes of action, regulate changes in the host transcriptome primarily through post-transcriptional gene silencing and occasionally through translational repression. Plant genetic resistance against hemipterans provides a model to explore the regulatory roles of sRNAs in plant defense. Aphid-induced sRNA expression in resistance genotypes delivers a new paradigm in understanding the regulation of R gene-mediated resistance in host plants. Unique sRNA profiles, including changes in sRNA biogenesis and expression can also provide insights into susceptibility to insect herbivores. Activation of phytohormone-mediated defense responses against insect herbivory is another hallmark of this interaction, and recent studies have shown that regulation of phytohormone signaling is under the control of sRNAs. Hemipterans feeding on resistant plants also show changes in insect sRNA profiles, possibly influencing insect development and reproduction. Changes in insect traits such as fecundity, host range, and resistance to insecticides are impacted by sRNAs and can directly contribute to the success of certain insect biotypes. In addition to causing direct damage to the host plant, hemipteran insects are often vectors of viral pathogens. Insect anti-viral RNAi machinery is activated to limit virus accumulation, suggesting a role in insect immunity. Virus-derived long sRNAs strongly resemble insect piRNAs, leading to the speculation that the piRNA

  20. Small RNA Regulators of Plant-Hemipteran Interactions: Micromanagers with Versatile Roles

    Science.gov (United States)

    Sattar, Sampurna; Thompson, Gary A.

    2016-01-01

    Non-coding small RNAs (sRNAs) in plants have important roles in regulating biological processes, including development, reproduction, and stress responses. Recent research indicates significant roles for sRNA-mediated gene silencing during plant-hemipteran interactions that involve all three of these biological processes. Plant responses to hemipteran feeding are determined by changes in the host transcriptome that appear to be fine-tuned by sRNAs. The role of sRNA in plant defense responses is complex. Different forms of sRNAs, with specific modes of action, regulate changes in the host transcriptome primarily through post-transcriptional gene silencing and occasionally through translational repression. Plant genetic resistance against hemipterans provides a model to explore the regulatory roles of sRNAs in plant defense. Aphid-induced sRNA expression in resistance genotypes delivers a new paradigm in understanding the regulation of R gene-mediated resistance in host plants. Unique sRNA profiles, including changes in sRNA biogenesis and expression can also provide insights into susceptibility to insect herbivores. Activation of phytohormone-mediated defense responses against insect herbivory is another hallmark of this interaction, and recent studies have shown that regulation of phytohormone signaling is under the control of sRNAs. Hemipterans feeding on resistant plants also show changes in insect sRNA profiles, possibly influencing insect development and reproduction. Changes in insect traits such as fecundity, host range, and resistance to insecticides are impacted by sRNAs and can directly contribute to the success of certain insect biotypes. In addition to causing direct damage to the host plant, hemipteran insects are often vectors of viral pathogens. Insect anti-viral RNAi machinery is activated to limit virus accumulation, suggesting a role in insect immunity. Virus-derived long sRNAs strongly resemble insect piRNAs, leading to the speculation that the piRNA

  1. An in vitro selection for small molecule induced switching RNA molecules.

    Science.gov (United States)

    Martini, Laura; Ellington, Andrew D; Mansy, Sheref S

    2016-08-15

    The selection of RNA and DNA aptamers now has a long history. However, the ability to directly select for conformational changes upon ligand binding has remained elusive. These difficulties have stymied attempts at making small molecule responsive strand displacement circuitry as well as synthetic riboswitches. Herein we present a detailed strand displacement based selection protocol to directly select for RNA molecules with switching activity. The library was based on a previously selected thiamine pyrophosphate riboswitch. The fully in vitro methodology gave sequences that showed strong strand displacement activity in the presence of thiamine pyrophosphate. Further, the selected sequences possessed riboswitch activity similar to that of natural riboswitches. The presented methodology should aid in the design of more complex, environmentally responsive strand displacement circuitry and in the selection of riboswitches responsive to toxic ligands. PMID:26899430

  2. Sequences more than 500 base pairs upstream of the human U3 small nuclear RNA gene stimulate the synthesis of U3 RNA in frog oocytes

    International Nuclear Information System (INIS)

    Small nuclear RNA (snRNA) genes contain strong promoters capable of initiating transcription once every 4 s. Studies on the human U1 snRNA gene, carried out in other laboratories, showed that sequences within 400 bp of the 5' flanking region are sufficient for maximal levels of transcription both in vivo and in frog oocytes [reviewed in Dahlberg and Lund (1988)]. The authors studied the expression of a human U3 snRNA gene by injecting 5' deletion mutants into frog oocytes. The results show that sequences more than 500 bp upstream of the U3 snRNA gene have a 2-3-fold stimulatory effect on the U3 snRNA synthesis. These results indicate that the human U3 snRNA gene is different from human U1 snRNA gene in containing regulatory elements more than 500 bp upstream. The U3 snRNA gene upstream sequences contain an AluI homologous sequence in the -1,200 region; these AluI sequences were transcribed in vitro and in frog oocytes but were not detectable in Hela cells

  3. A method for in vivo identification of bacterial small RNA-binding proteins.

    Science.gov (United States)

    Osborne, Jonathan; Djapgne, Louise; Tran, Bao Quoc; Goo, Young Ah; Oglesby-Sherrouse, Amanda G

    2014-12-01

    Small bacterial regulatory RNAs (sRNAs) have gained immense appreciation over the last decade for their roles in mediating posttranscriptional gene regulation of numerous physiological processes. Several proteins contribute to sRNA stability and regulation, most notably the Hfq RNA-binding protein. However, not all sRNAs rely on Hfq for their stability. It is therefore likely that other proteins contribute to the stability and function of certain bacterial sRNAs. Here, we describe a methodology for identifying in vivo-binding proteins of sRNAs, developed using the iron-responsive PrrF and PrrH sRNAs of Pseudomonas aeruginosa. RNA was isolated from iron-depleted cultures, which were irradiated to cross-link nucleoprotein complexes. Subsequently, PrrF- and PrrH-protein complexes were enriched using cDNA "bait", and enriched RNA-protein complexes were analyzed by tandem mass spectrometry to identify PrrF and PrrH associated proteins. This method identified Hfq as a potential PrrF- and PrrH-binding protein. Interestingly, Hfq was identified more often in samples probed with the PrrF cDNA "bait" as compared to the PrrH cDNA "bait", suggesting Hfq has a stronger binding affinity for the PrrF sRNAs in vivo. Hfq binding to the PrrF and PrrH sRNAs was validated by electrophoretic mobility shift assays with purified Hfq protein from P. aeruginosa. As such, this study demonstrates that in vivo cross-linking coupled with sequence-specific affinity chromatography and tandem mass spectrometry (SSAC-MS/MS) is an effective methodology for unbiased identification of bacterial sRNA-binding proteins.

  4. Suppression of sphingomyelin synthase 1 by small interference RNA is associated with enhanced ceramide production and apoptosis after photodamage

    OpenAIRE

    Separovic, Duska; Semaan, Louie; Tarca, Adi L.; Maitah, Ma’In Yehya Awad; Hanada, Kentaro; Bielawski, Jacek; Villani, Maristella; Luberto, Chiara

    2008-01-01

    We have shown that overexpression of SMS1, an enzyme that converts de novo ceramide into sphingomyelin, is accompanied by attenuated ceramide response and apoptotic resistance after photodamage with the photosensitizer Pc 4 (photodynamic therapy; PDT). To test whether SMS1 overexpression-related effects after PDT can be reversed, in this study SMS1 was downregulated in Jurkat T lymphoma/leukemia cells using small inhibitory RNA (siRNA) for SMS1. Compared to scrambled (control) siRNA-transfect...

  5. Identification of Novel MicroRNAs in Primates by Using the Synteny Information and Small RNA Deep Sequencing Data

    OpenAIRE

    Yuan, Zhidong; Liu, Hongde; Nie, Yumin; Ding, Suping; Yan, Mingli; Tan, Shuhua; Jin, Yuanchang; Sun, Xiao

    2013-01-01

    Current technologies that are used for genome-wide microRNA (miRNA) prediction are mainly based on BLAST tool. They often produce a large number of false positives. Here, we describe an effective approach for identifying orthologous pre-miRNAs in several primates based on syntenic information. Some of them have been validated by small RNA high throughput sequencing data. This approach uses the synteny information and experimentally validated miRNAs of human, and incorporates currently availab...

  6. Effects of down-regulation of clusterin by small interference RNA on human acute myeloid leukemia cells

    OpenAIRE

    Wang, Xiaoli; Liu, Ruidong; Wang, Yanxia; Cai, Hengjuan; Zhang, Lei

    2015-01-01

    Aims and background: Up-regulation of clusterin is associated with the survival and progression of various malignancies, and down-regulation of clusterin promotes apoptosis and inhibits invasion. The aim of this study was to explore the effect of clusterin small interference RNA (siRNA) on the proliferation, apoptosis and invasion of HL-60 acute myeloid leukemia (AML) cells. Methods: siRNA transfection was performed using Lipofectamine™2000 reagent. Relative protein expressions were quantifie...

  7. Application of small RNA technology for improved control of parasitic helminths.

    Science.gov (United States)

    Britton, Collette; Winter, Alan D; Marks, Neil D; Gu, Henry; McNeilly, Tom N; Gillan, Victoria; Devaney, Eileen

    2015-08-15

    Over the last decade microRNAs (miRNAs) and small interfering RNAs (siRNAs) have emerged as important regulators of post-transcriptional gene expression. miRNAs are short, non-coding RNAs that regulate a variety of processes including cancer, organ development and immune function. This class of small RNAs bind with partial complementarity to their target mRNA sequences, most often in the 3'UTR, to negatively regulate gene expression. In parasitic helminths, miRNAs are being increasingly studied for their potential roles in development and host-parasite interactions. The availability of genome data, combined with small RNA sequencing, has paved the way to profile miRNAs expressed at particular developmental stages for many parasitic helminths. While some miRNAs are conserved across species, others appear to be unique to specific parasites, suggesting important roles in adaptation and survival in the host environment. Some miRNAs are released from parasites, in exosomes or in protein complexes, and the potential effects of these on host immune function are being increasingly studied. In addition, release of miRNAs from schistosome and filarial parasites into host plasma can be exploited for the development of specific and sensitive diagnostic biomarkers of infection. Interfering with miRNA function, as well as silencing key components of the pathways they regulate, will progress our understanding of parasite development and provide a novel approach to therapeutic control. RNA interference (RNAi) by siRNAs has proven to be inconsistent in parasitic nematodes. However, the recent successes reported for schistosome and liver fluke RNAi, encourage further efforts to enhance delivery of RNA and improve in vitro culture systems and assays to monitor phenotypic effects in nematodes. These improvements are important for the establishment of reliable functional genomic platforms for novel drug and vaccine development. In this review we focus on the important roles of mi

  8. Binding of small interfering RNA molecules is crucial for RNA interference suppressor activity of rice hoja blanca virus NS3 in plants.

    Science.gov (United States)

    Hemmes, Hans; Kaaij, Lucas; Lohuis, Dick; Prins, Marcel; Goldbach, Rob; Schnettler, Esther

    2009-07-01

    The NS3 protein of rice hoja blanca virus represents a viral suppressor of RNA interference (RNAi) that sequesters small interfering (si)RNAs in vitro. To determine whether this siRNA binding property is the critical determinant for the suppressor activity of NS3, NS3 was altered by alanine point mutations and the resulting mutant proteins were tested for both siRNA binding ability and RNAi suppressor activity in plants. Alanine substitutions of lysine residues at positions 173-175 resulted in mutant proteins that lost both their affinity for siRNAs and their RNAi suppressor activity in planta. This indicates that siRNA binding of NS3 is indeed essential for the suppressor function of NS3 and that residues at positions 173-175 are involved in the siRNA binding and suppressor activities. PMID:19282433

  9. In human granulosa cells from small antral follicles, androgen receptor mRNA and androgen levels in follicular fluid correlate with FSH receptor mRNA

    DEFF Research Database (Denmark)

    Nielsen, M. E.; Rasmussen, I. A.; Kristensen, Stine Gry;

    2011-01-01

    RNA analysis (24 women). Expression of Androgen Receptor (AR) mRNA levels in granulosa cells, and of androstenedione and testosterone in FF, were correlated to the expression of FSH receptor (FSHR), LH receptor (LHR), CYP19 and anti-Müllerian Hormone-receptor2 (AMHR2) mRNA in the granulosa cells and to the FF...... with the expression of AMHR2, but did not correlate with any of the hormones in the FF. These data demonstrate an intimate association between AR expression in immature granulosa cells, and the expression of FSHR in normal small human antral follicles and between the FF levels of androgen and FSHR expression...

  10. MicroRNA Transcriptome in Swine Small Intestine during Weaning Stress

    OpenAIRE

    Tao, Xin; Xu, Ziwei

    2013-01-01

    MicroRNAs (miRNAs) play important roles in intestinal diseases; however, the role of miRNAs during weaning stress is unknown. In our study, six jejunal small RNA libraries constructed from weaning piglets at 1, 4 and 7 d after weaning (libraries W1, W4 and W7, respectively) and from suckling piglets on the same days as the weaning piglets (libraries S1, S4 and S7, respectively) were sequenced using Solexa high-throughput sequencing technology. Overall, 260 known swine miRNAs and 317 novel can...

  11. Elucidation of the physicochemical properties and potency of siRNA-loaded small-sized lipid nanoparticles for siRNA delivery.

    Science.gov (United States)

    Sato, Yusuke; Note, Yusuke; Maeki, Masatoshi; Kaji, Noritada; Baba, Yoshinobu; Tokeshi, Manabu; Harashima, Hideyoshi

    2016-05-10

    Because nanoparticles with diameters less than 50nm penetrate stromal-rich tumor tissues more efficiently, the synthesis of small-sized nanoparticles encapsulating short interfering RNA (siRNA) is important in terms of realizing novel siRNA medicine for the treatment of various cancers. Lipid nanoparticles (LNPs) are the leading systems for the delivery of siRNA in vivo. Limit size LNPs were successfully synthesized using a microfluidic mixing technique. However, the physicochemical properties and potential for in vivo siRNA delivery of the limit-size LNPs have not been examined in detail. In the present study, we prepared LNPs with different diameters from 32 to 67nm using a microfluidic mixing devise and examined the physicochemical properties of the particles and the potential for their use in delivering siRNA in vitro and in vivo to liver. Reducing the size of the LNPs causes poor-packing and an increased surface area, which result in their instability in serum. Moreover, it was revealed that the ability of endosomal escape (cytosolic siRNA release) of the smaller LNPs is subject to inhibition by serum compared to that of larger counterparts. Taken together, an increase in packing and avoiding the adsorption of serum components are key strategies for the development of next-generation highly potent and small-sized LNPs. PMID:26995758

  12. Down-regulation of survivin expression by small interfering RNA induces pancreatic cancer cell apoptosis and enhances its radiosensitivity

    Institute of Scientific and Technical Information of China (English)

    Hai-Tao Guan; Xing-Huan Xue; Zhi-Jun Dai; Xi-Jing Wang; Ang Li; Zhao-Yin Qin

    2006-01-01

    AIM: To investigate the inhibitory effect of small interfering RNA (siRNA) on the expression of survivin in pancreatic cancer cell line PC-2 and the role of siRNA in inducing PC-2 cell apoptosis and enhancing its radiosensitivity.METHODS: A siRNA plasmid expression vector against survivin was constructed and transfected into PC-2 cells with LipofectamineTM 2000. The down regulation of survivin expression was detected by semi-quantitive RT-PCR and immunohistochemical SP method and the role of siRNA in inducing PC-2 cell apoptosis and enhancing its radiosensitivity was detected by flow cytometry.RESULTS: The sequence-specific siRNA efficiently and specifically down-regulated the expression of survivin at both mRNA and protein levels. The expression inhibition ratio was 81.25% at mRNA level detected by semiquantitive RT-PCR and 74.24% at protein level detected by immunohistochemical method. Forty-eight hours after transfection,apoptosis was induced in 7.03% cells by siRNA and in 14.58% cells by siRNA combined with radiation.CONCLUSION: The siRNA plasmid expression vector against survivin can inhibit the expression of survivin in PC-2 cells efficiently and specifically. Inhibiting the expression of survivin can induce apoptosis of PC-2 cells and enhance its radiosensitivity significantly. RNAi against survivin is of potential value in gene tnerapy of pancreatic cancer.

  13. The Andes Hantavirus NSs Protein Is Expressed from the Viral Small mRNA by a Leaky Scanning Mechanism

    OpenAIRE

    Vera-Otarola, Jorge; Solis, Loretto; Soto-Rifo, Ricardo; Ricci, Emiliano P.; Pino, Karla; Tischler, Nicole D.; Ohlmann, Théophile; Darlix, Jean-Luc; López-Lastra, Marcelo

    2012-01-01

    The small mRNA (SmRNA) of all Bunyaviridae encodes the nucleocapsid (N) protein. In 4 out of 5 genera in the Bunyaviridae, the smRNA encodes an additional nonstructural protein denominated NSs. In this study, we show that Andes hantavirus (ANDV) SmRNA encodes an NSs protein. Data show that the NSs protein is expressed in the context of an ANDV infection. Additionally, our results suggest that translation initiation from the NSs initiation codon is mediated by ribosomal subunits that have bypa...

  14. Analysis of small RNA production patterns among the two potato spindle tuber viroid variants in tomato plants

    OpenAIRE

    Charith Raj Adkar-Purushothama; Jean-Pierre Perreault; Teruo Sano

    2015-01-01

    In order to analyze the production of small RNA (sRNA) by viroids upon infecting the plants, the tomato plants (Solanum lycopersicum cultivar Rutgers) were inoculated with the variants of Potato spindle tuber viroid (PSTVd). After 21-days of postinoculation, total RNA was extracted and subjected for deep-sequencing using Illumina HiSeq platform. The primers were trimmed and only 21- to 24-nt long sRNAs were filtered after quality check of the raw data. The filtered sRNA population was then ma...

  15. The plasma lncRNA acting as fingerprint in non-small-cell lung cancer.

    Science.gov (United States)

    Hu, Xiaodong; Bao, Jitao; Wang, Zhen; Zhang, Zigang; Gu, Peijie; Tao, Feng; Cui, Di; Jiang, Weilong

    2016-03-01

    Recent studies have indicated that long non-coding RNAs (lncRNAs) could act as non-invasive tumor markers in both diagnosis and predicting the prognosis. In this study, we focused to determine the expression of circulating lncRNAs in patients suffering from non-small-cell lung cancer (NSCLC), aiming to found the potential lncRNA as predictor. Twenty-one lncRNAs which previously identified were selected as candidate targets for subsequent circulating lncRNA assay. The candidate lncRNAs were validated by qRT-PCR arranged in the training and validation sets. Circulating SPRY4-IT1, ANRIL, and NEAT1 were significantly increased in plasma samples of NSCLC patients during training set and validation set. Receiver operating characteristic curve (ROC) analysis revealed that plasma ANRIL provided the highest diagnostic performance with an area under ROC curve value (AUC) of 0.798. Further combination with the three factors indicated a higher power (AUC, 0.876; sensitivity, 82.8 %; specificity, 92.3 %). The stableness detection of the three factors indicated that circulating SPRY4-IT1, ANRIL, and NEAT might serve as a predictor for the early warning of non-small-cell lung cancer. PMID:26453113

  16. A small RNA controls expression of the chitinase ChiA in Listeria monocytogenes

    OpenAIRE

    Nielsen, Jesper S.; Marianne Halberg Larsen; Eva Maria Sternkopf Lillebæk; Bergholz, Teresa M.; Mie H G Christiansen; Boor, Kathryn J.; Martin Wiedmann; Kallipolitis, Birgitte H.

    2011-01-01

    In recent years, more than 60 small RNAs (sRNAs) have been identified in the gram-positive human pathogen Listeria monocytogenes, but their putative roles and mechanisms of action remain largely unknown. The sRNA LhrA was recently shown to be a post-transcriptional regulator of a single gene, lmo0850, which encodes a small protein of unknown function. LhrA controls the translation and degradation of the lmo0850 mRNA by an antisense mechanism, and it depends on the RNA chaperone Hfq for effici...

  17. Chemical derivatives of a small molecule deubiquitinase inhibitor have antiviral activity against several RNA viruses.

    Directory of Open Access Journals (Sweden)

    Marta J Gonzalez-Hernandez

    Full Text Available Most antiviral treatment options target the invading pathogen and unavoidably encounter loss of efficacy as the pathogen mutates to overcome replication restrictions. A good strategy for circumventing drug resistance, or for pathogens without treatment options, is to target host cell proteins that are utilized by viruses during infection. The small molecule WP1130 is a selective deubiquitinase inhibitor shown previously to successfully reduce replication of noroviruses and some other RNA viruses. In this study, we screened a library of 31 small molecule derivatives of WP1130 to identify compounds that retained the broad-spectrum antiviral activity of the parent compound in vitro but exhibited improved drug-like properties, particularly increased aqueous solubility. Seventeen compounds significantly reduced murine norovirus infection in murine macrophage RAW 264.7 cells, with four causing decreases in viral titers that were similar or slightly better than WP1130 (1.9 to 2.6 log scale. Antiviral activity was observed following pre-treatment and up to 1 hour postinfection in RAW 264.7 cells as well as in primary bone marrow-derived macrophages. Treatment of the human norovirus replicon system cell line with the same four compounds also decreased levels of Norwalk virus RNA. No significant cytotoxicity was observed at the working concentration of 5 µM for all compounds tested. In addition, the WP1130 derivatives maintained their broad-spectrum antiviral activity against other RNA viruses, Sindbis virus, LaCrosse virus, encephalomyocarditis virus, and Tulane virus. Thus, altering structural characteristics of WP1130 can maintain effective broad-spectrum antiviral activity while increasing aqueous solubility.

  18. Small RNA sequencing for profiling microRNAs in long-term preserved formalin-fixed and paraffin-embedded non-small cell lung cancer tumor specimens.

    Directory of Open Access Journals (Sweden)

    Daniel H Buitrago

    Full Text Available The preservation of microRNAs in formalin-fixed and paraffin-embedded (FFPE tissue makes them particularly useful for biomarker studies. The utility of small RNA sequencing for microRNA expression profiling of FFPE samples has yet to be determined.Total RNA was extracted from de-paraffinized and proteinase K-treated FFPE specimens (15-20 years old of 8 human lung adenocarcinoma tumors by affinity chromatography on silica columns. MicroRNAs in the RNA preparations were quantified by the Illumina HiSeq 2000 sequencing platform with sequencing libraries prepared with the TruSeq Small RNA Sample Preparation Kit (version 2.0 to obtain unpaired reads of 50 b for small RNA fragments. MicroRNAs were also quantified using Agilent Human miRNA (release 16.0 microarrays that can detect 1,205 mature microRNAs and by quantitative reverse transcription (RT-PCR assays.Between 9.1-16.9 million reads were obtained by small RNA sequencing of extracted RNA samples. Of these, only 0.6-2.3% (mean = 1.5% represented microRNAs. The sequencing method detected 454-625 microRNAs/sample (mean = 550 compared with 200-349 (mean = 286 microRNAs detected by microarray. In Spearman correlation analyses, the average correlation coefficient for the 126 microRNAs detected in all samples by both methods was 0.37, and >0.5 for 63 microRNAs. In correlation analyses of the sequencing- and RT-PCR-based measurements, the coefficients were 0.19-0.95 (mean = 0.73 and >0.7, respectively, for 7 of 9 examined microRNAs. The average inter-replicate Spearman correlation coefficient for the sequencing method was 0.81.Small RNA sequencing can be used to obtain microRNA profiles of FFPE tissue specimens with performance characteristics similar to those of microarrays, in spite of the fragmentation of ribosomal and messenger RNAs that reduces the method's informative capacity. The accuracy of the method can conceivably be improved by increasing sequencing depth and/or depleting FFPE tissue RNAs of

  19. Small-angle X-ray scattering: a bridge between RNA secondary structures and three-dimensional topological structures

    Energy Technology Data Exchange (ETDEWEB)

    Fang, Xianyang [National Inst. of Health (NIH), Bethesda, MD (United States). National Cancer Inst., NCI Small Angle X-ray Scattering Core Facility; Stagno, Jason R. [National Inst. of Health (NIH), Bethesda, MD (United States). National Cancer Inst., Protein-Nucleic Acid Interaction Section, Structural Biophysics Lab.; Bhandari, Yuba R. [National Inst. of Health (NIH), Bethesda, MD (United States). National Cancer Inst., Protein-Nucleic Acid Interaction Section, Structural Biophysics Lab.; Zuo, Xiaobing [Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS); Wang, Yun-Xing [National Inst. of Health (NIH), Bethesda, MD (United States). National Cancer Inst., NCI Small Angle X-ray Scattering Core Facility; National Inst. of Health (NIH), Bethesda, MD (United States). National Cancer Inst., Protein-Nucleic Acid Interaction Section, Structural Biophysics Lab.

    2015-02-01

    Whereas the structures of small to medium-sized well folded RNA molecules often can be determined by either X-ray crystallography or NMR spectroscopy, obtaining structural information for large RNAs using experimental, computational, or combined approaches remains a major interest and challenge. RNA is very sensitive to small-angle X-ray scattering (SAXS) due to high electron density along phosphate-sugar backbones, whose scattering contribution dominates SAXS intensity. For this reason, SAXS is particularly useful in obtaining global RNA structural information that outlines backbone topologies and, therefore, molecular envelopes. Such information is extremely valuable in bridging the gap between the secondary structures and three-dimensional topological structures of RNAmolecules, particularly those that have proven difficult to study using other structuredetermination methods. Here we review published results of RNA topological structures derived from SAXS data or in combination with other experimental data, as well as details on RNA sample preparation for SAXS experiments.

  20. Mpn1, Mutated in Poikiloderma with Neutropenia Protein 1, Is a Conserved 3′-to-5′ RNA Exonuclease Processing U6 Small Nuclear RNA

    Directory of Open Access Journals (Sweden)

    Vadim Shchepachev

    2012-10-01

    Full Text Available Clericuzio-type poikiloderma with neutropenia (PN is a rare genodermatosis associated with mutations in the C16orf57 gene, which codes for the uncharacterized protein hMpn1. We show here that, in both fission yeasts and humans, Mpn1 processes the spliceosomal U6 small nuclear RNA (snRNA posttranscriptionally. In Mpn1-deficient cells, U6 molecules carry 3′ end polyuridine tails that are longer than those in normal cells and lack a terminal 2′,3′ cyclic phosphate group. In mpn1Δ yeast cells, U6 snRNA and U4/U6 di-small nuclear RNA protein complex levels are diminished, leading to precursor messenger RNA splicing defects, which are reverted by expression of either yeast or human Mpn1 and by overexpression of U6. Recombinant hMpn1 is a 3′-to-5′ RNA exonuclease that removes uridines from U6 3′ ends, generating terminal 2′,3′ cyclic phosphates in vitro. Finally, U6 degradation rates increase in mpn1Δ yeasts and in lymphoblasts established from individuals affected by PN. Our data indicate that Mpn1 promotes U6 stability through 3′ end posttranscriptional processing and implicate altered U6 metabolism as a potential mechanism for PN pathogenesis.

  1. A small RNA controls expression of the chitinase ChiA in Listeria monocytogenes.

    Directory of Open Access Journals (Sweden)

    Jesper S Nielsen

    Full Text Available In recent years, more than 60 small RNAs (sRNAs have been identified in the gram-positive human pathogen Listeria monocytogenes, but their putative roles and mechanisms of action remain largely unknown. The sRNA LhrA was recently shown to be a post-transcriptional regulator of a single gene, lmo0850, which encodes a small protein of unknown function. LhrA controls the translation and degradation of the lmo0850 mRNA by an antisense mechanism, and it depends on the RNA chaperone Hfq for efficient binding to its target. In the present study, we sought to gain more insight into the functional role of LhrA in L. monocytogenes. To this end, we determined the effects of LhrA on global-wide gene expression. We observed that nearly 300 genes in L. monocytogenes are either positively or negatively affected by LhrA. Among these genes, we identified lmo0302 and chiA as direct targets of LhrA, thus establishing LhrA as a multiple target regulator. Lmo0302 encodes a hypothetical protein with no known function, whereas chiA encodes one of two chitinases present in L. monocytogenes. We show here that LhrA acts as a post-transcriptional regulator of lmo0302 and chiA by interfering with ribosome recruitment, and we provide evidence that both LhrA and Hfq act to down-regulate the expression of lmo0302 and chiA. Furthermore, in vitro binding experiments show that Hfq stimulates the base pairing of LhrA to chiA mRNA. Finally, we demonstrate that LhrA has a negative effect on the chitinolytic activity of L. monocytogenes. In marked contrast to this, we found that Hfq has a stimulating effect on the chitinolytic activity, suggesting that Hfq plays multiple roles in the complex regulatory pathways controlling the chitinases of L. monocytogenes.

  2. Binding of small interfering RNA molecules is crucial for RNA interference suppressor activity of rice hoja blanca virus NS3 in plants

    NARCIS (Netherlands)

    Hemmes, J.C.; Kaaij, L.; Lohuis, H.; Prins, M.W.; Goldbach, R.W.; Schnettler, E.

    2009-01-01

    The NS3 protein of rice hoja blanca tenuivirus represents a viral suppressor of RNAi that sequesters small interfering (si)RNAs in vitro. To determine whether this siRNA binding property is the critical determinant for the suppressor activity of NS3, an alanine point mutational analysis was performe

  3. Caenorhabditis elegans pseudouridine synthase 1 activity in vivo: tRNA is a substrate, but not U2 small nuclear RNA.

    Science.gov (United States)

    Patton, Jeffrey R; Padgett, Richard W

    2003-06-01

    The formation of pseudouridine (Psi) from uridine is post-transcriptional and catalysed by pseudouridine synthases, several of which have been characterized from eukaryotes. Pseudouridine synthase 1 (Pus1p) has been well characterized from yeast and mice. In yeast, Pus1p has been shown to have dual substrate specificity, modifying uridines in tRNAs and at position 44 in U2 small nuclear RNA (U2 snRNA). In order to study the in vivo activity of a metazoan Pus1p, a knockout of the gene coding for the homologue of Pus1p in Caenorhabditis elegans was obtained. The deletion encompasses the first two putative exons and includes the essential aspartate that is required for activity in truA pseudouridine synthases. The locations of most modified nucleotides on small RNAs in C. elegans are not known, and the positions of Psi were determined on four tRNAs and U2 snRNA. The uridine at position 27 of tRNA(Val) (AAC), a putative Pus1p-modification site, was converted into Psi in the wild-type worms, but the tRNA(Val) (AAC) from mutant worms lacked the modification. Psi formation at positions 13, 32, 38 and 39, all of which should be modified by other pseudouridine synthases, was not affected by the loss of Pus1p. The absence of Pus1p in C. elegans had no effect on the modification of U2 snRNA in vivo, even though worm U2 snRNA has a Psi at position 45 (the equivalent of yeast U2 snRNA position 44) and at four other positions. This result was unexpected, given the known dual specificity of yeast Pus1p.

  4. Small RNA and transcriptome deep sequencing proffers insight into floral gene regulation in Rosa cultivars

    Directory of Open Access Journals (Sweden)

    Kim Jungeun

    2012-11-01

    Full Text Available Abstract Background Roses (Rosa sp., which belong to the family Rosaceae, are the most economically important ornamental plants—making up 30% of the floriculture market. However, given high demand for roses, rose breeding programs are limited in molecular resources which can greatly enhance and speed breeding efforts. A better understanding of important genes that contribute to important floral development and desired phenotypes will lead to improved rose cultivars. For this study, we analyzed rose miRNAs and the rose flower transcriptome in order to generate a database to expound upon current knowledge regarding regulation of important floral characteristics. A rose genetic database will enable comprehensive analysis of gene expression and regulation via miRNA among different Rosa cultivars. Results We produced more than 0.5 million reads from expressed sequences, totalling more than 110 million bp. From these, we generated 35,657, 31,434, 34,725, and 39,722 flower unigenes from Rosa hybrid: ‘Vital’, ‘Maroussia’, and ‘Sympathy’ and Rosa rugosa Thunb. , respectively. The unigenes were assigned functional annotations, domains, metabolic pathways, Gene Ontology (GO terms, Plant Ontology (PO terms, and MIPS Functional Catalogue (FunCat terms. Rose flower transcripts were compared with genes from whole genome sequences of Rosaceae members (apple, strawberry, and peach and grape. We also produced approximately 40 million small RNA reads from flower tissue for Rosa, representing 267 unique miRNA tags. Among identified miRNAs, 25 of them were novel and 242 of them were conserved miRNAs. Statistical analyses of miRNA profiles revealed both shared and species-specific miRNAs, which presumably effect flower development and phenotypes. Conclusions In this study, we constructed a Rose miRNA and transcriptome database, and we analyzed the miRNAs and transcriptome generated from the flower tissues of four Rosa cultivars. The database provides a

  5. Identification and expression profiling of Vigna mungo microRNAs from leaf small RNA transcriptome by deep sequencing.

    Science.gov (United States)

    Paul, Sujay; Kundu, Anirban; Pal, Amita

    2014-01-01

    MicroRNAs (miRNAs) represent a class of small non-coding RNA molecules that play a crucial role in post-transcriptional gene regulation. Several conserved and species-specific miRNAs have been characterized to date, predominantly from the plant species whose genome is well characterized. However, information on the variability of these regulatory RNAs in economically important but genetically less characterized crop species are limited. Vigna mungo is an important grain legume, which is grown primarily for its protein-rich edible seeds. miRNAs from this species have not been identified to date due to lack of genome sequence information. To identify miRNAs from V. mungo, a small RNA library was constructed from young leaves. High-throughput Illumina sequencing technology and bioinformatic analysis of the small RNA reads led to the identification of 66 miRNA loci represented by 45 conserved miRNAs belonging to 19 families and eight non-conserved miRNAs belonging to seven families. Besides, 13 novel miRNA candidates in V. mungo were also identified. Expression patterns of selected conserved, non-conserved, and novel miRNA candidates have been demonstrated in leaf, stem, and root tissues by quantitative polymerase chain reaction, and potential target genes were predicted for most of the conserved miRNAs. This information offers genomic resources for better understanding of miRNA mediated post-transcriptional gene regulation.

  6. Identification of novel microRNAs in primates by using the synteny information and small RNA deep sequencing data.

    Science.gov (United States)

    Yuan, Zhidong; Liu, Hongde; Nie, Yumin; Ding, Suping; Yan, Mingli; Tan, Shuhua; Jin, Yuanchang; Sun, Xiao

    2013-01-01

    Current technologies that are used for genome-wide microRNA (miRNA) prediction are mainly based on BLAST tool. They often produce a large number of false positives. Here, we describe an effective approach for identifying orthologous pre-miRNAs in several primates based on syntenic information. Some of them have been validated by small RNA high throughput sequencing data. This approach uses the synteny information and experimentally validated miRNAs of human, and incorporates currently available algorithms and tools to identify the pre-miRNAs in five other primates. First, we identified 929 potential pre-miRNAs in the marmoset in which miRNAs have not yet been reported. Then, we predicted the miRNAs in other primates, and we successfully re-identified most of the published miRNAs and found 721, 979, 650 and 639 new potential pre-miRNAs in chimpanzee, gorilla, orangutan and rhesus macaque, respectively. Furthermore, the miRNA transcriptome in the four primates have been re-analyzed and some novel predicted miRNAs have been supported by the small RNA sequencing data. Finally, we analyzed the potential functions of those validated miRNAs and explored the regulatory elements and transcription factors of some validated miRNA genes of interest. The results show that our approach can effectively identify novel miRNAs and some miRNAs that supported by small RNA sequencing data maybe play roles in the nervous system. PMID:24135875

  7. Identification and expression profiling of Vigna mungo microRNAs from leaf small RNA transcriptome by deep sequencing.

    Science.gov (United States)

    Paul, Sujay; Kundu, Anirban; Pal, Amita

    2014-01-01

    MicroRNAs (miRNAs) represent a class of small non-coding RNA molecules that play a crucial role in post-transcriptional gene regulation. Several conserved and species-specific miRNAs have been characterized to date, predominantly from the plant species whose genome is well characterized. However, information on the variability of these regulatory RNAs in economically important but genetically less characterized crop species are limited. Vigna mungo is an important grain legume, which is grown primarily for its protein-rich edible seeds. miRNAs from this species have not been identified to date due to lack of genome sequence information. To identify miRNAs from V. mungo, a small RNA library was constructed from young leaves. High-throughput Illumina sequencing technology and bioinformatic analysis of the small RNA reads led to the identification of 66 miRNA loci represented by 45 conserved miRNAs belonging to 19 families and eight non-conserved miRNAs belonging to seven families. Besides, 13 novel miRNA candidates in V. mungo were also identified. Expression patterns of selected conserved, non-conserved, and novel miRNA candidates have been demonstrated in leaf, stem, and root tissues by quantitative polymerase chain reaction, and potential target genes were predicted for most of the conserved miRNAs. This information offers genomic resources for better understanding of miRNA mediated post-transcriptional gene regulation. PMID:24138283

  8. Bcl-XL Small Interfering RNA Enhances Sensitivity of Hepg2 Hepatocellular Carcinoma Cells to 5-Fluorouracil and Hydroxycamptothecin

    Institute of Scientific and Technical Information of China (English)

    Xiao-Yong LEI; Miao ZHONG; Lan-Fang FENG; Bing-Yang ZHU; Sheng-Song TANG; Duan-Fang LIAO

    2006-01-01

    Changes in drug sensitivity in Bcl-XL small interfering RNA (siRNA) transfected Hepg2 hepatocellular carcinoma cells were investigated in this study. Bcl-XL siRNA and negative siRNA expression vector were constructed and stably transfected into Hepg2 cells. Reverse transcription (RT)-PCR, western blot and immunofluorescence were used to detect the target gene expression at mRNA and protein levels.Drug sensitivity of the cells to 5-fluorouracil (5-FU) and hydroxycamptothecin (HCPT) were evaluated with MTT. The Bcl-XL mRNA and protein expression levels in Bcl-XL siRNA transfectants were reduced compared with negative siRNA transfectants or mock cells. MTT results showed that Bcl-XL siRNA transfected cells have a higher cell inhibition rate than negative vector transfected cells or untreated cells after treatment with 13, 130, 1300 and 13,000 mg/L of 5-FU. Bcl-XL siRNA transfected cells also showed increased drug-sensitivity compared with negative vector transfected cells or untreated cells after treatment with 0.18, 0.36, 0.72 and 1.44 mg/L HCPT. Flow cytometry (FCM) results demonstrated that the sub-G1 population increased in the Bcl-XL siRNA group, compared with the negative siRNA group and untreated control group, after the addition of 5-FU (1300 mg/L) and HCPT (0.72 mg/L). siRNA targeting Bcl-XL gene can specifically down-regulate Bcl-XL expression in Hepg2 cells, and can increase spontaneous cell apoptosis and sensitize cells to 5-FU or HCPT.

  9. Detection of CEA mRNA on non-small lung cancer and it's significance

    Directory of Open Access Journals (Sweden)

    Guowen WANG

    2008-10-01

    Full Text Available Background and objective In recent years, many studies on micrometastasis in non-small cell lung cancer (NSCLC have been reported, this study is to investigate the effect of operation on micrometastasis from NSCLC andevaluate the relation between micrometastasis and clincopathological parameters. Methods The blood samples were taken from 70 cases of NSCLC and 18 patients with benign diseases at 3 intervals during the operation from peripheral vein. The transcription of carcinoembryonic antigen messenger ribonucleic acid (CEA mRNA was assayed by means of nested reverse transcriptase polymerase chain reaction (RT-PCR and micro-fluid chip. Results The CEA mRNA positive rates of all 3 time spots were as follows: 50% at beginning of the operation (Time 1, 62.8% at ligating the pulmonary vein (Time 2 and 57.1% at 1 h after ligating pulmonary vein (Time 3. There is significant difference between Time 1 and Time 2 (χ2=7.114, P <0.05. The positive rates of well-differentiation and middle-differentiation, stage Ⅰ and state Ⅱ, Tis, T1 and T2, N0 were significant less than non-differentiation and low-differentiation, stage Ⅲ and state Ⅳ, T3 and T4, N1,N2 and N3, respectively. No negative control samples was found to be positive, and no positive control samples was found to be negative. The sensitivity of our test was 10 cells/mL. Conclusion The cancer cells dissemination during operation was demonstrated indirectly in our study, the time of pulmonary vein ligation (earlier or later may affect the quantity of tumor cells released into circulation; The patients with lower differentiation, advanced TNM stage, larger tumor size and metastasis of lymph node have higher rates of metastasis in peripheral, so the detection of CEA mRNA can guide the therapy of NSCLC to a certain extent.

  10. Small Interference RNA Targeting TLR4 Gene Effectively Attenuates Pulmonary Inflammation in a Rat Model

    Directory of Open Access Journals (Sweden)

    Feixiang Wu

    2012-01-01

    Full Text Available Objective. The present study was to investigate the feasibility of adenovirus-mediated small interference RNA (siRNA targeting Toll-like receptor 4 (TLR4 gene in ameliorating lipopolysaccharide- (LPS- induced acute lung injury (ALI. Methods. In vitro, alveolar macrophages (AMs were treated with Ad-siTLR4 and Ad-EFGP, respectively, for 12 h, 24 h, and 48 h, and then with LPS (100 ng/mL for 2 h, and the function and expression of TLR4 were evaluated. In vivo, rats received intratracheal injection of 300 μL of normal saline (control group, 300 μL of Ad-EGFP (Ad-EGFP group, or 300 μL of Ad-siTLR4 (Ad-siTLR4 group and then were intravenously treated with LPS (50 mg/kg to induce ALI. Results. Ad-siTLR4 treatment significantly reduced TLR4 expression and production of proinflammatory cytokines following LPS treatment both in vitro and in vivo. Significant alleviation of tissue edema, microvascular protein leakage, and neutrophil infiltration was observed in the AdsiTLR4-treated animals. Conclusion. TLR4 plays a critical role in LPS-induced ALI, and transfection of Ad-siTLR4 can effectively downregulate TLR4 expression in vitro and in vivo, accompanied by alleviation of LPS-induced lung injury. These findings suggest that TLR4 may serve as a potential target in the treatment of ALI and RNA interfering targeting TLR4 expression represents a therapeutic strategy.

  11. Small interfering RNA targeted to secretory clusterin blocks tumor growth, motility, and invasion in breast cancer

    Institute of Scientific and Technical Information of China (English)

    Zhaohe Niu; Xinhui Li; Bin Hu; Rong Li; Ligang Wang; Lilin Wu; Xingang Wang

    2012-01-01

    Clusterin/apolipoprotein J (Clu) is a ubiquitously expressed secreted heterodimeric glycoprotein that is implicated in several physiological processes.It has been reported that the elevated level of secreted clusterin (sClu) protein is associated with poor survival in breast cancer patients and can induce metastasis in rodent models.In this study,we investigated the effects of sClu inhibition with small interfering RNAs (siRNAs) on cell motility,invasion,and growth in vitro and in vivo.MDA-MB-231 cells were transfected with pSuper-siRNA/sClu.Cell survival and proliferation were examined by 3-(4,5-dimethyl-thiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and clonogenic survival assay.The results showed that sClu silencing significantly inhibited the proliferation of MDA-MB-231 cells.The invasion and migration ability were also dramatically decreased,which was detected by matrigel assays.TUNEL staining and caspase-3 activity assay demonstrated that sClu silencing also could increase the apoptosis rate of cells,resulting in the inhibition of cell growth.We also determined the effects of sClu silencing on tumor growth and metastatic progression in an orthotopic breast cancer model.The results showed that orthotopic primary tumors derived from MDA-MB-231/pSuper sClu siRNA cells grew significantly slower than tumors derived from parental MDA-MB-231 or MDA-MB-231/pSuper scramble siRNA cells,and metastasize less to the lungs.These data suggest that secretory clusterin plays a significant role in tumor growth and metastatic progression.Knocking-down sClu gene expression may provide a valuable method for breast cancer therapy.

  12. RNA-Seq quantification of the human small airway epithelium transcriptome

    Directory of Open Access Journals (Sweden)

    Hackett Neil R

    2012-02-01

    Full Text Available Abstract Background The small airway epithelium (SAE, the cell population that covers the human airway surface from the 6th generation of airway branching to the alveoli, is the major site of lung disease caused by smoking. The focus of this study is to provide quantitative assessment of the SAE transcriptome in the resting state and in response to chronic cigarette smoking using massive parallel mRNA sequencing (RNA-Seq. Results The data demonstrate that 48% of SAE expressed genes are ubiquitous, shared with many tissues, with 52% enriched in this cell population. The most highly expressed gene, SCGB1A1, is characteristic of Clara cells, the cell type unique to the human SAE. Among other genes expressed by the SAE are those related to Clara cell differentiation, secretory mucosal defense, and mucociliary differentiation. The high sensitivity of RNA-Seq permitted quantification of gene expression related to infrequent cell populations such as neuroendocrine cells and epithelial stem/progenitor cells. Quantification of the absolute smoking-induced changes in SAE gene expression revealed that, compared to ubiquitous genes, more SAE-enriched genes responded to smoking with up-regulation, and those with the highest basal expression levels showed most dramatic changes. Smoking had no effect on SAE gene splicing, but was associated with a shift in molecular pattern from Clara cell-associated towards the mucus-secreting cell differentiation pathway with multiple features of cancer-associated molecular phenotype. Conclusions These observations provide insights into the unique biology of human SAE by providing quantit-ative assessment of the global transcriptome under physiological conditions and in response to the stress of chronic cigarette smoking.

  13. Limitations of mRNA amplification from small-size cell samples

    Directory of Open Access Journals (Sweden)

    Myklebost Ola

    2005-10-01

    Full Text Available Abstract Background Global mRNA amplification has become a widely used approach to obtain gene expression profiles from limited material. An important concern is the reliable reflection of the starting material in the results obtained. This is especially important with extremely low quantities of input RNA where stochastic effects due to template dilution may be present. This aspect remains under-documented in the literature, as quantitative measures of data reliability are most often lacking. To address this issue, we examined the sensitivity levels of each transcript in 3 different cell sample sizes. ANOVA analysis was used to estimate the overall effects of reduced input RNA in our experimental design. In order to estimate the validity of decreasing sample sizes, we examined the sensitivity levels of each transcript by applying a novel model-based method, TransCount. Results From expression data, TransCount provided estimates of absolute transcript concentrations in each examined sample. The results from TransCount were used to calculate the Pearson correlation coefficient between transcript concentrations for different sample sizes. The correlations were clearly transcript copy number dependent. A critical level was observed where stochastic fluctuations became significant. The analysis allowed us to pinpoint the gene specific number of transcript templates that defined the limit of reliability with respect to number of cells from that particular source. In the sample amplifying from 1000 cells, transcripts expressed with at least 121 transcripts/cell were statistically reliable and for 250 cells, the limit was 1806 transcripts/cell. Above these thresholds, correlation between our data sets was at acceptable values for reliable interpretation. Conclusion These results imply that the reliability of any amplification experiment must be validated empirically to justify that any gene exists in sufficient quantity in the input material. This

  14. Small RNA molecules and regulation of spermatogenesis%小RNA分子与精子发生调控

    Institute of Scientific and Technical Information of China (English)

    孟雅楠; 孟丽军; 宋亚娟; 刘美玲; 张秀军

    2011-01-01

    Small RNA molecules (small RNAs) have recently emerged as important regulators of gene expression at the post-transcriptional or translation level.Significant progtess has recently been made in utilizing small RNAs to elucidate the molecular mechanisms regulating spermatogenesis.There are three major small RNAs: small interfering RNAs (siRNAs),microRNAs (miRNAs).and piwi-interacting RNAs (piRNAs).Small-RNAs have diverse biological functions in meiosis and spermatogenesis, In vitro or in vivo, use of siRNA to knockdown genes is a way to study the function of genes of intrest in spermatogenesis.miRNA can be involved in the regulation of mitosis, meiosis, and postmeiosis in spermatogenesis.piRNAs are mainly involved in regulating the process of meiosis and postmeiosis, and repressing retrotransposon transposition in male germline cells.In this paper, we reviewed recent works on the synthesis, mechanism, function, and Outlook of smaIIRNAs.%近来研究发现小RNA(small RNAs)可作为转录后及翻译水平上基因表达调节的重要调节因子,利用小RNA来阐明调节精子发生的分子机制取得了显著进展.这些小RNA主要分为3类,即小干扰RNA(siRNA),微小RNA(miRNA)以及与piwi蛋白相互作用的RNA(piRNA).在减数分裂和精子发生过程中,小RNA具有多种生物学功能,如利用siRNA体外转染或体内注射来敲低特定基因从而研究该基因在精子发生过程中的作用;miRNA可能参与精子发生中有丝、减数及后减数分裂阶段的基因表达调节;piRNA主要参与调节雄性生殖细胞减数及后减数分裂的过程,在精子发生中起抑制反转录转座子(retrotransposons)的作用.文章对小RNAs合成、作用机制、功能及展望等最新进展进行了综述.

  15. Small RNA in situ hybridization in Caenorhabditis elegans, combined with RNA-seq, identifies germline-enriched microRNAs.

    Science.gov (United States)

    McEwen, Tamara J; Yao, Qiuming; Yun, Sijung; Lee, Chin-Yung; Bennett, Karen L

    2016-10-15

    Over four hundred different microRNAs (miRNAs) have been identified in the genome of the model organism the nematode Caenorhabditis elegans. As the germline is dedicated to the preservation of each species, and almost half of all the cells in an adult nematode are germline, it is likely that regulatory miRNAs are important for germline development and maintenance. In C. elegans the miR35 family has strong maternal effects, contributing to normal embryogenesis and to adult fecundity. To determine whether any particular miRNAs are greatly enriched in the C. elegans germline we used RNA-seq to compare the miRNA populations in several germline-defective strains of adult C. elegans worms, including glp-4(germline proliferation-4), glh-1(germline helicase-1) and dcr-1(dicer-1). Statistical analyses of RNA-seq comparisons identified 13 miRNAs that are germline-enriched, including seven members of the well-studied miR35 family that were reduced as much as 1000-fold in TaqMan qRT PCR miRNA assays. Along with the miR35s, six others: miR-56 (a member of the miR51 family),-70, -244, -260 , -788 and -4813, none of which previously considered as such, were also identified by RNA-seq as germline-enriched candidates. We went on to develop a successful miRNA in situ hybridization protocol for C. elegans, revealing miR35s specifically concentrate during oogenesis in the pachytene region of the gonad, and persist throughout early embryogenesis, while in adult animals neither let-7 nor miR-228 has a germline-bias.

  16. Genome-wide search reveals a novel GacA-regulated small RNA in Pseudomonas species

    Directory of Open Access Journals (Sweden)

    Kay Elisabeth

    2008-04-01

    Full Text Available Abstract Background Small RNAs (sRNAs are widespread among bacteria and have diverse regulatory roles. Most of these sRNAs have been discovered by a combination of computational and experimental methods. In Pseudomonas aeruginosa, a ubiquitous Gram-negative bacterium and opportunistic human pathogen, the GacS/GacA two-component system positively controls the transcription of two sRNAs (RsmY, RsmZ, which are crucial for the expression of genes involved in virulence. In the biocontrol bacterium Pseudomonas fluorescens CHA0, three GacA-controlled sRNAs (RsmX, RsmY, RsmZ regulate the response to oxidative stress and the expression of extracellular products including biocontrol factors. RsmX, RsmY and RsmZ contain multiple unpaired GGA motifs and control the expression of target mRNAs at the translational level, by sequestration of translational repressor proteins of the RsmA family. Results A combined computational and experimental approach enabled us to identify 14 intergenic regions encoding sRNAs in P. aeruginosa. Eight of these regions encode newly identified sRNAs. The intergenic region 1698 was found to specify a novel GacA-controlled sRNA termed RgsA. GacA regulation appeared to be indirect. In P. fluorescens CHA0, an RgsA homolog was also expressed under positive GacA control. This 120-nt sRNA contained a single GGA motif and, unlike RsmX, RsmY and RsmZ, was unable to derepress translation of the hcnA gene (involved in the biosynthesis of the biocontrol factor hydrogen cyanide, but contributed to the bacterium's resistance to hydrogen peroxide. In both P. aeruginosa and P. fluorescens the stress sigma factor RpoS was essential for RgsA expression. Conclusion The discovery of an additional sRNA expressed under GacA control in two Pseudomonas species highlights the complexity of this global regulatory system and suggests that the mode of action of GacA control may be more elaborate than previously suspected. Our results also confirm that

  17. RelA protein stimulates the activity of RyhB small RNA by acting on RNA-binding protein Hfq

    OpenAIRE

    Argaman, Liron; Elgrably-Weiss, Maya; Hershko, Tal; Vogel, Jörg; Altuvia, Shoshy

    2012-01-01

    The conserved RNA-binding protein Hfq and its associated small regulatory RNAs (sRNAs) are increasingly recognized as the players of a large network of posttranscriptional control of gene expression in Gram-negative bacteria. The role of Hfq in this network is to facilitate base pairing between sRNAs and their trans-encoded target mRNAs. Although the number of known sRNA–mRNA interactions has grown steadily, cellular factors that influence Hfq, the mediator of these interactions, have remaine...

  18. INFLUENCE OF p53 SMALL DOUBLE STRANDED RNA INTERFERENCE ON HEPATOMA CELL LINE SK-HEP-1

    Institute of Scientific and Technical Information of China (English)

    CAO Xiao-zhe; ZHU Ming-hua; ZHU Zhi; FENG Fei; ZHAO Mei-lan; CHEN Ying; LIU Xiao-hong

    2005-01-01

    Objective: The effects on cell-cycle and p53 expression in hepatoma cell line SK-Hep-1 were explored by transfecting exogenous p53 small double stranded RNA (dsRNA) into the SK-Hep-1 cells. Methods: p53 dsRNA and EGFP dsRNA were synthesized. SK-Hep-1 (wtp53) cell line was transfected with 200 ng and 400 ng p53 dsRNA or EGFP and EGFP+EGFP dsRNA (as positive control) or 9% NaCl (as blank control) by liposome transfection technique. Flow cytometry was adopted to measure the effects of p53 dsRNA on cell cycle. Expression of p53 protein was detected by Western-Blotting at 48 h after transfecting p53 dsRNA. Results: The number of G0-G1 phase SK-Hep-1 cells, which were transfected with 200 ng p53 dsRNA, was decreased by 52.53% comparing with the control, and decreased by 50.29% (P0.05) comparing with the positive control cells transfected with same dosage of EGFP+EGFP dsRNA. After 48 h, p53 protein expression was not detected in the SK-Hep-1 cells transfected with p53 dsRNA. Conclusion: p53 dsRNA can obviously improve the proliferation of SK-Hep-1 cells, and suppress p53 protein expression of SK-Hep-1 cells, the former may be related to of the latter.

  19. Reducing acetate excretion from E. coli K-12 by over-expressing the small RNA sgrS

    OpenAIRE

    Negrete, Alejandro; Majdalani, Nadim; Phue, Je Nie; Shiloach, Joseph

    2011-01-01

    When exposed to the non-metabolized glucose derivative alpha methyl glucoside, both E. coli K-12 (JM109 and MG1655) and E. coli B (BL21) respond by reducing the concentration of the mRNA of the ptsG gene which is responsible for the biosynthesis of the glucose transporter EIICBglu. This occurs through the over-expression of the non-coding small RNA SgrS, which interacts specifically with the mRNA of the ptsG gene and prevents its translation. However, when these bacteria are exposed to a gluc...

  20. Small interfering RNA targeting of S phase kinase-interacting protein 2 inhibits cell proliferation of pterygium fibroblasts

    OpenAIRE

    Su, Ying; Wang, Feng; Qi, Hu; Zhao, Shi Guang; Li, Xue; Cui, Hao

    2011-01-01

    Purpose Fibroblast cell proliferation is major reason for recurrence of pterygia. In the present study, we investigated if small interfering RNA (siRNA)-mediated gene silencing of S phase-kinase-interacting protein 2 (Skp2) can be employed to inhibit protein 27 kinase inhibition protein 1 (p27kip1 ) down-regulation in pterygium fibroblast cells (PFC) in vitro and in vivo. Methods A plasmid containing transgenes encoding Skp2 siRNA was used to decreasing the high constitutive levels of Skp2 pr...

  1. Identification and expression profiling of Vigna mungo microRNAs from leaf small RNA transcriptome by deep sequencing

    Institute of Scientific and Technical Information of China (English)

    Sujay Paul; Anirban Kundu; Amita Pal

    2014-01-01

    MicroRNAs (miRNAs) represent a class of small non-coding RNA molecules that play a crucial role in post-transcriptional gene regulation. Several conserved and species-specific miRNAs have been characterized to date, predominantly from the plant species whose genome is well characterized. However, information on the variability of these regulatory RNAs in economically important but genetically less characterized crop species are limited. Vigna mungo is an important grain legume, which is grown primarily for its protein-rich edible seeds. miRNAs from this species have not been identified to date due to lack of genome sequence information. To identify miRNAs from V. mungo, a small RNA library was constructed from young leaves. High-throughput Illumina sequencing technology and bioinformat-ic analysis of the small RNA reads led to the identification of 66 miRNA loci represented by 45 conserved miRNAs belonging to 19 families and eight non-conserved miRNAs belonging to seven families. Besides, 13 novel miRNA candidates in V. mungo were also identified. Expression patterns of selected conserved, non-conserved, and novel miRNA candidates have been demonstrated in leaf, stem, and root tissues by quantitative polymerase chain reaction, and potential target genes were predicted for most of the conserved miRNAs. This information offers genomic resour-ces for better understanding of miRNA mediated post-transcriptional gene regulation.

  2. Differential RNA-seq of Vibrio cholerae identifies the VqmR small RNA as a regulator of biofilm formation.

    Science.gov (United States)

    Papenfort, Kai; Förstner, Konrad U; Cong, Jian-Ping; Sharma, Cynthia M; Bassler, Bonnie L

    2015-02-17

    Quorum sensing (QS) is a process of cell-to-cell communication that enables bacteria to transition between individual and collective lifestyles. QS controls virulence and biofilm formation in Vibrio cholerae, the causative agent of cholera disease. Differential RNA sequencing (RNA-seq) of wild-type V. cholerae and a locked low-cell-density QS-mutant strain identified 7,240 transcriptional start sites with ∼ 47% initiated in the antisense direction. A total of 107 of the transcripts do not appear to encode proteins, suggesting they specify regulatory RNAs. We focused on one such transcript that we name VqmR. vqmR is located upstream of the vqmA gene encoding a DNA-binding transcription factor. Mutagenesis and microarray analyses demonstrate that VqmA activates vqmR transcription, that vqmR encodes a regulatory RNA, and VqmR directly controls at least eight mRNA targets including the rtx (repeats in toxin) toxin genes and the vpsT transcriptional regulator of biofilm production. We show that VqmR inhibits biofilm formation through repression of vpsT. Together, these data provide to our knowledege the first global annotation of the transcriptional start sites in V. cholerae and highlight the importance of posttranscriptional regulation for collective behaviors in this human pathogen.

  3. The small RNA content of human sperm reveals pseudogene-derived piRNAs complementary to protein-coding genes

    Science.gov (United States)

    Pantano, Lorena; Jodar, Meritxell; Bak, Mads; Ballescà, Josep Lluís; Tommerup, Niels; Oliva, Rafael; Vavouri, Tanya

    2015-01-01

    At the end of mammalian sperm development, sperm cells expel most of their cytoplasm and dispose of the majority of their RNA. Yet, hundreds of RNA molecules remain in mature sperm. The biological significance of the vast majority of these molecules is unclear. To better understand the processes that generate sperm small RNAs and what roles they may have, we sequenced and characterized the small RNA content of sperm samples from two human fertile individuals. We detected 182 microRNAs, some of which are highly abundant. The most abundant microRNA in sperm is miR-1246 with predicted targets among sperm-specific genes. The most abundant class of small noncoding RNAs in sperm are PIWI-interacting RNAs (piRNAs). Surprisingly, we found that human sperm cells contain piRNAs processed from pseudogenes. Clusters of piRNAs from human testes contain pseudogenes transcribed in the antisense strand and processed into small RNAs. Several human protein-coding genes contain antisense predicted targets of pseudogene-derived piRNAs in the male germline and these piRNAs are still found in mature sperm. Our study provides the most extensive data set and annotation of human sperm small RNAs to date and is a resource for further functional studies on the roles of sperm small RNAs. In addition, we propose that some of the pseudogene-derived human piRNAs may regulate expression of their parent gene in the male germline. PMID:25904136

  4. Small RNA and A-to-I Editing in Autism Spectrum Disorders

    Science.gov (United States)

    Eran, Alal

    One in every 88 children is diagnosed with Autism Spectrum Disorders (ASDs), a set of neurodevelopmental conditions characterized by social impairments, communication deficits, and repetitive behavior. ASDs have a substantial genetic component, but the specific cause of most cases remains unknown. Understanding gene-environment interactions underlying ASD is essential for improving early diagnosis and identifying critical targets for intervention and prevention. Towards this goal, we surveyed adenosine-to-inosine (A-to-I) RNA editing in autistic brains. A-to-I editing is an epigenetic mechanism that fine-tunes synaptic function in response to environmental stimuli, shown to modulate complex behavior in animals. We used ultradeep sequencing to quantify A-to-I receding of candidate synaptic genes in postmortem cerebella from individuals with ASD and neurotypical controls. We found unexpectedly wide distributions of human A-to-I editing levels, whose extremes were consistently populated by individuals with ASD. We correlated A-to-I editing with isoform usage, identified clusters of correlated sites, and examined differential editing patterns. Importantly, we found that individuals with ASD commonly use a dysfunctional form of the editing enzyme ADARB1. We next profiled small RNAs thought to regulate A-to-I editing, which originate from one of the most commonly altered loci in ASD, 15q11. Deep targeted sequencing of SNORD115 and SNORD116 transcripts enabled their high-resolution detection in human brains, and revealed a strong gender bias underlying their expression. The consistent 2-fold upregulation of 15q11 small RNAs in male vs. female cerebella could be important in delineating the role of this locus in ASD, a male dominant disorder. Overall, these studies provide an accurate population-level view of small RNA and A-to-I editing in human cerebella, and suggest that A-to-I editing of synaptic genes may be informative for assessing the epigenetic risk for autism

  5. plantDARIO: web based quantitative and qualitative analysis of small RNA-seq data in plants.

    Science.gov (United States)

    Patra, Deblina; Fasold, Mario; Langenberger, David; Steger, Gerhard; Grosse, Ivo; Stadler, Peter F

    2014-01-01

    High-throughput sequencing techniques have made it possible to assay an organism's entire repertoire of small non-coding RNAs (ncRNAs) in an efficient and cost-effective manner. The moderate size of small RNA-seq datasets makes it feasible to provide free web services to the research community that provide many basic features of a small RNA-seq analysis, including quality control, read normalization, ncRNA quantification, and the prediction of putative novel ncRNAs. DARIO is one such system that so far has been focussed on animals. Here we introduce an extension of this system to plant short non-coding RNAs (sncRNAs). It includes major modifications to cope with plant-specific sncRNA processing. The current version of plantDARIO covers analyses of mapping files, small RNA-seq quality control, expression analyses of annotated sncRNAs, including the prediction of novel miRNAs and snoRNAs from unknown expressed loci and expression analyses of user-defined loci. At present Arabidopsis thaliana, Beta vulgaris, and Solanum lycopersicum are covered. The web tool links to a plant specific visualization browser to display the read distribution of the analyzed sample. The easy-to-use platform of plantDARIO quantifies RNA expression of annotated sncRNAs from different sncRNA databases together with new sncRNAs, annotated by our group. The plantDARIO website can be accessed at http://plantdario.bioinf.uni-leipzig.de/. PMID:25566282

  6. Quantification of Small Non-Coding RNAs Allows an Accurate Comparison of miRNA Expression Profiles

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    Andrea Masotti

    2009-01-01

    Full Text Available MicroRNAs (miRNAs are highly conserved ∼22-mer RNA molecules, encoded by plants and animals that regulate the expression of genes binding to the 3′-UTR of specific target mRNAs. The amount of miRNAs in a total RNA sample depends on the recovery efficiency that may be significantly affected by the different purification methods employed. Traditional approaches may be inefficient at recovering small RNAs, and common spectrophotometric determination is not adequate to quantify selectively these low molecular weight (LMW species from total RNA samples. Here, we describe the use of qualitative and quantitative lab-on-a-chip tools for the analysis of these LMW RNA species. Our data emphasize the close correlation of LMW RNAs with the expression levels of some miRNAs. We therefore applied our result to the comparison of some miRNA expression profiles in different tissues. Finally, the methods we used in this paper allowed us to analyze the efficiency of extraction protocols, to study the small (but significant differences among various preparations and to allow a proper comparison of some miRNA expression profiles in various tissues.

  7. MicroRNA-944 Affects Cell Growth by Targeting EPHA7 in Non-Small Cell Lung Cancer

    Science.gov (United States)

    Liu, Minxia; Zhou, Kecheng; Cao, Yi

    2016-01-01

    MicroRNAs (miRNAs) have critical roles in lung tumorigenesis and development. To determine aberrantly expressed miRNAs involved in non-small cell lung cancer (NSCLC) and investigate pathophysiological functions and mechanisms, we firstly carried out small RNA deep sequencing in NSCLC cell lines (EPLC-32M1, A549 and 801D) and a human immortalized cell line 16HBE, we then studied miRNA function by cell proliferation and apoptosis. cDNA microarray, luciferase reporter assay and miRNA transfection were used to investigate interaction between the miRNA and target gene. miR-944 was significantly down-regulated in NSCLC and had many putative targets. Moreover, the forced expression of miR-944 significantly inhibited the proliferation of NSCLC cells in vitro. By integrating mRNA expression data and miR-944-target prediction, we disclosed that EPHA7 was a potential target of miR-944, which was further verified by luciferase reporter assay and microRNA transfection. Our data indicated that miR-944 targets EPHA7 in NSCLC and regulates NSCLC cell proliferation, which may offer a new mechanism underlying the development and progression of NSCLC. PMID:27681722

  8. Production of virus-derived ping-pong-dependent piRNA-like small RNAs in the mosquito soma.

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    Elaine M Morazzani

    2012-01-01

    Full Text Available The natural maintenance cycles of many mosquito-borne pathogens require establishment of persistent non-lethal infections in the invertebrate host. The mechanism by which this occurs is not well understood, but we have previously shown that an antiviral response directed by small interfering RNAs (siRNAs is important in modulating the pathogenesis of alphavirus infections in the mosquito. However, we report here that infection of mosquitoes with an alphavirus also triggers the production of another class of virus-derived small RNAs that exhibit many similarities to ping-pong-dependent piwi-interacting RNAs (piRNAs. However, unlike ping-pong-dependent piRNAs that have been described previously from repetitive elements or piRNA clusters, our work suggests production in the soma. We also present evidence that suggests virus-derived piRNA-like small RNAs are capable of modulating the pathogenesis of alphavirus infections in dicer-2 null mutant mosquito cell lines defective in viral siRNA production. Overall, our results suggest that a non-canonical piRNA pathway is present in the soma of vector mosquitoes and may be acting redundantly to the siRNA pathway to target alphavirus replication.

  9. Comparison of dengue virus type 2-specific small RNAs from RNA interference-competent and -incompetent mosquito cells.

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    Jaclyn C Scott

    Full Text Available The exogenous RNA interference (RNAi pathway is an important antiviral defense against arboviruses in mosquitoes, and virus-specific small interfering (siRNAs are key components of this pathway. Understanding the biogenesis of siRNAs in mosquitoes could have important ramifications in using RNAi to control arbovirus transmission. Using deep sequencing technology, we characterized dengue virus type 2 (DENV2-specific small RNAs produced during infection of Aedes aegypti mosquitoes and A. aegypti Aag2 cell cultures and compared them to those produced in the C6/36 Aedes albopictus cell line. We show that the size and mixed polarity of virus-specific small RNAs from DENV-infected A. aegypti cells indicate that they are products of Dicer-2 (Dcr2 cleavage of long dsRNA, whereas C6/36 cells generate DENV2-specific small RNAs that are longer and predominantly positive polarity, suggesting that they originate from a different small RNA pathway. Examination of virus-specific small RNAs after infection of the two mosquito cell lines with the insect-only flavivirus cell fusing agent virus (CFAV corroborated these findings. An in vitro assay also showed that Aag2 A. aegypti cells are capable of siRNA production, while C6/36 A. albopictus cells exhibit inefficient Dcr2 cleavage of long dsRNA. Defective expression or function of Dcr2, the key initiator of the RNAi pathway, might explain the comparatively robust growth of arthropod-borne viruses in the C6/36 cell line, which has been used frequently as a surrogate for studying molecular interactions between arboviruses and cells of their mosquito hosts.

  10. Inducing effect of PGRs on small regulatory si/miRNA in resistance to sugar beet cyst nematode.

    Science.gov (United States)

    Tsygankova, V A; Stefanovska, T R; Galkin, A P; Ponomarenko, S P; Blume, Ya B

    2012-01-01

    Sugar beet cyst nematode Heterodera schachtii Schmidt is an economically important plant parasite of sugar beet in Ukraine. The pest control options are limited. Sugar beet cyst nematode resistant varieties are not available on the market. Carbamate and organophosphate pesticides have been banned due to the high toxicity. The problem is aggravated by continuously increasing of oilseed rape (which is suitable host for H. schachtii) growing area due to biofuel demands. Several studies' results indicate that PGRs have role in management of plant parasitic nematodes but for sugar beet it is not studied well. We had an objective- studying of the role of four compositional PGRs created based of avermectin in suppression of sugar beet cyst nematode population on sugar beet and oilseed rape caused by enhancing of endogenous si/miRNA complementary to H. schachtii mRNA. Laboratory study was conducted in 2011 with using method DOT-blot hybridization si/miRNA with mRNA and by testing inhibitory activity in cell free system protein biosynthesis. That was shown that application of the PGRs enhances sugar beet and oilseeds rape plant immune-protective properties and resistance against plant-parasitic nematode Heterodera schochtii through enhancement of synthesis of small regulatory si/miRNA related (complementary) to an mRNA structure of the parasitic organisms. As a result, translation of mRNA of the nematode is blocked and causes the mortality of plant parasite juveniles.

  11. Inhibition of hepatocelluar carcinoma MAT2A and MAT2beta gene expressions by single and dual small interfering RNA

    Directory of Open Access Journals (Sweden)

    Sun Quan

    2008-11-01

    Full Text Available Abstract RNA interference (RNAi has been successfully applied in suppression of hepatic cancer genes. In hepatocelluar carcinoma cell, one methionine adenosyltransferase (MAT isozyme, MATII was found to have two catalytic subunits which were encoded by MAT2A and MAT2β respectively. During tumorigeness of hepatocelluar carcinoma, expressions of the two genes were discovered to be increased combining with a switch of MAT (form MATI to MATII, To figure out the role played by MATII in hepatic cancer, In this study, for the first time we established a dual small interfering RNA (siRNA expression system, which could simultaneously express two different siRNA molecules specifically targeting two genes. To test the effectiveness of this system, we applied this approach to express simultaneously two different siRNA duplexes that specifically target MAT2A and MAT2β genes of hepatocelluar carcinoma respectively in HepG2 cell. Results indicated that dual siRNA could simultaneously inhibit the expression of MAT2A and MAT2β gene by 89.5% and 97.8% respectively, In addition, dual siRNA molecules were able to significantly suppress growth of hepatocelluar carcinoma cell in vitro as well as induce apoptosis which was involved in arrest cell cycle at the G1/S checkpoint and the expressions of p21, p27 and Bax.

  12. Circulating U2 small nuclear RNA fragments as a novel diagnostic biomarker for pancreatic and colorectal adenocarcinoma.

    Science.gov (United States)

    Baraniskin, Alexander; Nöpel-Dünnebacke, Stefanie; Ahrens, Maike; Jensen, Steffen Grann; Zöllner, Hannah; Maghnouj, Abdelouahid; Wos, Alexandra; Mayerle, Julia; Munding, Johanna; Kost, Dennis; Reinacher-Schick, Anke; Liffers, Sven; Schroers, Roland; Chromik, Ansgar M; Meyer, Helmut E; Uhl, Waldemar; Klein-Scory, Susanne; Weiss, Frank U; Stephan, Christian; Schwarte-Waldhoff, Irmgard; Lerch, Markus M; Tannapfel, Andrea; Schmiegel, Wolff; Andersen, Claus Lindbjerg; Hahn, Stephan A

    2013-01-15

    Improved non-invasive strategies for early cancer detection are urgently needed to reduce morbidity and mortality. Non-coding RNAs, such as microRNAs and small nucleolar RNAs, have been proposed as biomarkers for non-invasive cancer diagnosis. Analyzing serum derived from nude mice implanted with primary human pancreatic ductal adenocarcinoma (PDAC), we identified 15 diagnostic microRNA candidates. Of those miR-1246 was selected based on its high abundance in serum of tumor carrying mice. Subsequently, we noted a cross reactivity of the established miR-1246 assays with RNA fragments derived from U2 small nuclear RNA (RNU2-1). Importantly, we found that the assay signal discriminating tumor from controls was derived from U2 small nuclear RNA (snRNA) fragments (RNU2-1f) and not from miR-1246. In addition, we observed a remarkable stability of RNU2-1f in serum and provide experimental evidence that hsa-miR-1246 is likely a pseudo microRNA. In a next step, RNU2-1f was measured by qRT-PCR and normalized to cel-54 in 191 serum/plasma samples from PDAC and colorectal carcinoma (CRC) patients. In comparison to 129 controls, we were able to classify samples as cancerous with a sensitivity and specificity of 97.7% [95% CI = (87.7, 99.9)] and 90.6% [95% CI = (80.7, 96.5)], respectively [area under the ROC curve 0.972]. Of note, patients with CRC were detected with our assay as early as UICC Stage II with a sensitivity of 81%. In conclusion, this is the first report showing that fragments of U2 snRNA are highly stable in serum and plasma and may serve as novel diagnostic biomarker for PDAC and CRC for future prospective screening studies. PMID:22907602

  13. Identification of Novel MicroRNAs in Primates by Using the Synteny Information and Small RNA Deep Sequencing Data

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    Xiao Sun

    2013-10-01

    Full Text Available Current technologies that are used for genome-wide microRNA (miRNA prediction are mainly based on BLAST tool. They often produce a large number of false positives. Here, we describe an effective approach for identifying orthologous pre-miRNAs in several primates based on syntenic information. Some of them have been validated by small RNA high throughput sequencing data. This approach uses the synteny information and experimentally validated miRNAs of human, and incorporates currently available algorithms and tools to identify the pre-miRNAs in five other primates. First, we identified 929 potential pre-miRNAs in the marmoset in which miRNAs have not yet been reported. Then, we predicted the miRNAs in other primates, and we successfully re-identified most of the published miRNAs and found 721, 979, 650 and 639 new potential pre-miRNAs in chimpanzee, gorilla, orangutan and rhesus macaque, respectively. Furthermore, the miRNA transcriptome in the four primates have been re-analyzed and some novel predicted miRNAs have been supported by the small RNA sequencing data. Finally, we analyzed the potential functions of those validated miRNAs and explored the regulatory elements and transcription factors of some validated miRNA genes of interest. The results show that our approach can effectively identify novel miRNAs and some miRNAs that supported by small RNA sequencing data maybe play roles in the nervous system.

  14. Comparative Small RNA Analysis of Pollen Development in Autotetraploid and Diploid Rice.

    Science.gov (United States)

    Li, Xiang; Shahid, Muhammad Qasim; Wu, Jinwen; Wang, Lan; Liu, Xiangdong; Lu, Yonggen

    2016-01-01

    MicroRNAs (miRNAs) play key roles in plant reproduction. However, knowledge on microRNAome analysis in autotetraploid rice is rather limited. Here, high-throughput sequencing technology was employed to analyze miRNAomes during pollen development in diploid and polyploid rice. A total of 172 differentially expressed miRNAs (DEM) were detected in autotetraploid rice compared to its diploid counterpart, and 57 miRNAs were specifically expressed in autotetraploid rice. Of the 172 DEM, 115 and 61 miRNAs exhibited up- and down-regulation, respectively. Gene Ontology analysis on the targets of up-regulated DEM showed that they were enriched in transport and membrane in pre-meiotic interphase, reproduction in meiosis, and nucleotide binding in single microspore stage. osa-miR5788 and osa-miR1432-5p_R+1 were up-regulated in meiosis and their targets revealed interaction with the meiosis-related genes, suggesting that they may involve in the genes regulation associated with the chromosome behavior. Abundant 24 nt siRNAs associated with transposable elements were found in autotetraploid rice during pollen development; however, they significantly declined in diploid rice, suggesting that 24 nt siRNAs may play a role in pollen development. These findings provide a foundation for understanding the effect of polyploidy on small RNA expression patterns during pollen development that cause pollen sterility in autotetraploid rice. PMID:27077850

  15. Comparative Small RNA Analysis of Pollen Development in Autotetraploid and Diploid Rice

    Directory of Open Access Journals (Sweden)

    Xiang Li

    2016-04-01

    Full Text Available MicroRNAs (miRNAs play key roles in plant reproduction. However, knowledge on microRNAome analysis in autotetraploid rice is rather limited. Here, high-throughput sequencing technology was employed to analyze miRNAomes during pollen development in diploid and polyploid rice. A total of 172 differentially expressed miRNAs (DEM were detected in autotetraploid rice compared to its diploid counterpart, and 57 miRNAs were specifically expressed in autotetraploid rice. Of the 172 DEM, 115 and 61 miRNAs exhibited up- and down-regulation, respectively. Gene Ontology analysis on the targets of up-regulated DEM showed that they were enriched in transport and membrane in pre-meiotic interphase, reproduction in meiosis, and nucleotide binding in single microspore stage. osa-miR5788 and osa-miR1432-5p_R+1 were up-regulated in meiosis and their targets revealed interaction with the meiosis-related genes, suggesting that they may involve in the genes regulation associated with the chromosome behavior. Abundant 24 nt siRNAs associated with transposable elements were found in autotetraploid rice during pollen development; however, they significantly declined in diploid rice, suggesting that 24 nt siRNAs may play a role in pollen development. These findings provide a foundation for understanding the effect of polyploidy on small RNA expression patterns during pollen development that cause pollen sterility in autotetraploid rice.

  16. Distribution and mRNA Expression of BAMBI in Non-small-cell Lung Cancer

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    Shen MIAO

    2009-03-01

    Full Text Available Background and objective BAMBI structure is similar with that of the receptor Ⅰof TGF-β, it broadly participates in the control of TGF-β signaling. The aim of this study is to investigate the expression and its significance of BAMBI in non-small cell lung cancer (NSCLC and explore the relation between BAMBI and clinical and pathological factors of NSCLC. Methods Sixty-three cases with NSCLC and adjacent normal tissue specimens were used for immunohistochemical assay. Thirty-one fresh lung cancer tissue specimens and surrounding normal lung tissue specimens was preserved for RT-PCR in -70 ℃ after quick-frozen in liquid nitrogen immediately. Results The level of BAMBI mRNA in cancer tissues was higher than that in the corresponding adjacent tissues (0.358±0.135 vs 0.249±0.129, with the difference being statistically significant (P =0.003. BAMBI protein expressed mainly in the membrane and the cytoplasm close to the membrane, its expression in the cancer tissue was higher than that in the adjacent tissues, the difference was significant (P <0.01. Expression of BAMBI in the cancer tissue was higher than that in the adjacent tissues, and the expression of BAMBI in adenocarcinoma of lung is higher than that in squamous carcinoma. Conclusion The expressions of BAMBI significantly increase in NSCLC. It might be a common affair in carcinogenesis of NSCLC.

  17. Efficient protection and transfection of small interfering RNA by cationic shell-crosslinked knedel-like nanoparticles.

    Science.gov (United States)

    Shen, Yuefei; Fang, Huafeng; Zhang, Ke; Shrestha, Ritu; Wooley, Karen L; Taylor, John-Stephen A

    2013-04-01

    Despite the great potential of small interfering RNA (siRNA) as a therapeutic agent, progress in this area has been hampered by a lack of efficient biocompatible transfection agents. Recently, cationic shell-crosslinked knedel-like nanoparticles (cSCKs) were found to possess lower cytotoxicity and better transfection ability for phosphorothioate ODNs and plasmid DNA than the commonly used cationic lipid-based agent Lipofectamine. To determine the usefulness of cSCKs for siRNA transfection, a small library of cSCKs with varying percentage of primary and tertiary amines was assessed for its ability to bind to siRNA, inhibit siRNA degradation in human serum, and to transfect HeLa and mouse macrophage cell lines. The silencing efficiency in HeLa cells was greatest with the cSCK with 100% primary amines (pa100) as determined by their viability following transfection with cytotoxic and non-cytotoxic siRNAs. cSCK-pa100 showed greater silencing efficiency than Lipofectamine 2000 in the HeLa cells, as well in 293T and human bronchial epithelial (HEK) cells, but was comparable in human bronchial epithelial (BEAS-2B) cells and human mammary epithelial (MCF10a) cells. cSCK-pa100 also showed greater silencing of iNOS expression than Lipofectamine 2000 in a mouse macrophage cell line, and provided greater protection from serum degradation, demonstrating its potential usefulness as an siRNA transfection agent. The siRNA silencing of iNOS at lower concentrations of siRNA could be enhanced by complexation with the fusogenic GALA peptide, which was shown to enhance endosomal escape following uptake.

  18. Downregulation of matrix metalloproteinase-2 (MMP-2) utilizing adenovirus-mediated transfer of small interfering RNA (siRNA) in a novel spinal metastatic melanoma model.

    Science.gov (United States)

    Tsung, Andrew J; Kargiotis, Odysseas; Chetty, Chandramu; Lakka, Sajani S; Gujrati, Meena; Spomar, Daniel G; Dinh, Dzung H; Rao, Jasti S

    2008-03-01

    Matrix metalloproteinases (MMPs) comprise a class of secreted zinc-dependent endopeptidases implicated in the metastatic potential of tumor cells due to their ability to degrade the extracellular matrix (ECM) and basement membrane. Matrix metalloproteinase-2 (MMP-2) has been detected in high levels and correlates with invasiveness in human melanoma. We have studied the effect of adenovirus-mediated transfer of small interfering RNA (siRNA) against MMP-2 in the human melanoma cell line A2058. The delivery of these double-stranded RNA molecules represents an efficient technology in silencing disease-causing genes with known sequences at the post-transcriptional level. siRNA against MMP-2 mRNA (Ad-MMP-2) was found to decrease MMP-2 protein expression and activity in melanoma cells as demonstrated by western blotting and gelatin zymography. Furthermore, infection of cells with Ad-MMP-2 inhibited cellular migration and invasion as indicated by spheroid and matrigel assays. We also observed dose-dependent suppression of vascular network formation in an angiogenesis assay. Finally, we developed a nude mouse spinal metastatic model to investigate the local effects of tumor metastasis. Intravenous tail vein injection with Ad-MMP-2 on days 5, 9 and 11 after tumor implantation resulted in complete retention of neurological function as compared to control and scrambled vector (Ad-SV)-treated groups that showed complete paraplegia by day 14+/-2 days. Hematoxylin and eosin staining revealed decreased tumor size in the Ad-MMP-2-treated animals. This novel experimental model revealed that adenoviral-mediated transfer of RNA interference against MMP-2 results in the retention of neurological function and significantly inhibited tumor growth.

  19. Identification and profiling of novel microRNAs in the Brassica rapa genome based on small RNA deep sequencing

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    Kim Bumjin

    2012-11-01

    Full Text Available Abstract Background MicroRNAs (miRNAs are one of the functional non-coding small RNAs involved in the epigenetic control of the plant genome. Although plants contain both evolutionary conserved miRNAs and species-specific miRNAs within their genomes, computational methods often only identify evolutionary conserved miRNAs. The recent sequencing of the Brassica rapa genome enables us to identify miRNAs and their putative target genes. In this study, we sought to provide a more comprehensive prediction of B. rapa miRNAs based on high throughput small RNA deep sequencing. Results We sequenced small RNAs from five types of tissue: seedlings, roots, petioles, leaves, and flowers. By analyzing 2.75 million unique reads that mapped to the B. rapa genome, we identified 216 novel and 196 conserved miRNAs that were predicted to target approximately 20% of the genome’s protein coding genes. Quantitative analysis of miRNAs from the five types of tissue revealed that novel miRNAs were expressed in diverse tissues but their expression levels were lower than those of the conserved miRNAs. Comparative analysis of the miRNAs between the B. rapa and Arabidopsis thaliana genomes demonstrated that redundant copies of conserved miRNAs in the B. rapa genome may have been deleted after whole genome triplication. Novel miRNA members seemed to have spontaneously arisen from the B. rapa and A. thaliana genomes, suggesting the species-specific expansion of miRNAs. We have made this data publicly available in a miRNA database of B. rapa called BraMRs. The database allows the user to retrieve miRNA sequences, their expression profiles, and a description of their target genes from the five tissue types investigated here. Conclusions This is the first report to identify novel miRNAs from Brassica crops using genome-wide high throughput techniques. The combination of computational methods and small RNA deep sequencing provides robust predictions of miRNAs in the genome. The

  20. A negative regulation loop of long noncoding RNA HOTAIR and p53 in non-small-cell lung cancer

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    Zhai N

    2016-09-01

    Full Text Available Nailiang Zhai,1 Yongfu Xia,1 Rui Yin,2 Jinping Liu,3 Fuquan Gao1 1Department of Respiratory Medicine, Affiliated Hospital of Binzhou Medical University, 2Department of Respiratory Medicine, People’s Hospital of Binzhou City, 3Department of Pharmacology, Binzhou Medical University, Binzhou, Shandong, People’s Republic of China Abstract: Non-small-cell lung cancer (NSCLC is one of the leading causes of cancer-related death worldwide, and the 5-year survival rate is still low despite advances in diagnosis and therapeutics. A long noncoding RNA (lncRNA HOX antisense intergenic RNA (HOTAIR has been revealed to play important roles in NSCLC carcinogenesis but the detailed mechanisms are still unclear. In the current study, we aimed to investigate the regulation between the lncRNA HOTAIR and p53 in the NSCLC patient samples and cell lines. Our results showed that HOTAIR expression was significantly higher in the cancer tissues than that in the adjacent normal tissue, and was negatively correlated with p53 functionality rather than expression. When p53 was overexpressed in A549 cells, the lncRNA HOTAIR expression was downregulated, and the cell proliferation rate and cell invasion capacity decreased as a consequence. We identified two binding sites of p53 on the promoter region of HOTAIR, where the p53 protein would bind to and suppress the HOTAIR mRNA transcription. Inversely, overexpression of lncRNA HOTAIR inhibited the expression of p53 in A549 cells. Mechanistic studies revealed that HOTAIR modified the promoter of p53 and enhanced histone H3 lysine 27 trimethylation (H3K27me3. These studies identified a specific negative regulation loop of lncRNA HOTAIR and p53 in NSCLC cells, which revealed a new understanding of tumorigenesis in p53 dysfunction NSCLC cells. Keywords: NSCLC, LncRNA HOTAIR, p53, negative loop

  1. Effect of silencing Bcl-2 expression by small interfering RNA on radiosensitivity of gastric cancer BGC823 cells

    Institute of Scientific and Technical Information of China (English)

    Hong-Tao Liu; Chun-Lei Lu

    2013-01-01

    Objective: To explore the influence of silencing Bcl-2 expression by small interfering RNA (siRNA) on Bcl-2 protein expression, cell apoptosis rate and radiosensitivity of gastric cancer BGC823 cells. Methods: siRNA segment for Bcl-2 gene was designed and synthesized, then was induced into gastric cancer BGC 823 cells by liposome transfection. Bcl-2 protein expression was detected by Western Blotting. After X radiation, flow cytometry and clone forming assay were used to determine the effects of RNA interference on BGC823 cell apoptosis rate and radiosensitivity.Result:After the transfection of Bcl-2 siRNA, the positive expression rate of Bcl-2 protein in BGC823 cells was (35.45±2.35)%. Compared with the control group and negative siRNA transfection group, the rate was significantly decreased (P<0.01). The apoptosis rate of BGC823-RNAi cell was (10.81±0.91)%, which was significantly higher than the control group and negative siRNA transfection group (P<0.01). After 48h X radiation, the apoptosis rate of BGC823-RNAi was (28.91 ±1.40)%, which was significantly higher than the control group and the group without radiation (P<0.01). During clone forming assay D0, Dq and SF2 values in Bcl-2 siRNA1 transfection group were all lower than those in the control group. The radiosensitivity ratio was 1.28 (the ratio of D0) and 1.60 (the ratio of Dq). Conclusions: Specific siRNA of Bcl-2 gene can effectively inhibit the expression of Bcl-2 gene, enhance the radiosensitivity and apoptosis of gastric cancer BGC823 cells, having good clinical application perspective.

  2. Translational regulation of gene expression by an anaerobically induced small non-coding RNA in Escherichia coli

    DEFF Research Database (Denmark)

    Boysen, Anders; Møller-Jensen, Jakob; Kallipolitis, Birgitte H.;

    2010-01-01

    Small non-coding RNAs (sRNA) have emerged as important elements of gene regulatory circuits. In enterobacteria such as Escherichia coli and Salmonella many of these sRNAs interact with the Hfq protein, an RNA chaperone similar to mammalian Sm-like proteins and act in the post-transcriptional regu......Small non-coding RNAs (sRNA) have emerged as important elements of gene regulatory circuits. In enterobacteria such as Escherichia coli and Salmonella many of these sRNAs interact with the Hfq protein, an RNA chaperone similar to mammalian Sm-like proteins and act in the post......S, possesses signatures of base-pairing RNAs, and we show by employing global proteomic and transcriptomic profiling that the expression of multiple genes is negatively regulated by the sRNA. Intriguingly, many of these genes encode enzymes with "aerobic" functions or enzymes linked to oxidative stress....... Furthermore, in previous work most of the potential target genes have been shown to be repressed by FNR through an undetermined mechanism. Collectively, our results provide insight into the mechanism by which FNR negatively regulates genes such as sodA, sodB, cydDC, and metE, thereby demonstrating...

  3. Small Molecule Binding, Docking, and Characterization of the Interaction between Pth1 and Peptidyl-tRNA

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    Mary C. Hames

    2013-11-01

    Full Text Available Bacterial Pth1 is essential for viability. Pth1 cleaves the ester bond between the peptide and nucleotide of peptidyl-tRNA generated from aborted translation, expression of mini-genes, and short ORFs. We have determined the shape of the Pth1:peptidyl-tRNA complex using small angle neutron scattering. Binding of piperonylpiperazine, a small molecule constituent of a combinatorial synthetic library common to most compounds with inhibitory activity, was mapped to Pth1 via NMR spectroscopy. We also report computational docking results, modeling piperonylpiperazine binding based on chemical shift perturbation mapping. Overall these studies promote Pth1 as a novel antibiotic target, contribute to understanding how Pth1 interacts with its substrate, advance the current model for cleavage, and demonstrate feasibility of small molecule inhibition.

  4. Small Interfering RNA Targeting MDR1 Inhibits Ovarian Cancer Growth and Increases Efficacy of Chemotherapy in vivo

    Institute of Scientific and Technical Information of China (English)

    Fu-jun Liu; Guo-lan Gao; Kai-jia Tu; Li-qun Yu; Jun Gao

    2009-01-01

    Objective: To further validate a knockdown approach for circumventing the multidrug resistance gene (MDR1), we used small interfering RNA(siRNA) targeting MDR1 gene to inhibit the expression of MDR1 gene and P-glycoprotein(P-gp) in vivo.Methods: Ascite tumor xenografts were established by implanting human ovarian carcinoma cells SKOV3/AR intraperitoneally into the nude mice. The mice were randomized into the following three treatment groups with each group six mice respectively: Taxol, Taxol with lipofectamine and Taxol with siRNA/MDR1- lipofectamine intraperitoneal injection. The tumor growth rate and the ascite growth rate of mice were investigated. The expressions of MDR1 gene and P-gp in mice were determined by reverse transcription-polymerase chain reaction(RT-PCR) and immunohistochemistry respctively.Results: The growth of tumors and ascites in mice treated with Taxol and siRNA/MDR1- lipofectamine was significantly inhibited compared with those in mice of other groups. After 28 days' treatment, the average tumor weight and ascite volume decreased by 43.6% and 29.7% in the group treated with Taxol and siRNA/MDR1-lipofectamine compared with these treated with Taxol alone (P<0.001). The expressions of MDR1 gene and P-gp in the group treated with Taxol and siRNA/MDR1-lipofectamine were also decreased compared with those in the group treated with Taxol alone (P<0.001).Conclusion: Small interfering RNA targeting-MDR1 can effectively and specifically suppress the expression of MDR1(P-glycoprotein) and inhibit ovarian cancer growth in vivo.

  5. De novo transcriptome and small RNA analysis of two Chinese willow cultivars reveals stress response genes in Salix matsudana.

    Directory of Open Access Journals (Sweden)

    Guodong Rao

    Full Text Available Salix matsudana Koidz. is a deciduous, rapidly growing, and drought resistant tree and is one of the most widely distributed and commonly cultivated willow species in China. Currently little transcriptomic and small RNAomic data are available to reveal the genes involve in the stress resistant in S. matsudana. Here, we report the RNA-seq analysis results of both transcriptome and small RNAome data using Illumina deep sequencing of shoot tips from two willow variants(Salix. matsudana and Salix matsudana Koidz. cultivar 'Tortuosa'. De novo gene assembly was used to generate the consensus transcriptome and small RNAome, which contained 106,403 unique transcripts with an average length of 944 bp and a total length of 100.45 MB, and 166 known miRNAs representing 35 miRNA families. Comparison of transcriptomes and small RNAomes combined with quantitative real-time PCR from the two Salix libraries revealed a total of 292 different expressed genes(DEGs and 36 different expressed miRNAs (DEMs. Among the DEGs and DEMs, 196 genes and 24 miRNAs were up regulated, 96 genes and 12 miRNA were down regulated in S. matsudana. Functional analysis of DEGs and miRNA targets showed that many genes were involved in stress resistance in S. matsudana. Our global gene expression profiling presents a comprehensive view of the transcriptome and small RNAome which provide valuable information and sequence resources for uncovering the stress response genes in S. matsudana. Moreover the transcriptome and small RNAome data provide a basis for future study of genetic resistance in Salix.

  6. Inhibition of PARP1 by small interfering RNA enhances docetaxel activity against human prostate cancer PC3 cells

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Wenqi, E-mail: wwqwml@163.com [Department of Urology, Minimally Invasive Surgery Center, The First Affiliated Hospital of Guangzhou Medical University, Guangdong Key Laboratory of Urology (China); Kong, Zhenzhen; Duan, Xiaolu; Zhu, Hanliang; Li, Shujue; Zeng, Shaohua; Liang, Yeping [Department of Urology, Minimally Invasive Surgery Center, The First Affiliated Hospital of Guangzhou Medical University, Guangdong Key Laboratory of Urology (China); Iliakis, George [Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, Essen (Germany); Gui, Zhiming [Department of Urology, The Affiliated Hospital of Guangdong Medical College (China); Yang, Dong [Department of Urology, Minimally Invasive Surgery Center, The First Affiliated Hospital of Guangzhou Medical University, Guangdong Key Laboratory of Urology (China)

    2013-12-06

    Highlights: •PARP1 siRNA enhances docetaxel’s activity against PC3 cells. •PARP1 siRNA enhances docetaxel’s activity against EGFR/Akt/FOXO1 pathway. •PARP1 siRNA and PARP1 inhibitor differently affect the phosphorylation and expression of FOXO1. -- Abstract: Though poly(ADP-ribose) polymerase 1 (PARP1) inhibitors have benefits in combination with radiotherapy in prostate cancers, few is known about the exactly role and underlying mechanism of PARP1 in combination with chemotherapy agents. Here our data revealed that inhibition of PARP1 by small interfering RNA (siRNA) could enhance docetaxel’s activity against PC3 cells, which is associated with an accelerate repression of EGF/Akt/FOXO1 signaling pathway. Our results provide a novel role of PARP1 in transcription regulation of EGFR/Akt/FOXO1 signaling pathway and indicate that PARP1 siRNA combined with docetaxel can be an innovative treatment strategy to potentially improve outcomes in CRPC patients.

  7. Inhibition of PARP1 by small interfering RNA enhances docetaxel activity against human prostate cancer PC3 cells

    International Nuclear Information System (INIS)

    Highlights: •PARP1 siRNA enhances docetaxel’s activity against PC3 cells. •PARP1 siRNA enhances docetaxel’s activity against EGFR/Akt/FOXO1 pathway. •PARP1 siRNA and PARP1 inhibitor differently affect the phosphorylation and expression of FOXO1. -- Abstract: Though poly(ADP-ribose) polymerase 1 (PARP1) inhibitors have benefits in combination with radiotherapy in prostate cancers, few is known about the exactly role and underlying mechanism of PARP1 in combination with chemotherapy agents. Here our data revealed that inhibition of PARP1 by small interfering RNA (siRNA) could enhance docetaxel’s activity against PC3 cells, which is associated with an accelerate repression of EGF/Akt/FOXO1 signaling pathway. Our results provide a novel role of PARP1 in transcription regulation of EGFR/Akt/FOXO1 signaling pathway and indicate that PARP1 siRNA combined with docetaxel can be an innovative treatment strategy to potentially improve outcomes in CRPC patients

  8. Experimental RNomics in Aquifex aeolicus: identification of small non-coding RNAs and the putative 6S RNA homolog

    OpenAIRE

    Willkomm, Dagmar K; Minnerup, Jens; Hüttenhofer, Alexander; Roland K. Hartmann

    2005-01-01

    By an experimental RNomics approach, we have generated a cDNA library from small RNAs expressed from the genome of the hyperthermophilic bacterium Aquifex aeolicus. The library included RNAs that were antisense to mRNAs and tRNAs as well as RNAs encoded in intergenic regions. Substantial steady-state levels in A.aeolicus cells were confirmed for several of the cloned RNAs by northern blot analysis. The most abundant intergenic RNA of the library was identified as the 6S RNA homolog of A.aeoli...

  9. Nucleotide sequence of a crustacean 18S ribosomal RNA gene and secondary structure of eukaryotic small subunit ribosomal RNAs.

    Science.gov (United States)

    Nelles, L; Fang, B L; Volckaert, G; Vandenberghe, A; De Wachter, R

    1984-12-11

    The primary structure of the gene for 18 S rRNA of the crustacean Artemia salina was determined. The sequence has been aligned with 13 other small ribosomal subunit RNA sequences of eukaryotic, archaebacterial, eubacterial, chloroplastic and plant mitochondrial origin. Secondary structure models for these RNAs were derived on the basis of previously proposed models and additional comparative evidence found in the alignment. Although there is a general similarity in the secondary structure models for eukaryotes and prokaryotes, the evidence seems to indicate a different topology in a central area of the structures.

  10. Application of X-ray absorption spectroscopy and anomalous small angle scattering to RNA polymerase

    International Nuclear Information System (INIS)

    X-ray absorption spectroscopy is ideally suited for the investigation of the electronic structure and the local environment (≤∝5 A) of specific atoms in biomolecules. While the edge region provides information about the valence state of the absorbing atom, the chemical identity of neighboring atoms, and the coordination geometry, the EXAFS region contains information about the number and average distance of neighboring atoms and their relative disorder. The development of sensitive detection methods has allowed studies using near-physiological concentrations (as low as ∝100 μM). With careful choice of model compounds, judicious use of fitting procedures, and consideration of the results of biochemical and other spectrOScopic results, this data has provided pivotal information about the structures of these active sites which store energy in their conformation changes or ligand exchanges. Although the application of anomalous small angle scattering to biomolecules has occurred more recently, it clearly provides a method of determining distances between active sites that are outside the range of X-ray absorption spectroscopy. The wavelength dependence of the X-ray scattering power varies rapidly near the edge of the absorbing atom in both amplitude and phase. This behavior selectively alters the contribution of the absorbing atom to the scattering pattern. The structure-function relationship of the intermediate states provide the key to understanding the mechanisms of these complex molecules. It is this precise structural information about the active sites that is not obtainable by other spectroscopic techniques. Combination of these techniques offers a unique approach to the determination of the organization of active sites in biomolecules, especially metalloenzymes. Application of these methods to the substrate and template binding sites of RNA polymerase which contain zinc atoms demonstrates the versatility of this approach. (orig.)

  11. MicroRNA expression and clinical outcome of small cell lung cancer.

    Directory of Open Access Journals (Sweden)

    Jih-Hsiang Lee

    Full Text Available The role of microRNAs in small-cell lung carcinoma (SCLC is largely unknown. miR-34a is known as a p53 regulated tumor suppressor microRNA in many cancer types. However, its therapeutic implication has never been studied in SCLC, a cancer type with frequent dysfunction of p53. We investigated the expression of a panel of 7 microRNAs (miR-21, miR-29b, miR-34a/b/c, miR-155, and let-7a in 31 SCLC tumors, 14 SCLC cell lines, and 26 NSCLC cell lines. We observed significantly lower miR-21, miR-29b, and miR-34a expression in SCLC cell lines than in NSCLC cell lines. The expression of the 7 microRNAs was unrelated to SCLC patients' clinical characteristics and was neither prognostic in term of overall survival or progression-free survival nor predictive of treatment response. Overexpression or downregulation of miR-34a did not influence SCLC cell viability. The expression of these 7 microRNAs also did not predict in vitro sensitivity to cisplatin or etoposide in SCLC cell lines. Overexpression or downregulation of miR-34a did not influence sensitivity to cisplatin or etoposide in SCLC cell lines. In contrast to downregulation of the miR-34a target genes cMET and Axl by overexpression of miR-34a in NSCLC cell lines, the intrinsic expression of cMET and Axl was low in SCLC cell lines and was not influenced by overexpression of miR-34a. Our results suggest that the expression of the 7 selected microRNAs are not prognostic in SCLC patients, and miR-34a is unrelated to the malignant behavior of SCLC cells and is unlikely to be a therapeutic target.

  12. Dual function of the McaS small RNA in controlling biofilm formation

    DEFF Research Database (Denmark)

    Jørgensen, Mikkel Girke; Thomason, Maureen K.; Havelund, Johannes;

    2013-01-01

    , and biofilm formation. Moreover, ectopic McaS expression leads to induction of two additional CsrA-repressed genes encoding diguanylate cyclases. Collectively, our study shows that McaS is a dual-function sRNA with roles in the two major post-transcriptional regulons controlled by the RNA-binding proteins Hfq...

  13. Non-viral nanoparticle delivers small interfering RNA to macrophages in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Mei Zhang

    Full Text Available Macrophages are increasingly being viewed as therapeutic target for various cancers and many inflammatory diseases. Sequence specific gene reduction by siRNA represents an attractive approach to modulate macrophage function. However, delivery of the therapeutic siRNA into macrophages by non-viral nanoparticles has been a major technical challenge. In this study, we developed a glucan-based siRNA carrier system (BG34-10-Re-I and demonstrated that the BG34-10-Re-I can effectively assemble siRNA into uniformly distributed nanoparticles of the novel core-shell structure. The BG34-10-Re-I/siRNA nanoparticles effectively reduced gene expression of macrophage migration inhibitory factor (MIF in primary macrophages at both protein and mRNA level. The nanoparticles also mediated a sustained reduction of MIF within primary macrophages. Moreover, systemic injection of the nanoparticles into the Balb/c mice bearing 4T1 mammary tumors resulted in the MIF reduction in tumor-associated macrophages. Mechanistic studies demonstrated that the glucan-shell and the siRNA-core structure contribute to the effective delivery of MIF siRNA to macrophages both in vitro and in vivo. This study represents the first development of the primary macrophage MIF gene targeted non-viral nanoparticle system for both in vitro and in vivo applications.

  14. Cotranscriptional Chromatin Remodeling by Small RNA Species: An HTLV-1 Perspective

    Directory of Open Access Journals (Sweden)

    Nishat Aliya

    2012-01-01

    Full Text Available Cell type specificity of human T cell leukemia virus 1 has been proposed as a possible reason for differential viral outcome in primary target cells versus secondary. Through chromatin remodeling, the HTLV-1 transactivator protein Tax interacts with cellular factors at the chromosomally integrated viral promoter to activate downstream genes and control viral transcription. RNA interference is the host innate defense mechanism mediated by short RNA species (siRNA or miRNA that regulate gene expression. There exists a close collaborative functioning of cellular transcription factors with miRNA in order to regulate the expression of a number of eukaryotic genes including those involved in suppression of cell growth, induction of apoptosis, as well as repressing viral replication and propagation. In addition, it has been suggested that retroviral latency is influenced by chromatin alterations brought about by miRNA. Since Tax requires the assembly of transcriptional cofactors to carry out viral gene expression, there might be a close association between miRNA influencing chromatin alterations and Tax-mediated LTR activation. Herein we explore the possible interplay between HTLV-1 infection and miRNA pathways resulting in chromatin reorganization as one of the mechanisms determining HTLV-1 cell specificity and viral fate in different cell types.

  15. The major transcripts of the kinetoplast Trypanosoma brucei are very small ribosomal RNA's.

    NARCIS (Netherlands)

    I.C. Eperon; J.W.G. Janssen; J.H.J. Hoeijmakers (Jan); P. Borst (Piet)

    1983-01-01

    textabstractThe nucleotide sequence has been determined of a 2.2 kb segment of kinetoplast DNA, which encodes the major mitochondrial transcripts (12S and 9S) of Trypanosoma brucei. The sequence shows that the 12S RNA is a large subunit rRNA, although sufficiently unusual for resistance to chloramph

  16. Immunity to tomato yellow leaf curl virus in transgenic tomato is associated with accumulation of transgene small RNA.

    Science.gov (United States)

    Leibman, Diana; Prakash, Shanmugam; Wolf, Dalia; Zelcer, Aaron; Anfoka, Ghandi; Haviv, Sabrina; Brumin, Marina; Gaba, Victor; Arazi, Tzahi; Lapidot, Moshe; Gal-On, Amit

    2015-11-01

    Gene silencing is a natural defense response of plants against invading RNA and DNA viruses. The RNA post-transcriptional silencing system has been commonly utilized to generate transgenic crop plants that are "immune" to plant virus infection. Here, we applied this approach against the devastating DNA virus tomato yellow leaf curl virus (TYLCV) in its host tomato (Solanum lycopersicum L.). To generate broad resistance to a number of different TYLCV viruses, three conserved sequences (the intergenic region [NCR], V1-V2 and C1-C2 genes) from the genome of the severe virus (TYLCV) were synthesized as a single insert and cloned into a hairpin configuration in a binary vector, which was used to transform TYLCV-susceptible tomato plants. Eight of 28 independent transgenic tomato lines exhibited immunity to TYLCV-Is and to TYLCV-Mld, but not to tomato yellow leaf curl Sardinia virus, which shares relatively low sequence homology with the transgene. In addition, a marker-free (nptII-deleted) transgenic tomato line was generated for the first time by Agrobacterium-mediated transformation without antibiotic selection, followed by screening of 1180 regenerated shoots by whitefly-mediated TYLCV inoculation. Resistant lines showed a high level of transgene-siRNA (t-siRNA) accumulation (22% of total small RNA) with dominant sizes of 21 nt (73%) and 22 nt (22%). The t-siRNA displayed hot-spot distribution ("peaks") along the transgene, with different distribution patterns than the viral-siRNA peaks observed in TYLCV-infected tomato. A grafting experiment demonstrated the mobility of 0.04% of the t-siRNA from transgenic rootstock to non-transformed scion, even though scion resistance against TYLCV was not achieved.

  17. Measuring expression levels of small regulatory RNA molecules from body fluids and formalin-fixed, paraffin-embedded samples.

    Science.gov (United States)

    Gyongyosi, Adrienn; Docs, Otto; Czimmerer, Zsolt; Orosz, Laszlo; Horvath, Attila; Török, Olga; Mehes, Gabor; Nagy, Laszlo; Balint, Balint L

    2014-01-01

    MicroRNAs are involved in the regulation of various pathophysiological processes such as immune regulation and cancer. Next-generation sequencing methods enable us to monitor their presence in various types of samples but we need flexible methods for validating datasets generated by high-throughput methods. Here we describe the detailed protocols to be used with our MiRNA Primer Design Tool assay design system. The presented methods allow the flexible design of the oligonucleotides needed for the RT-qPCR detection of any variant of small regulatory RNA molecules from virtually any species. This method can be used to measure miRNA levels from formalin-fixed, paraffin-embedded (FFPE) samples and various body fluids. As an example, we show the results of the hsa-miR-515-3p, hsa-miR-325, and hsa-miR-155 quantification using a specific UPL probe (Universal Probe Library) and a stem-loop RT-qPCR assay. The small nucleolar RNA RNU43 is used as endogenous control for normalization of the results. Urine from healthy pregnant women and FFPE samples from patients diagnosed with colorectal cancer and treated with antibody-based anti-EGFR monotherapy were used as samples.

  18. Bridging small interfering RNA with giant therapeutic outcomes using nanometric liposomes.

    Science.gov (United States)

    Singh, Yuvraj; Tomar, Sandeep; Khan, Shariq; Meher, Jaya Gopal; Pawar, Vivek K; Raval, Kavit; Sharma, Komal; Singh, Pankaj K; Chaurasia, Mohini; Surendar Reddy, B; Chourasia, Manish K

    2015-12-28

    The scope of RNAi based therapeutics is unquestionable. However, if we dissect the current trend of clinical trials for afore mentioned drug class, some stark trends appear: 1) naked siRNA only exerts influence in topical mode whilst systemic delivery requires a carrier and 2) even after two decades of extensive efforts, not even a single siRNA containing product is commercially available. It was therefore felt that a perspective simplifying the unique intricacies of working with a merger of siRNA and liposomes from a pharmaceutical viewpoint could draw the attention of a wider array of interested researchers. We begin from the beginning and attempt to conduit the gap between theoretical logic and experimental/actual constraints. This, in turn could stimulate the next generation of investigators, gearing them to tackle the conundrum, which is siRNA delivery.

  19. Transcriptome landscape of Lactococcus lactis reveals many novel RNAs including a small regulatory RNA involved in carbon uptake and metabolism.

    Science.gov (United States)

    van der Meulen, Sjoerd B; de Jong, Anne; Kok, Jan

    2016-01-01

    RNA sequencing has revolutionized genome-wide transcriptome analyses, and the identification of non-coding regulatory RNAs in bacteria has thus increased concurrently. Here we reveal the transcriptome map of the lactic acid bacterial paradigm Lactococcus lactis MG1363 by employing differential RNA sequencing (dRNA-seq) and a combination of manual and automated transcriptome mining. This resulted in a high-resolution genome annotation of L. lactis and the identification of 60 cis-encoded antisense RNAs (asRNAs), 186 trans-encoded putative regulatory RNAs (sRNAs) and 134 novel small ORFs. Based on the putative targets of asRNAs, a novel classification is proposed. Several transcription factor DNA binding motifs were identified in the promoter sequences of (a)sRNAs, providing insight in the interplay between lactococcal regulatory RNAs and transcription factors. The presence and lengths of 14 putative sRNAs were experimentally confirmed by differential Northern hybridization, including the abundant RNA 6S that is differentially expressed depending on the available carbon source. For another sRNA, LLMGnc_147, functional analysis revealed that it is involved in carbon uptake and metabolism. L. lactis contains 13% leaderless mRNAs (lmRNAs) that, from an analysis of overrepresentation in GO classes, seem predominantly involved in nucleotide metabolism and DNA/RNA binding. Moreover, an A-rich sequence motif immediately following the start codon was uncovered, which could provide novel insight in the translation of lmRNAs. Altogether, this first experimental genome-wide assessment of the transcriptome landscape of L. lactis and subsequent sRNA studies provide an extensive basis for the investigation of regulatory RNAs in L. lactis and related lactococcal species. PMID:26950529

  20. Suppression of Breast Cancer Cell Migration by Small Interfering RNA Delivered by Polyethylenimine-Functionalized Graphene Oxide

    Science.gov (United States)

    Huang, Yuan-Pin; Hung, Chao-Ming; Hsu, Yi-Chiang; Zhong, Cai-Yan; Wang, Wan-Rou; Chang, Chi-Chang; Lee, Mon-Juan

    2016-05-01

    The carbon-based nanomaterial graphene can be chemically modified to associate with various molecules such as chemicals and biomolecules and developed as novel carriers for drug and gene delivery. In this study, a nonviral gene transfection reagent was produced by functionalizing graphene oxide (GO) with a polycationic polymer, polyethylenimine (PEI), to increase the biocompatibility of GO and to transfect small interfering RNA (siRNA) against C-X-C chemokine receptor type 4 (CXCR4), a biomarker associated with cancer metastasis, into invasive breast cancer cells. PEI-functionalized GO (PEI-GO) was a homogeneous aqueous solution that remained in suspension during storage at 4 °C for at least 6 months. The particle size of PEI-GO was 172 ± 4.58 and 188 ± 5.00 nm at 4 and 25 °C, respectively, and increased slightly to 262 ± 17.6 nm at 37 °C, but remained unaltered with time. Binding affinity of PEI-GO toward siRNA was assessed by electrophoretic mobility shift assay (EMSA), in which PEI-GO and siRNA were completely associated at a PEI-GO:siRNA weight ratio of 2:1 and above. The invasive breast cancer cell line, MDA-MB-231, was transfected with PEI-GO in complex with siRNAs against CXCR4 (siCXCR4). Suppression of the mRNA and protein expression of CXCR4 by the PEI-GO/siCXCR4 complex was confirmed by real-time PCR and western blot analysis. In addition, the metastatic potential of MDA-MB-231 cells was attenuated by the PEI-GO/siCXCR4 complex as demonstrated in wound healing assay. Our results suggest that PEI-GO is effective in the delivery of siRNA and may contribute to targeted gene therapy to suppress cancer metastasis.

  1. Key importance of small RNA binding for the activity of a glycine-tryptophan (GW) motif-containing viral suppressor of RNA silencing.

    Science.gov (United States)

    Pérez-Cañamás, Miryam; Hernández, Carmen

    2015-01-30

    Viruses express viral suppressors of RNA silencing (VSRs) to counteract RNA silencing-based host defenses. Although virtually all stages of the antiviral silencing pathway can be inhibited by VSRs, small RNAs (sRNAs) and Argonaute (AGO) proteins seem to be the most frequent targets. Recently, GW/WG motifs of some VSRs have been proposed to dictate their suppressor function by mediating interaction with AGO(s). Here we have studied the VSR encoded by Pelargonium line pattern virus (family Tombusviridae). The results show that p37, the viral coat protein, blocks RNA silencing. Site-directed mutagenesis of some p37 sequence traits, including a conserved GW motif, allowed generation of suppressor-competent and -incompetent molecules and uncoupling of the VSR and particle assembly capacities. The engineered mutants were used to assess the importance of p37 functions for viral infection and the relative contribution of diverse molecular interactions to suppressor activity. Two main conclusions can be drawn: (i) the silencing suppression and encapsidation functions of p37 are both required for systemic Pelargonium line pattern virus infection, and (ii) the suppressor activity of p37 relies on the ability to bind sRNAs rather than on interaction with AGOs. The data also caution against potential misinterpretations of results due to overlap of sequence signals related to distinct protein properties. This is well illustrated by mutation of the GW motif in p37 that concurrently affects nucleolar localization, efficient interaction with AGO1, and sRNA binding capability. These concomitant effects could have been overlooked in other GW motif-containing suppressors, as we exemplify with the orthologous p38 of turnip crinkle virus. PMID:25505185

  2. The stress-related, rhizobial small RNA RcsR1 destabilizes the autoinducer synthase encoding mRNA sinI in Sinorhizobium meliloti.

    Science.gov (United States)

    Baumgardt, Kathrin; Šmídová, Klára; Rahn, Helen; Lochnit, Günter; Robledo, Marta; Evguenieva-Hackenberg, Elena

    2016-05-01

    Quorum sensing is a cell density-dependent communication system of bacteria relying on autoinducer molecules. During the analysis of the post-transcriptional regulation of quorum sensing in the nitrogen fixing plant symbiont Sinorhizobium meliloti, we predicted and verified a direct interaction between the 5'-UTR of sinI mRNA encoding the autoinducer synthase and a small RNA (sRNA), which we named RcsR1. In vitro, RcsR1 prevented cleavage in the 5'-UTR of sinI by RNase E and impaired sinI translation. In line with low ribosomal occupancy and transcript destabilization upon binding of RcsR1 to sinI, overproduction of RcsR1 in S. meliloti resulted in lower level and shorter half-life of sinI mRNA, and in decreased autoinducer amount. Although RcsR1 can influence quorum sensing via sinI, its level did not vary at different cell densities, but decreased under salt stress and increased at low temperature. We found that RcsR1 and its stress-related expression pattern, but not the interaction with sinI homologs, are conserved in Sinorhizobium, Rhizobium and Agrobacterium. Consistently, overproduction of RcsR1 in S. meliloti and Agrobacterium tumefaciens inhibited growth at high salinity. We identified conserved targets of RcsR1 and showed that most conserved interactions and the effect on growth under salt stress are mediated by the first stem-loop of RcsR1, while its central part is responsible for the species-specific interaction with sinI. We conclude that RcsR1 is an ancient, stress-related riboregulator in rhizobia and propose that it links stress responses to quorum sensing in S. meliloti. PMID:26588798

  3. Differentially expressed genes in repairing irradiation-induced damage in mouse intestinal tract by foreign mouse small intestinal RNA

    International Nuclear Information System (INIS)

    To study the differentially expressed genes correlative to irradiation-induced damage in mouse intestinal tract and its repair by foreign mouse small intestinal RNA. Divide 90 mice into 4 groups randomly. For the experimental groups, each mouse was given 40μg foreign mouse small intestinal RNA, and for the control groups, 0.4 mL of 0.9% NaCl. The intestinal specimens were collected form each group 6h, 12h, 24h, 4d and 8d after 60Co γ-rays irradiation respectively. The differentially expressed genes in the tested and control groups were checked and cloned by long-distance PCR based on subtractive hybridization. 165 differential gene clones were obtained, of which 75 named as AXCZI-75 maybe were correlated with the irradiation-induced damage in mouse intestinal tract; and 90 named as XCZ1-90 maybe were correlated with repairing of the damaged mouse intestinal tract by foreign mouse small intestinal RNA

  4. The small RNA content of human sperm reveals pseudogene-derived piRNAs complementary to protein-coding genes

    DEFF Research Database (Denmark)

    Pantano, Lorena; Jodar, Meritxell; Bak, Mads;

    2015-01-01

    -specific genes. The most abundant class of small noncoding RNAs in sperm are PIWI-interacting RNAs (piRNAs). Surprisingly, we found that human sperm cells contain piRNAs processed from pseudogenes. Clusters of piRNAs from human testes contain pseudogenes transcribed in the antisense strand and processed...... that generate sperm small RNAs and what roles they may have, we sequenced and characterized the small RNA content of sperm samples from two human fertile individuals. We detected 182 microRNAs, some of which are highly abundant. The most abundant microRNA in sperm is miR-1246 with predicted targets among sperm...... into small RNAs. Several human protein-coding genes contain antisense predicted targets of pseudogene-derived piRNAs in the male germline and these piRNAs are still found in mature sperm. Our study provides the most extensive data set and annotation of human sperm small RNAs to date and is a resource...

  5. A Salmonella small non-coding RNA facilitates bacterial invasion and intracellular replication by modulating the expression of virulence factors.

    Directory of Open Access Journals (Sweden)

    Hao Gong

    2011-09-01

    Full Text Available Small non-coding RNAs (sRNAs that act as regulators of gene expression have been identified in all kingdoms of life, including microRNA (miRNA and small interfering RNA (siRNA in eukaryotic cells. Numerous sRNAs identified in Salmonella are encoded by genes located at Salmonella pathogenicity islands (SPIs that are commonly found in pathogenic strains. Whether these sRNAs are important for Salmonella pathogenesis and virulence in animals has not been reported. In this study, we provide the first direct evidence that a pathogenicity island-encoded sRNA, IsrM, is important for Salmonella invasion of epithelial cells, intracellular replication inside macrophages, and virulence and colonization in mice. IsrM RNA is expressed in vitro under conditions resembling those during infection in the gastrointestinal tract. Furthermore, IsrM is found to be differentially expressed in vivo, with higher expression in the ileum than in the spleen. IsrM targets the mRNAs coding for SopA, a SPI-1 effector, and HilE, a global regulator of the expression of SPI-1 proteins, which are major virulence factors essential for bacterial invasion. Mutations in IsrM result in disregulation of expression of HilE and SopA, as well as other SPI-1 genes whose expression is regulated by HilE. Salmonella with deletion of isrM is defective in bacteria invasion of epithelial cells and intracellular replication/survival in macrophages. Moreover, Salmonella with mutations in isrM is attenuated in killing animals and defective in growth in the ileum and spleen in mice. Our study has shown that IsrM sRNA functions as a pathogenicity island-encoded sRNA directly involved in Salmonella pathogenesis in animals. Our results also suggest that sRNAs may represent a distinct class of virulence factors that are important for bacterial infection in vivo.

  6. Small activating RNA induces myogenic differentiation of rat adipose-derived stem cells by upregulating MyoD

    Directory of Open Access Journals (Sweden)

    Chenghe Wang

    2015-08-01

    Full Text Available ABSTRACTPurpose:RNA activation (RNAa is a mechanism of gene activation triggered by promoter-targeted small double stranded RNAs (dsRNAs, also known as small activating RNAs (saRNAs. Myogenic regulatory factor MyoD is regarded as the master activator of myogenic differentiation cascade by binding to enhancer of muscle specific genes. Stress urinary incontinence (SUI is a condition primarily resulted from urethral sphincter deficiency. It is thus expected that by promoting differentiation of adipose-derived stem cells (ADSCs into myoblasts by activating MyoD gene through RNAa may offer benefits to SUI.Materials and Methods:Rats ADSCs were isolated, proliferated in vitro, and identified by flow cytometry. Purified ADSCs were then transfected with a MyoD saRNA or control transfected. Real-time polymerase chain reaction (RT-PCR and western blotting were used to detect MyoD mRNA and protein expression, respectively. Immunocytochemical staining was applied to determine the expression of desmin protein in transfected cells. Cell viability was measured by using CellTiter 96® AQueous One Solution Cell Proliferation Assay kit.Results:Transfection of a MyoD saRNA (dsMyoD into ADSCs significantly induced the expression of MyoD at both the mRNA and protein levels, and inhibited cell proliferation. Desmin protein expression was detected in dsMyoD treated ADSCs 2 weeks later.Conclusion:Our findings show that RNAa mediated overexpression of MyoD can promote transdifferentiation of ADSCs into myoblasts and may help treat stress urinary incontinence (SUI–a condition primarily resulted from urethral sphincter deficiency.

  7. Constructionof the recombinant adenovirus vectors of CALB2 gene and small interfering RNA, and application in testicular Leydig cells

    Institute of Scientific and Technical Information of China (English)

    Luo Jian; Wang Jing; Liu Shan; Sun Xue-ping; Gao Chao; Gao Li; Yang Xiao-yu; Liu Jia-yin; Cui Yu-gui

    2011-01-01

    Objective:To construct the recombinant adenovirus vectors of calretinin (CALB2) gene and small interfering RNA (siRNA),for over-expression or knock-down of CALB2,as the basis of functional investigation of CALB2 in testicular Leydig cells.Methods:The cDNA sequence of CALB2 was cloned by the reverse transcriptive polymerase chain reaction (RT-PCR).A CALB2 gene fragment was sub-cloned into adenovirus shuttle plasmid pAdTrack-CMV to construct the shuttle plasmid pAdTrack-CALB2.Then it was transformed into BJ5183 cells with the adenoviral backbone pAdEasy-1 to obtain the homologous recombinant AdCMV-CALB2.The recombinant AdCMV-CALB2 was further packaged and amplificated in AD293 cells.The expression of CALB2 protein in AD293 cells was detected by Western blotting.CALB2 protein was over-expressed in mouse Leydig cell line (MLTC-1 cells) by the constructed AdCMVCALB2.CALB2 gene siRNA recombinant adenovirus vector (Ad-H1-siRNA/CALB2 was also constructed simultaneously.Its efficacy was detected in AD293 cells by Western blotting.Results:The CALB2 gene recombinant adenovirus vector AdCMV-CALB2 and the CALB2 gene siRNA recombinant adenovirus vector Ad-H1-siRNA/CALB2 were constructed successfully by endonulease digestion and sequencing.AD293 cells infected with AdCMV-CALB2 or Ad-H1-SiRNA/CALB2 significantly expressed GFP protein.The expression of CALB2 protein was significantly up-regulated in AD293 cells infected with AdCMV-CALB2 plasmids,while the expression of CALB2 protein was down-regulated by 60% in the CALB2 cells infected with Ad-H1SiRNA/CALB2.MLTC-1 cells did not markedly express CALLB2 protein,while MLTC-1 cells infected with AdCMV-CALB2 expressed CALB2 protein at a high level.Conclusions:The recombinant adenovirus vectors of AdCMV-CALB2 and Ad-H1-SiRNA/CALB2 were successfully constructed.Both vectors effectively expressed in AD293.CALB2 protein was over-expressed in the cultured MLTC-1 cells by AdCMV-CALB2.These vectors of CALB2 gene and Leydig cell line are

  8. Inhibition of Proliferation of Human Hela Cells by Small Interference RNA against Pokemon Gene

    Institute of Scientific and Technical Information of China (English)

    DENG Yi-jing; NI Bing; JIANG Man; YANG Di; LI Fan; WU Yu-zhang

    2008-01-01

    Objective:The transcriptional repressor Pokemon(encoded by the Zbtb7 gene)is a critical factor in oncogenesis.Pokemon overexpression leads to overt oncogenic transformation both in vitro and in vivo in transgenic mice. The objective of this study was to investigate the effect of retrovirus expressing the siRNA targeting Pokemon in human cervical cancer cells. Methods:We constructed and identified the recombinant retrovirus particle expressing siRNA of Pokemon gene,and then testified the suppression of recombinant plasmid and evaluated the gene-silencing effect. Results:We got the positive evaluation from colony forming experiment we found that the retrovirus expressing siRNA targeting Pokemon had repressing effect. Conclusion:Our work provides basis for the study of suppression effect of retrovirus in vivo and the design of the target-complex.

  9. Transdermal Delivery of Small Interfering RNA with Elastic Cationic Liposomes in Mice

    Directory of Open Access Journals (Sweden)

    Yoshiyuki Hattori

    2013-01-01

    Full Text Available We developed elastic cationic liposomal vectors for transdermal siRNA delivery. These liposomes were prepared with 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP as a cationic lipid and sodium cholate (NaChol or Tween 80 as an edge activator. When NaChol or Tween 80 was included at 5, 10, and 15% (w/w into DOTAP liposomal formulations (C5-, C10-, and C15-liposomes and T5-, T10-, and T15-liposomes, C15- and T10-liposomes showed 2.4- and 2.7-fold-higher elasticities than DOTAP liposome, respectively. Although the sizes of all elastic liposomes prepared in this study were about 80–90 nm, the sizes of C5-, C10- and C15-liposome/siRNA complexes (lipoplexes were about 1,700–1,800 nm, and those of T5-, T10-, and T15-lipoplexes were about 550–780 nm. Their elastic lipoplexes showed strong gene suppression by siRNA without cytotoxicity when transfected into human cervical carcinoma SiHa cells. Following skin application of the fluorescence-labeled lipoplexes in mice, among the elastic lipoplexes, C15- and T5-lipoplexes showed effective penetration of siRNA into skin, compared with DOTAP lipoplex and free siRNA solution. These data suggest that elastic cationic liposomes containing an appropriate amount of NaChol or Tween 80 as an edge activator could deliver siRNA transdermally.

  10. Silencing of MGMT with small interference RNA reversed resistance in human BCUN-resistant glioma cell lines

    Institute of Scientific and Technical Information of China (English)

    XIE Si-ming; FANG Mao; GUO Hui; ZHONG Xue-yun

    2011-01-01

    Background Our previous study had cloned two glioma cell lines SWOZ1 and SWOZ2 isolated from parental glioma cell line SWO38.The 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) resistance of SWOZ1 was higher than that of SWOZ2.Since O6-methylguanine-DNA methyltransferase (MGMT) was thought to be closely related to BCNU resistance in glioma,this study aimed to explore the function of MGMT in glioma resistant to BCNU.Methods A BCNU resistant glioma cell line SWOZ2-BCNU was established.The expression of MGMT was detected in SWOZ1,SWOZ2 and SWOZ2-BCNU.Small interferencing RNA targeting MGMT was used to silence the expression of MGMT in resistant cell lines SWOZ1 and SWOZ2-BCNU.The cytotoxicity of BCNU to these cells was measured using the cell counting kit-8 assay.Statistical analysis was carried out by one-way analysis of variance in statistical package SPSS 13.0.Results The resistance of SWOZ1 and SWOZ2-BCNU against BCNU was 4.9-fold and 5.3-fold higher than that of SWOZ2.The results of quantitative RT-PCR and Western blotting confirmed that MGMT was both significantly increased in SWOZ1 and SWOZ2-BCNU compared to SOWZ2.After transfection with small interferencing RNA targeting MGMT,a decreased level of MGMT mRNA expression in SWOZ1 and SWOZ2-BCNU for more than 75% compared to negative control was found and confirmed by Western blotting.As a result,the resistance against BCNU was reversed for about 50% both in the BCNU-resistant cell lines SWOZ1 and SWOZ2-BCNU.Conclusions Silencing MGMT with specific small interferencing RNA can reverse the BCNU resistant phenotype in these glioma cell lines.MGMT may play an important role both in intrinsic and acquired BCNU-resistance in glioma.

  11. Use of electrophoretic mobility to determine the secondary structure of a small antisense RNA.

    OpenAIRE

    Jacques, J P; Susskind, M M

    1991-01-01

    Natural antisense RNAs have stem-loop (hairpin) secondary structures that are important for their function. The sar antisense RNA of phage P22 is unusual: the 3' half of the molecule forms an extensive stem-loop, but potential structures for the 5' half are not predicted to be thermodynamically stable. We devised a novel method to determine the secondary structure of sar RNA by examining the electrophoretic mobility on non-denaturing gels of numerous sar mutants. The results show that the wil...

  12. Small Interfering RNA Targeted to ASPP2 Promotes Progression of Experimental Proliferative Vitreoretinopathy

    Directory of Open Access Journals (Sweden)

    Xiao-Li Chen

    2016-01-01

    Full Text Available Background. Epithelial-mesenchymal transition (EMT of retinal pigment epithelium (RPE is vital in proliferative vitreoretinopathy (PVR development. Apoptosis-stimulating proteins of p53 (ASPP2 have recently been reported to participate in EMT. However, the role of ASPP2 in PVR pathogenesis has not been identified. Methods. Immunohistochemistry was used to investigate the expression of ASPP2 in epiretinal membranes of PVR patients. ARPE-19 cells were transfected with ASPP2-siRNA, followed with measurement of cell cytotoxicity, proliferation, and migration ability. EMT markers and related inflammatory and fibrosis cytokines were measured by western blot or flow cytometry. Additionally, PVR rat models were induced by intravitreal injection of ARPE-19 cells transfected with ASPP2-siRNA and evaluated accordingly. Results. Immunofluorescence analysis revealed less intense expression of ASPP2 in PVR membranes. ASPP2 knockdown facilitated the proliferation and migration of RPE cells and enhanced the expression of mesenchymal markers such as alpha smooth muscle actin, fibronectin, and ZEB1. Meanwhile, ASPP2-siRNA increased EMT-related and inflammatory cytokines, including TGF-β, CTGF, VEGF, TNF-α, and interleukins. PVR severities were more pronounced in the rat models with ASPP2-siRNA treatment. Conclusions. ASPP2 knockdown promoted EMT of ARPE-19 cells in vitro and exacerbated the progression of experimental PVR in vivo, possibly via inflammatory and fibrosis cytokines.

  13. DeAnnIso: a tool for online detection and annotation of isomiRs from small RNA sequencing data.

    Science.gov (United States)

    Zhang, Yuanwei; Zang, Qiguang; Zhang, Huan; Ban, Rongjun; Yang, Yifan; Iqbal, Furhan; Li, Ao; Shi, Qinghua

    2016-07-01

    Small RNA (sRNA) Sequencing technology has revealed that microRNAs (miRNAs) are capable of exhibiting frequent variations from their canonical sequences, generating multiple variants: the isoforms of miRNAs (isomiRs). However, integrated tool to precisely detect and systematically annotate isomiRs from sRNA sequencing data is still in great demand. Here, we present an online tool, DeAnnIso (Detection and Annotation of IsomiRs from sRNA sequencing data). DeAnnIso can detect all the isomiRs in an uploaded sample, and can extract the differentially expressing isomiRs from paired or multiple samples. Once the isomiRs detection is accomplished, detailed annotation information, including isomiRs expression, isomiRs classification, SNPs in miRNAs and tissue specific isomiR expression are provided to users. Furthermore, DeAnnIso provides a comprehensive module of target analysis and enrichment analysis for the selected isomiRs. Taken together, DeAnnIso is convenient for users to screen for isomiRs of their interest and useful for further functional studies. The server is implemented in PHP + Perl + R and available to all users for free at: http://mcg.ustc.edu.cn/bsc/deanniso/ and http://mcg2.ustc.edu.cn/bsc/deanniso/. PMID:27179030

  14. Ancient Origin of the U2 Small Nuclear RNA Gene-Targeting Non-LTR Retrotransposons Utopia.

    Science.gov (United States)

    Kojima, Kenji K; Jurka, Jerzy

    2015-01-01

    Most non-long terminal repeat (non-LTR) retrotransposons encoding a restriction-like endonuclease show target-specific integration into repetitive sequences such as ribosomal RNA genes and microsatellites. However, only a few target-specific lineages of non-LTR retrotransposons are distributed widely and no lineage is found across the eukaryotic kingdoms. Here we report the most widely distributed lineage of target sequence-specific non-LTR retrotransposons, designated Utopia. Utopia is found in three supergroups of eukaryotes: Amoebozoa, SAR, and Opisthokonta. Utopia is inserted into a specific site of U2 small nuclear RNA genes with different strength of specificity for each family. Utopia families from oomycetes and wasps show strong target specificity while only a small number of Utopia copies from reptiles are flanked with U2 snRNA genes. Oomycete Utopia families contain an "archaeal" RNase H domain upstream of reverse transcriptase (RT), which likely originated from a plant RNase H gene. Analysis of Utopia from oomycetes indicates that multiple lineages of Utopia have been maintained inside of U2 genes with few copy numbers. Phylogenetic analysis of RT suggests the monophyly of Utopia, and it likely dates back to the early evolution of eukaryotes. PMID:26556480

  15. High Sensitivity RT-qPCR Assay of Nonlabeled siRNA in Small Blood Volume for Pharmacokinetic Studies: Application to Survivin siRNA.

    Science.gov (United States)

    Yeung, Bertrand Z; Lu, Ze; Wientjes, Guillaume M; Au, Jessie L-S

    2015-11-01

    RNAi therapeutics provide an opportunity to correct faulty genes, and several RNAi have entered clinical evaluation. The existing quantification methods typically use radioactivity- or fluorescence-labeled RNAi, require large blood volumes, and/or have a limited dynamic detection range. We established a quantitative reverse transcriptase real-time polymerase chain reaction (RT-qPCR) assay to measure RNAi; the model analyte was survivin siRNA (siSurvivin). A second siRNA was used as the internal standard. The three major steps were (a) extraction of the two siRNAs from blood or water, (b) synthesis of their cDNA by poly-A extension, and (c) qPCR of cDNA. Standard curves were established. Utility of the assay was demonstrated in a pharmacokinetic study where all 12 samples for the blood concentration-time profile were obtained from a single mouse given an intravenous dose of 1 nmole siSurvivin (prepared as lipoplex with pegylated cationic liposomes). The RT-qPCR assay was sensitive (lower detection limit of 100 fM) and had a 5 × 107-fold dynamic range and low sample volume requirement (10 μL). The 16-point standard curves constructed using whole blood samples were linear (R (2) > 0.98). The intraday and interday variations for the slopes were ≤6%, although the variations for accuracy and precision at individual concentrations were substantially higher (58-145%). Standard curves prepared with water in place of blood showed similar results (water may be used when blood is not available. The current RT-qPCR assay enabled the measurement of nonlabeled siRNA in small volume of blood samples. PMID:26286676

  16. Determination of low-energy structures of a small RNA hairpin using Monte Carlo–based techniques

    Indian Academy of Sciences (India)

    Sudhanshu Shanker; Pradipta Bandyopadhyay

    2012-07-01

    The energy landscape of RNA is known to be extremely rugged, and hence finding low-energy structures starting from a random structure is a challenging task for any optimization algorithm. In the current work, we have investigated the ability of one Monte Carlo–based optimization algorithm, Temperature Basin Paving, to explore the energy landscape of a small RNA T-loop hairpin. In this method, the history of the simulation is used to increase the probability of states less visited in the simulation. It has been found that using both energy and end-to-end distance as the biasing parameters in the simulation, the partially folded structure of the hairpin starting from random structures could be obtained.

  17. Phylogenetic position of three Condylostoma species(Protozoa,Ciliophora,Heterotricheal inferred from the small subunit rRNA gene sequence

    Institute of Scientific and Technical Information of China (English)

    Wenbo Guo; Shan Gao; Weibo Song; Khaled A.S.A1-Rasheid; Chen Shao; Miao Miao; Saleh A.A1-Farraj; Saleh A.A1-Qurishy; Zigui Chen; Zhenzhen Yi

    2008-01-01

    The systematically poorly known ciliate genus Conadylostoma was erected by Vincent in 1826.About 10 morphotypes have been reported,but any molecular investigations concerning this group SO far are lacking.In this work,the small subunit ribosomal RNA (SS rRNA)gene of three marine Conaylostoma species was sequenced,by which the phylogenetic trees were constructed by distance-matrix,maximum parsimony and Bayesian inference methods.The results show that(1)all the trees have similar topologies with high supports;(2)Condylostoma is mostly related to the genus Condylostentor;and(3)three Condylostoma species as well as Conadylostentor auriculatus cluster together and form a sister group with other heterotrichs.This is moderately consistent with the assessment of phylo-genetic relationships of Conaylostoma-related heterotrichs from the morphological information.The phylogenetic relationship of some other related heterotrichs,Peritromus,Fotlictllina,Stentor and Blepharisma,has been also discussed.

  18. Genome-wide Annotation, Identification, and Global Transcriptomic Analysis of Regulatory or Small RNA Gene Expression in Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Ronan K. Carroll

    2016-02-01

    Full Text Available In Staphylococcus aureus, hundreds of small regulatory or small RNAs (sRNAs have been identified, yet this class of molecule remains poorly understood and severely understudied. sRNA genes are typically absent from genome annotation files, and as a consequence, their existence is often overlooked, particularly in global transcriptomic studies. To facilitate improved detection and analysis of sRNAs in S. aureus, we generated updated GenBank files for three commonly used S. aureus strains (MRSA252, NCTC 8325, and USA300, in which we added annotations for >260 previously identified sRNAs. These files, the first to include genome-wide annotation of sRNAs in S. aureus, were then used as a foundation to identify novel sRNAs in the community-associated methicillin-resistant strain USA300. This analysis led to the discovery of 39 previously unidentified sRNAs. Investigating the genomic loci of the newly identified sRNAs revealed a surprising degree of inconsistency in genome annotation in S. aureus, which may be hindering the analysis and functional exploration of these elements. Finally, using our newly created annotation files as a reference, we perform a global analysis of sRNA gene expression in S. aureus and demonstrate that the newly identified tsr25 is the most highly upregulated sRNA in human serum. This study provides an invaluable resource to the S. aureus research community in the form of our newly generated annotation files, while at the same time presenting the first examination of differential sRNA expression in pathophysiologically relevant conditions.

  19. IGS Minisatellites Useful for Race Differentiation in Colletotrichum lentis and a Likely Site of Small RNA Synthesis Affecting Pathogenicity.

    Directory of Open Access Journals (Sweden)

    Jonathan Durkin

    Full Text Available Colletotrichum lentis is a fungal pathogen of lentil in Canada but rarely reported elsewhere. Two races, Ct0 and Ct1, have been identified using differential lines. Our objective was to develop a PCR-probe differentiating these races. Sequences of the translation elongation factor 1α (tef1α, RNA polymerase II subunit B2 (rpb2, ATP citrate lyase subunit A (acla, and internal transcribed spacer (ITS regions were monomorphic, while the intergenic spacer (IGS region showed length polymorphisms at two minisatellites of 23 and 39 nucleotides (nt. A PCR-probe (39F/R amplifying the 39 nt minisatellite was developed which subsequently revealed 1-5 minisatellites with 1-12 repeats in C. lentis. The probe differentiated race Ct1 isolates having 7, 9 or 7+9 repeats from race Ct0 having primarily 2 or 4 repeats, occasionally 5, 6, or 8, but never 7 or 9 repeats. These isolates were collected between 1991 and 1999. In a 2012 survey isolates with 2 and 4 repeats increased from 34% to 67%, while isolated with 7 or 9 repeats decreased from 40 to 4%, likely because Ct1 resistant lentil varieties had been grown. The 39 nt repeat was identified in C. gloeosporioides, C. trifolii, Ascochyta lentis, Sclerotinia sclerotiorum and Botrytis cinerea. Thus, the 39F/R PCR probe is not species specific, but can differentiate isolates based on repeat number. The 23 nt minisatellite in C. lentis exists as three length variants with ten sequence variations differentiating race Ct0 having 14 or 19 repeats from race Ct1 having 17 repeats, except for one isolate. RNA-translation of 23 nt repeats forms hairpins and has the appropriate length to suggest that IGS could be a site of small RNA synthesis, a hypothesis that warrants further investigation. Small RNA from fungal plant pathogens able to silence genes either in the host or pathogen thereby aiding infection have been reported.

  20. TAXONOMIC STATUS OF CAR BACILLUS BASED ON THE SMALL SUBUNIT RIBOSOMAL RNA SEQUENCES

    Institute of Scientific and Technical Information of China (English)

    魏强; TsujiM; TakahashiT; IshiharaC; ItohT

    1995-01-01

    In an attempt to identify the taxonomic relationship between CAR bacillus and other bacteria, the SSU rRNA gene sequences of two CAR bacillus strains, CBM and CBR isolated from mice and rats respectively were used in the present studies. The SSU rRNA gene sequences, approximately 1.5 kb in size amplified from genomic DNAs from both strains, were determined and 96. 8% homologies were found to exist be-tween them. Those sequences were aligned to most euhacteria with a computer search showing high homol-ogy with those of Flavobacter/Flexibacter species especially closed to Fx. sanai and Ft. ferrugineum. Phylogenetic analysts indicated that CAR bacillus belongs to a species close to Fx. sancti and Ft. ferrug-imum subdivision.

  1. Gifsy-1 Prophage IsrK with Dual Function as Small and Messenger RNA Modulates Vital Bacterial Machineries.

    Science.gov (United States)

    Hershko-Shalev, Tal; Odenheimer-Bergman, Ahuva; Elgrably-Weiss, Maya; Ben-Zvi, Tamar; Govindarajan, Sutharsan; Seri, Hemda; Papenfort, Kai; Vogel, Jörg; Altuvia, Shoshy

    2016-04-01

    While an increasing number of conserved small regulatory RNAs (sRNAs) are known to function in general bacterial physiology, the roles and modes of action of sRNAs from horizontally acquired genomic regions remain little understood. The IsrK sRNA of Gifsy-1 prophage of Salmonella belongs to the latter class. This regulatory RNA exists in two isoforms. The first forms, when a portion of transcripts originating from isrK promoter reads-through the IsrK transcription-terminator producing a translationally inactive mRNA target. Acting in trans, the second isoform, short IsrK RNA, binds the inactive transcript rendering it translationally active. By switching on translation of the first isoform, short IsrK indirectly activates the production of AntQ, an antiterminator protein located upstream of isrK. Expression of antQ globally interferes with transcription termination resulting in bacterial growth arrest and ultimately cell death. Escherichia coli and Salmonella cells expressing AntQ display condensed chromatin morphology and localization of UvrD to the nucleoid. The toxic phenotype of AntQ can be rescued by co-expression of the transcription termination factor, Rho, or RNase H, which protects genomic DNA from breaks by resolving R-loops. We propose that AntQ causes conflicts between transcription and replication machineries and thus promotes DNA damage. The isrK locus represents a unique example of an island-encoded sRNA that exerts a highly complex regulatory mechanism to tune the expression of a toxic protein. PMID:27057757

  2. Small RNA interference-mediated gene silencing of heparanase abolishes the invasion, metastasis and angiogenesis of gastric cancer cells

    International Nuclear Information System (INIS)

    Heparanase facilitates the invasion and metastasis of cancer cells, and is over-expressed in many kinds of malignancies. Our studies indicated that heparanase was frequently expressed in advanced gastric cancers. The aim of this study is to determine whether silencing of heparanase expression can abolish the malignant characteristics of gastric cancer cells. Three heparanase-specific small interfering RNA (siRNAs) were designed, synthesized, and transfected into cultured gastric cancer cell line SGC-7901. Heparanase expression was measured by RT-PCR, real-time quantitative PCR and Western blot. Cell proliferation was detected by MTT colorimetry and colony formation assay. The in vitro invasion and metastasis of cancer cells were measured by cell adhesion assay, scratch assay and matrigel invasion assay. The angiogenesis capabilities of cancer cells were measured by tube formation of endothelial cells. Transfection of siRNA against 1496-1514 bp of encoding regions resulted in reduced expression of heparanase, which started at 24 hrs and lasted for 120 hrs post-transfection. The siRNA-mediated silencing of heparanase suppressed the cellular proliferation of SGC-7901 cells. In addition, the in vitro invasion and metastasis of cancer cells were attenuated after knock-down of heparanase. Moreover, transfection of heparanase-specific siRNA attenuated the in vitro angiogenesis of cancer cells in a dose-dependent manner. These results demonstrated that gene silencing of heparanase can efficiently abolish the proliferation, invasion, metastasis and angiogenesis of human gastric cancer cells in vitro, suggesting that heparanase-specific siRNA is of potential values as a novel therapeutic agent for human gastric cancer

  3. Efficient delivery of small interfering RNA into injured spinal cords in rats by photomechanical waves

    Science.gov (United States)

    Ando, Takahiro; Sato, Shunichi; Toyooka, Terushige; Kobayashi, Hiroaki; Nawashiro, Hiroshi; Ashida, Hiroshi; Obara, Minoru

    2011-03-01

    In the central nervous system, lack of axonal regeneration leads to permanent functional disabilities. In spinal cord injury (SCI), the over-expressions of intermediate filament (IF) proteins, such as glial fibrillary acidic protein (GFAP) and vimentin, are mainly involved in glial scar formation; these proteins work as both physical and biochemical barriers to axonal regeneration. Thus, silencing of these IF proteins would be an attractive strategy to treat SCI. In this study, we first attempted to deliver fluorescent probe-labeled siRNAs into injured spinal cords in rats by applying photomechanical waves (PMWs) to examine the capability of PMWs as a tool for siRNA delivery. Intense fluorescence from siRNAs was observed in much broader regions in the spinal cords with PMW application when compared with those with siRNA injection alone. Based on this result, we delivered siRNAs for GFAP and vimentin into injured spinal tissues in rats by applying PMWs. The treatment resulted in efficient silencing of the proteins at five days after SCI and a decrease of the cavity area in the injured tissue at three weeks after SCI when compared with those with siRNA injection alone. These results demonstrate the capability of PMWs for efficient delivery of siRNAs into injured spinal cords and treatment of SCIs.

  4. The 3′ Untranslated Region of the Andes Hantavirus Small mRNA Functionally Replaces the Poly(A) Tail and Stimulates Cap-Dependent Translation Initiation from the Viral mRNA

    OpenAIRE

    Vera-Otarola, Jorge; Soto-Rifo, Ricardo; Ricci, Emiliano P.; Ohlmann, Théophile; Darlix, Jean-Luc; López-Lastra, Marcelo

    2010-01-01

    In the process of translation of eukaryotic mRNAs, the 5′ cap and the 3′ poly(A) tail interact synergistically to stimulate protein synthesis. Unlike its cellular counterparts, the small mRNA (SmRNA) of Andes hantavirus (ANDV), a member of the Bunyaviridae, lacks a 3′ poly(A) tail. Here we report that the 3′ untranslated region (3′UTR) of the ANDV SmRNA functionally replaces a poly(A) tail and synergistically stimulates cap-dependent translation initiation from the viral mRNA. Stimulation of ...

  5. Suppression of Breast Cancer Cell Migration by Small Interfering RNA Delivered by Polyethylenimine-Functionalized Graphene Oxide.

    Science.gov (United States)

    Huang, Yuan-Pin; Hung, Chao-Ming; Hsu, Yi-Chiang; Zhong, Cai-Yan; Wang, Wan-Rou; Chang, Chi-Chang; Lee, Mon-Juan

    2016-12-01

    The carbon-based nanomaterial graphene can be chemically modified to associate with various molecules such as chemicals and biomolecules and developed as novel carriers for drug and gene delivery. In this study, a nonviral gene transfection reagent was produced by functionalizing graphene oxide (GO) with a polycationic polymer, polyethylenimine (PEI), to increase the biocompatibility of GO and to transfect small interfering RNA (siRNA) against C-X-C chemokine receptor type 4 (CXCR4), a biomarker associated with cancer metastasis, into invasive breast cancer cells. PEI-functionalized GO (PEI-GO) was a homogeneous aqueous solution that remained in suspension during storage at 4 °C for at least 6 months. The particle size of PEI-GO was 172 ± 4.58 and 188 ± 5.00 nm at 4 and 25 °C, respectively, and increased slightly to 262 ± 17.6 nm at 37 °C, but remained unaltered with time. Binding affinity of PEI-GO toward siRNA was assessed by electrophoretic mobility shift assay (EMSA), in which PEI-GO and siRNA were completely associated at a PEI-GO:siRNA weight ratio of 2:1 and above. The invasive breast cancer cell line, MDA-MB-231, was transfected with PEI-GO in complex with siRNAs against CXCR4 (siCXCR4). Suppression of the mRNA and protein expression of CXCR4 by the PEI-GO/siCXCR4 complex was confirmed by real-time PCR and western blot analysis. In addition, the metastatic potential of MDA-MB-231 cells was attenuated by the PEI-GO/siCXCR4 complex as demonstrated in wound healing assay. Our results suggest that PEI-GO is effective in the delivery of siRNA and may contribute to targeted gene therapy to suppress cancer metastasis. PMID:27173676

  6. siRNA-induced in vivo downregulation of L-type calcium channels in rat small mesenteric arteries

    DEFF Research Database (Denmark)

    Møller, Kate; Aalkjær, Christian; Matchkov, Vladimir

    2009-01-01

    Previous gene deletion studies have provided insight into the critical role of L-type voltage-gated Ca2+ channels (Cav1.2) in regulation of blood pressure. Homozygous knockout is, however, lethal but this limitation can be overcome by a small RNA interference (siRNA). A specific downregulation of...

  7. Comparison of Serum MicroRNA21 and Tumor Markers in Diagnosis of Early Non-Small Cell Lung Cancer

    OpenAIRE

    Mingzhong Sun; Jiangxiang Song; Zhongwei Zhou; Rong Zhu; Hao Jin; Yuqiao Ji; Qiang Lu; Huixiang Ju

    2016-01-01

    Objective. To compare the clinical value of serum microRNA21 (miR21) and other tumor markers in early diagnosis of non-small cell lung cancer (NSCLC). Methods. Serums carcinoembryonic antigen (CEA), cytokeratin 19 fragment (CYFRA21-1), neuron-specific enolase (NSE), and miR21 were detected in 50 NSCLC cases and 60 healthy control individuals. Results. Average serums miR21, CEA, NSE, and CYFRA21-1 levels were significantly higher in the case group than in control group (P < 0.01). Analysis of ...

  8. Comprehensive Small RNA-Seq of Adeno-Associated Virus (AAV)-Infected Human Cells Detects Patterns of Novel, Non-Coding AAV RNAs in the Absence of Cellular miRNA Regulation

    Science.gov (United States)

    Stutika, Catrin; Mietzsch, Mario; Gogol-Döring, Andreas; Weger, Stefan; Sohn, Madlen; Chen, Wei; Heilbronn, Regine

    2016-01-01

    Most DNA viruses express small regulatory RNAs, which interfere with viral or cellular gene expression. For adeno-associated virus (AAV), a small ssDNA virus with a complex biphasic life cycle miRNAs or other small regulatory RNAs have not yet been described. This is the first comprehensive Illumina-based RNA-Seq analysis of small RNAs expressed by AAV alone or upon co-infection with helper adenovirus or HSV. Several hotspots of AAV-specific small RNAs were detected mostly close to or within the AAV-ITR and apparently transcribed from the newly identified anti-p5 promoter. An additional small RNA hotspot was located downstream of the p40 promoter, from where transcription of non-coding RNAs associated with the inhibition of adenovirus replication were recently described. Parallel detection of known Ad and HSV miRNAs indirectly validated the newly identified small AAV RNA species. The predominant small RNAs were analyzed on Northern blots and by human argonaute protein-mediated co-immunoprecipitation. None of the small AAV RNAs showed characteristics of bona fide miRNAs, but characteristics of alternative RNA processing indicative of differentially regulated AAV promoter-associated small RNAs. Furthermore, the AAV-induced regulation of cellular miRNA levels was analyzed at different time points post infection. In contrast to other virus groups AAV infection had virtually no effect on the expression of cellular miRNA, which underscores the long-established concept that wild-type AAV infection is apathogenic. PMID:27611072

  9. Comprehensive Small RNA-Seq of Adeno-Associated Virus (AAV)-Infected Human Cells Detects Patterns of Novel, Non-Coding AAV RNAs in the Absence of Cellular miRNA Regulation.

    Science.gov (United States)

    Stutika, Catrin; Mietzsch, Mario; Gogol-Döring, Andreas; Weger, Stefan; Sohn, Madlen; Chen, Wei; Heilbronn, Regine

    2016-01-01

    Most DNA viruses express small regulatory RNAs, which interfere with viral or cellular gene expression. For adeno-associated virus (AAV), a small ssDNA virus with a complex biphasic life cycle miRNAs or other small regulatory RNAs have not yet been described. This is the first comprehensive Illumina-based RNA-Seq analysis of small RNAs expressed by AAV alone or upon co-infection with helper adenovirus or HSV. Several hotspots of AAV-specific small RNAs were detected mostly close to or within the AAV-ITR and apparently transcribed from the newly identified anti-p5 promoter. An additional small RNA hotspot was located downstream of the p40 promoter, from where transcription of non-coding RNAs associated with the inhibition of adenovirus replication were recently described. Parallel detection of known Ad and HSV miRNAs indirectly validated the newly identified small AAV RNA species. The predominant small RNAs were analyzed on Northern blots and by human argonaute protein-mediated co-immunoprecipitation. None of the small AAV RNAs showed characteristics of bona fide miRNAs, but characteristics of alternative RNA processing indicative of differentially regulated AAV promoter-associated small RNAs. Furthermore, the AAV-induced regulation of cellular miRNA levels was analyzed at different time points post infection. In contrast to other virus groups AAV infection had virtually no effect on the expression of cellular miRNA, which underscores the long-established concept that wild-type AAV infection is apathogenic. PMID:27611072

  10. Caenorhabditis elegans RSD-2 and RSD-6 promote germ cell immortality by maintaining small interfering RNA populations.

    Science.gov (United States)

    Sakaguchi, Aisa; Sarkies, Peter; Simon, Matt; Doebley, Anna-Lisa; Goldstein, Leonard D; Hedges, Ashley; Ikegami, Kohta; Alvares, Stacy M; Yang, Liwei; LaRocque, Jeannine R; Hall, Julie; Miska, Eric A; Ahmed, Shawn

    2014-10-14

    Germ cells are maintained in a pristine non-aging state as they proliferate over generations. Here, we show that a novel function of the Caenorhabditis elegans RNA interference proteins RNAi spreading defective (RSD)-2 and RSD-6 is to promote germ cell immortality at high temperature. rsd mutants cultured at high temperatures became progressively sterile and displayed loss of small interfering RNAs (siRNAs) that target spermatogenesis genes, simple repeats, and transposons. Desilencing of spermatogenesis genes occurred in late-generation rsd mutants, although defective spermatogenesis was insufficient to explain the majority of sterility. Increased expression of repetitive loci occurred in both germ and somatic cells of late-generation rsd mutant adults, suggesting that desilencing of many heterochromatic segments of the genome contributes to sterility. Nuclear RNAi defective (NRDE)-2 promotes nuclear silencing in response to exogenous double-stranded RNA, and our data imply that RSD-2, RSD-6, and NRDE-2 function in a common transgenerational nuclear silencing pathway that responds to endogenous siRNAs. We propose that RSD-2 and RSD-6 promote germ cell immortality at stressful temperatures by maintaining transgenerational epigenetic inheritance of endogenous siRNA populations that promote genome silencing. PMID:25258416

  11. Circulating U2 small nuclear RNA fragments as a novel diagnostic biomarker for pancreatic and colorectal adenocarcinoma

    DEFF Research Database (Denmark)

    Baraniskin, Alexander; Nöpel-Dünnebacke, Stefanie; Ahrens, Maike;

    2013-01-01

    Improved non-invasive strategies for early cancer detection are urgently needed to reduce morbidity and mortality. Non-coding RNAs, such as microRNAs and small nucleolar RNAs, have been proposed as biomarkers for non-invasive cancer diagnosis. Analyzing serum derived from nude mice implanted with...... primary human pancreatic ductal adenocarcinoma (PDAC), we identified 15 diagnostic microRNA candidates. Of those miR-1246 was selected based on its high abundance in serum of tumor carrying mice. Subsequently, we noted a cross reactivity of the established miR-1246 assays with RNA fragments derived from U...... that hsa-miR-1246 is likely a pseudo microRNA. In a next step, RNU2-1f was measured by qRT-PCR and normalized to cel-54 in 191 serum/plasma samples from PDAC and colorectal carcinoma (CRC) patients. In comparison to 129 controls, we were able to classify samples as cancerous with a sensitivity and...

  12. HmsB, a small RNA, enhances biofilm formation in Yersinia pestis

    Directory of Open Access Journals (Sweden)

    Dongsheng eZhou

    2014-12-01

    Full Text Available The hmsHFRS operon is responsible for biosynthesis and translocation of biofilm matrix exopolysaccharide. Yersinia pestis expresses the two sole diguanylate cyclases HmsT and HmsD and the sole phosphodiesterase HmsP, which are specific for biosynthesis and degradation, respectively, of 3’,5’-cyclic diguanosine monophosphate (c-di-GMP, a second messenger promoting exopolysaccharide production. In this work, the phenotypic assays indicates that Y. pestis sRNA HmsB enhances the production of c-di-GMP, exopolysaccharide, and biofilm. Further gene regulation experiments disclose that HmsB stimulates the expression of hmsB, hmsCDE, hmsT, and hmsHFRS but represses that of hmsP. HmsB most likely acts as a major activator of biofilm formation in Y. pestis. This is the first report of regulation of Yersinia biofilm formation by a sRNA. Data presented here will promote us to gain a deeper understanding of the complex regulatory circuits controlling Yersinia biofilm formation.

  13. The 5' spreading of small RNAs in Dictyostelium discoideum depends on the RNA-dependent RNA polymerase RrpC and on the dicer-related nuclease DrnB.

    Directory of Open Access Journals (Sweden)

    Stephan Wiegand

    Full Text Available RNA interference (RNAi is a gene-regulatory mechanism in eukarya that is based on the presence of double stranded RNA and that can act on both, the transcription or post-transcriptional level. In many species, RNA-dependent RNA polymerases (RdRPs are required for RNAi. To study the function of the three RdRPs in the amoeba Dictyostelium discoideum, we have deleted the encoding genes rrpA, rrpB and rrpC in all possible combinations. In these strains, expression of either antisense or hairpin RNA constructs against the transgene lacZ resulted in a 50% reduced β-Galactosidase activity. Northern blots surprisingly revealed unchanged lacZ mRNA levels, indicative of post-transcriptional regulation. Only in rrpC knock out strains, low levels of β-gal small interfering RNAs (siRNAs could be detected in antisense RNA expressing strains. In contrast to this, and at considerably higher levels, all hairpin RNA expressing strains featured β-gal siRNAs. Spreading of the silencing signal to mRNA sequences 5' of the original hairpin trigger was observed in all strains, except when the rrpC gene or that of the dicer-related nuclease DrnB was deleted. Thus, our data indicate that transitivity of an RNA silencing signal exists in D. discoideum and that it requires the two enzymes RrpC and DrnB.

  14. Deep sequencing of maize small RNAs reveals a diverse set of microRNA in dry and imbibed seeds.

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    Detao Li

    Full Text Available Seed germination plays a pivotal role during the life cycle of plants. As dry seeds imbibe water, the resumption of energy metabolism and cellular repair occur and miRNA-mediated gene expression regulation is involved in the reactivation events. This research was aimed at understanding the role of miRNA in the molecular control during seed imbibition process. Small RNA libraries constructed from dry and imbibed maize seed embryos were sequenced using the Illumina platform. Twenty-four conserved miRNA families were identified in both libraries. Sixteen of them showed significant expression differences between dry and imbibed seeds. Twelve miRNA families, miR156, miR159, miR164, miR166, miR167, miR168, miR169, miR172, miR319, miR393, miR394 and miR397, were significantly down-regulated; while four families, miR398, miR408, miR528 and miR529, were significantly up-regulated in imbibed seeds compared to that in dry seeds. Furthermore, putative novel maize miRNAs and their target genes were predicted. Target gene GO analysis was performed for novel miRNAs that were sequenced more than 50 times in the normalized libraries. The result showed that carbohydrate catabolic related genes were specifically enriched in the dry seed, while in imbibed seed target gene enrichment covered a broad range of functional categories including genes in amino acid biosynthesis, isomerase activity, ligase activity and others. The sequencing results were partially validated by quantitative RT-PCR for both conserved and novel miRNAs and the predicted target genes. Our data suggested that diverse and complex miRNAs are involved in the seed imbibition process. That miRNA are involved in plant hormone regulation may play important roles during the dry-imbibed seed transition.

  15. Diverse mechanisms of post-transcriptional repression by the small RNA regulator of glucose-phosphate stress.

    Science.gov (United States)

    Bobrovskyy, Maksym; Vanderpool, Carin K

    2016-01-01

    The Escherichia coli small RNA SgrS controls a metabolic stress response that occurs upon accumulation of certain glycolytic intermediates. SgrS base pairs with and represses translation of ptsG and manXYZ mRNAs, which encode sugar transporters, and activates translation of yigL mRNA, encoding a sugar phosphatase. This study defines four new genes as direct targets of E. coli SgrS. These new targets, asd, adiY, folE and purR, encode transcription factors or enzymes of diverse metabolic pathways, including aspartate semialdehyde dehydrogenase, arginine decarboxylase gene activator, GTP cyclohydrolase I and a repressor of purine biosynthesis, respectively. SgrS represses translation of each of the four target mRNAs via distinct mechanisms. SgrS binding sites overlapping the Shine-Dalgarno sequences of adiY and folE mRNAs suggest that SgrS pairing with these targets directly occludes ribosome binding and prevents translation initiation. SgrS binding within the purR coding sequence recruits the RNA chaperone Hfq to directly repress purR translation. Two separate SgrS binding sites were found on asd mRNA, and both are required for full translational repression. Ectopic overexpression of asd, adiY and folE is specifically detrimental to cells experiencing glucose-phosphate stress, suggesting that SgrS-dependent repression of the metabolic functions encoded by these targets promotes recovery from glucose-phosphate stress.

  16. INHIBITION OF PROLIFERATION OF HUMAN BREAST CANCER MCF-7 CELLS BY SMALL INTERFERENCE RNA AGAINST LRP16 GENE

    Institute of Scientific and Technical Information of China (English)

    韩为东; 赵亚力; 李琦; 母义明; 李雪; 宋海静; 陆祖谦

    2004-01-01

    Objective: Our previous studies have firstly demonstrated that 17(-E2 up-regulates LRP16 gene expression in human breast cancer MCF-7 cells, and ectopic expression of the LRP16 gene promotes MCF-7 cells proliferation. Here, the effects of the LRP16 gene expression on growth of MCF-7 human breast cancer cells and the mechanism were further studied by establishing two stably LRP16-inhibitory MCR-7 cell lines. Methods: Hairpin small interference RNA (siRNA) strategy, by which hairpin siRNA was released by U6 promoter and was mediated by pLPC-based retroviral vector, was adopted to knockdown endogenous LRP16 level in MCF-7 cells. And the hairpin siRNA against green fluorescence protein (GFP) was used as the negative control. The suppressant efficiency of the LRP16 gene expression was confirmed by Nothern blot. Cell proliferation assay and soft agar colony formation assay were used to determine the status of the cells proliferation. Cell cycle checkpoints including cyclin E and cyclin D1 were examined by Western blot. Results: The results from cell proliferation assays suggested that down-regulation of LRP16 gene expression is capable of inhibiting MCF-7 breast cancer cell growth and down-regulation of the LRP16 gene expression is able to inhibit anchorage-independent growth of breast cancer cells in soft agar. We also demonstrated that cyclin E and cyclin D1 proteins were much lower in the LRP16-inhibitory cells than in the control cells. Conclusion: These data suggest that LRP16 gene play an important role in MCF-7 cells proliferation by regulating the pathway of the G1/S transition and may function as an important modulator in regulating the process of tumorigenesis in human breast.

  17. Regulation of heterochromatin assembly on unpaired chromosomes during Caenorhabditis elegans meiosis by components of a small RNA-mediated pathway.

    Directory of Open Access Journals (Sweden)

    Xingyu She

    2009-08-01

    Full Text Available Many organisms have a mechanism for down regulating the expression of non-synapsed chromosomes and chromosomal regions during meiosis. This phenomenon is thought to function in genome defense. During early meiosis in Caenorhabditis elegans, unpaired chromosomes (e.g., the male X chromosome become enriched for a modification associated with heterochromatin and transcriptional repression, dimethylation of histone H3 on lysine 9 (H3K9me2. This enrichment requires activity of the cellular RNA-directed RNA polymerase, EGO-1. Here we use genetic mutation, RNA interference, immunofluorescence microscopy, fluorescence in situ hybridization, and molecular cloning methods to identify and analyze three additional regulators of meiotic H3K9me2 distribution: CSR-1 (a Piwi/PAZ/Argonaute protein, EKL-1 (a Tudor domain protein, and DRH-3 (a DEAH/D-box helicase. In csr-1, ekl-1, and drh-3 mutant males, we observed a reduction in H3K9me2 accumulation on the unpaired X chromosome and an increase in H3K9me2 accumulation on paired autosomes relative to controls. We observed a similar shift in H3K9me2 pattern in hermaphrodites that carry unpaired chromosomes. Based on several assays, we conclude that ectopic H3K9me2 accumulates on paired and synapsed chromosomes in these mutants. We propose alternative models for how a small RNA-mediated pathway may regulate H3K9me2 accumulation during meiosis. We also describe the germline phenotypes of csr-1, ekl-1, and drh-3 mutants. Our genetic data suggest that these factors, together with EGO-1, participate in a regulatory network to promote diverse aspects of development.

  18. Posttranscriptional down-regulation of small ribosomal subunit proteins correlates with reduction of 18S rRNA in RPS19 deficiency.

    Science.gov (United States)

    Badhai, Jitendra; Fröjmark, Anne-Sophie; Razzaghian, Hamid Reza; Davey, Edward; Schuster, Jens; Dahl, Niklas

    2009-06-18

    Ribosomal protein S19 (RPS19) is mutated in patients with Diamond-Blackfan anemia (DBA). We hypothesized that decreased levels of RPS19 lead to a coordinated down-regulation of other ribosomal (r-)proteins at the subunit level. We show that small interfering RNA (siRNA) knock-down of RPS19 results in a relative decrease of small subunit (SSU) r-proteins (S20, S21 and S24) when compared to large subunit (LSU) r-proteins (L3, L9, L30 and L38). This correlates with a relative decrease in 18S rRNA with respect to 28S rRNA. The r-protein mRNA levels remain relatively unchanged indicating a post transcriptional regulation of r-proteins at the level of subunit formation.

  19. Small indels induced by CRISPR/Cas9 in the 5' region of microRNA lead to its depletion and Drosha processing retardance.

    Science.gov (United States)

    Jiang, Qian; Meng, Xing; Meng, Lingwei; Chang, Nannan; Xiong, Jingwei; Cao, Huiqing; Liang, Zicai

    2014-01-01

    MicroRNA knockout by genome editing technologies is promising. In order to extend the application of the technology and to investigate the function of a specific miRNA, we used CRISPR/Cas9 to deplete human miR-93 from a cluster by targeting its 5' region in HeLa cells. Various small indels were induced in the targeted region containing the Drosha processing site and seed sequences. Interestingly, we found that even a single nucleotide deletion led to complete knockout of the target miRNA with high specificity. Functional knockout was confirmed by phenotype analysis. Furthermore, de novo microRNAs were not found by RNA-seq. Nevertheless, expression of the pri-microRNAs was increased. When combined with structural analysis, the data indicated that biogenesis was impaired. Altogether, we showed that small indels in the 5' region of a microRNA result in sequence depletion as well as Drosha processing retard.

  20. A small yeast RNA inhibits HCV IRES mediated translation and inhibits replication of poliovirus in vivo

    Institute of Scientific and Technical Information of China (English)

    Xue-Song Liang; Jian-Qi Lian; Yong-Xing Zhou; Qing-He Nie; Chun-Qiu Hao

    2003-01-01

    AIM: To investigate the anti-virus infection activity of internal ribosome entry site (IRES) specific inhibitor RNA (IRNA).METHODS: IRNA eukaryotic vector pcRz-IRNA or mIRNA eukaryotic vector pcRz-mIRNA was tansfected into human hepatocarcinoma cells (HHCC), then selected with neomycin G418 for 4 to 8 weeks, and then infected with polio virus vaccinas line. The cytopethogenesis effect was investigated and the cell extract was collected. At last the polio virus titer of different cells was determined by plaque assay.RESULTS: Constitutive expression of IRNA was not detrimental to cell growth. HCV IRES-mediated capindependent translation was markedly inhibited in cells constitutively expressing IRNA compared to control hepatoma cells. However, cap-dependent translation was not significantly affected in these cell line. Additionally, HHCC cells constitutively expressing IRNA became refractory to infection of polio virus.CONCLUSION: IRES specific IRNA can inhibit HCV IRES mediated translation and poliovirus replication.

  1. The roles of histone modifications and small RNA in centromere function.

    Science.gov (United States)

    Ekwall, Karl

    2004-01-01

    Here, epigenetic regulation of centromeric chromatin in fission yeast (Schizosaccharomyces pombe) is reviewed, focussing on the role of histone modifications and the link to RNA interference (RNAi). Fission yeast centromeres are organized into two structurally and functionally distinct domains, both of which are required for centromere function. The central core domain anchors the kinetochore structure while the flanking heterochromatin domain is important for sister centromere cohesion. The chromatin structure of both domains is regulated epigenetically. In the central core domain, the histone H3 variant Cnp1(CENP-A) plays a key role. In the flanking heterochromatin domain, histones are kept underacetylated by the histone deacetylases (HDACs) Clr3, Clr6 and Sir2, and methylated by Clr4 methyltransferase (HMTase) to create a specific binding site for the Swi6 protein. Swi6 then directly mediates cohesin binding to the centromeric heterochromatin. Recently, a surprising link was made between heterochromatin formation and RNAi. PMID:15289661

  2. The small RNA transcriptome of Pseudomonas syringae pathovar tomato DC3000

    Science.gov (United States)

    Small non-coding RNAs (ncRNAs) are regarded as important global regulators in prokaryotes and play critical roles in a variety of metabolic and cellular processes. Pseudomonas syringae pathovar tomato strain DC3000 (DC3000) is an important plant pathogenic bacterium that causes bacterial speck of to...

  3. Polymorphisms in miRNA binding site: new insight into small cell lung cancer susceptibility

    Institute of Scientific and Technical Information of China (English)

    Hong-yu LIU; Jun CHEN

    2011-01-01

    Lung cancer is a leading cause in cancer-related deaths with less than 15% five-year survival worldwide.Small cell lung cancer (SCLC),which accounts for about 15%-18% of lung cancer,carries the worst prognosis within the lung cancer patients.SCLC differs from other lung cancers,so called non-small cell lung cancers (NSCLCs),in the specifically clinical and biologic characteristics.It exhibits aggressive behavior,with rapid growth,early spread to distant sites.Although exquisite sensitive to chemotherapy and radiation,SCLC recurs rapidly with only 5% of patients surviving five years and frequent association with distinct paraneoplastic syndromes[1].

  4. Small RNA profiling of influenza A virus-infected cells identifies miR-449b as a regulator of histone deacetylase 1 and interferon beta.

    Directory of Open Access Journals (Sweden)

    William A Buggele

    Full Text Available The mammalian antiviral response relies on the alteration of cellular gene expression, to induce the production of antiviral effectors and regulate their activities. Recent research has indicated that virus infections can induce the accumulation of cellular microRNA (miRNA species that influence the stability of host mRNAs and their protein products. To determine the potential for miRNA regulation of cellular responses to influenza A virus infection, small RNA profiling was carried out using next generation sequencing. Comparison of miRNA expression profiles in uninfected human A549 cells to cells infected with influenza A virus strains A/Udorn/72 and A/WSN/33, revealed virus-induced changes in miRNA abundance. Gene expression analysis identified mRNA targets for a cohort of highly inducible miRNAs linked to diverse cellular functions. Experiments demonstrate that the histone deacetylase, HDAC1, can be regulated by influenza-inducible miR-449b, resulting in altered mRNA and protein levels. Expression of miR-449b enhances virus and poly(I:C activation of the IFNβ promoter, a process known to be negatively regulated by HDAC1. These findings demonstrate miRNA induction by influenza A virus infection and elucidate an example of miRNA control of antiviral gene expression in human cells, defining a role for miR-449b in regulation of HDAC1 and antiviral cytokine signaling.

  5. The cell cycle- and insulin-signaling-inhibiting miRNA expression pattern of very small embryonic-like stem cells contributes to their quiescent state.

    Science.gov (United States)

    Maj, Magdalena; Schneider, Gabriela; Ratajczak, Janina; Suszynska, Malwina; Kucia, Magda; Ratajczak, Mariusz Z

    2015-08-01

    Murine Oct4(+), very small embryonic-like stem cells (VSELs), are a quiescent stem cell population that requires a supportive co-culture layer to proliferate and/or to differentiate in vitro. Gene expression studies have revealed that the quiescence of these cells is due to changes in expression of parentally imprinted genes, including genes involved in cell cycle regulation and insulin and insulin-like growth factor signaling (IIS). To investigate the role of microRNAs (miRNAs) in VSEL quiescence, we performed miRNA studies in highly purified VSELs and observed a unique miRNA expression pattern in these cells. Specifically, we observed significant differences in the expression of certain miRNA species (relative to a reference cell population), including (i) miRNA-25_1 and miRNA-19 b, whose downregulation has the effect of upregulating cell cycle checkpoint genes and (ii) miRNA-675-3 p and miRNA-675-5 p, miRNA-292-5 p, miRNA-184, and miRNA-125 b, whose upregulation attenuates IIS. These observations are important for understanding the biology of these cells and for developing efficient ex vivo expansion strategies for VSELs isolated from adult tissues.

  6. The cell cycle- and insulin-signaling-inhibiting miRNA expression pattern of very small embryonic-like stem cells contributes to their quiescent state

    Science.gov (United States)

    Maj, Magdalena; Schneider, Gabriela; Ratajczak, Janina; Suszynska, Malwina; Kucia, Magda

    2015-01-01

    Murine Oct4+, very small embryonic-like stem cells (VSELs), are a quiescent stem cell population that requires a supportive co-culture layer to proliferate and/or to differentiate in vitro. Gene expression studies have revealed that the quiescence of these cells is due to changes in expression of parentally imprinted genes, including genes involved in cell cycle regulation and insulin and insulin-like growth factor signaling (IIS). To investigate the role of microRNAs (miRNAs) in VSEL quiescence, we performed miRNA studies in highly purified VSELs and observed a unique miRNA expression pattern in these cells. Specifically, we observed significant differences in the expression of certain miRNA species (relative to a reference cell population), including (i) miRNA-25_1 and miRNA-19 b, whose downregulation has the effect of upregulating cell cycle checkpoint genes and (ii) miRNA-675-3 p and miRNA-675-5 p, miRNA-292-5 p, miRNA-184, and miRNA-125 b, whose upregulation attenuates IIS. These observations are important for understanding the biology of these cells and for developing efficient ex vivo expansion strategies for VSELs isolated from adult tissues. PMID:25966979

  7. Footprints of a trypanosomatid RNA world: pre-small subunit rRNA processing by spliced leader addition trans-splicing

    Directory of Open Access Journals (Sweden)

    Mario Gustavo Mayer

    2012-06-01

    Full Text Available The addition of a capped mini-exon [spliced leader (SL] through trans-splicing is essential for the maturation of RNA polymerase (pol II-transcribed polycistronic pre-mRNAs in all members of the Trypanosomatidae family. This process is an inter-molecular splicing reaction that follows the same basic rules of cis-splicing reactions. In this study, we demonstrated that mini-exons were added to precursor ribosomal RNA (pre-rRNA are transcribed by RNA pol I, including the 5' external transcribed spacer (ETS region. Additionally, we detected the SL-5'ETS molecule using three distinct methods and located the acceptor site between two known 5'ETS rRNA processing sites (A' and A1 in four different trypanosomatids. Moreover, we detected a polyadenylated 5'ETS upstream of the trans-splicing acceptor site, which also occurs in pre-mRNA trans-splicing. After treatment with an indirect trans-splicing inhibitor (sinefungin, we observed SL-5'ETS decay. However, treatment with 5-fluorouracil (a precursor of RNA synthesis that inhibits the degradation of pre-rRNA led to the accumulation of SL-5'ETS, suggesting that the molecule may play a role in rRNA degradation. The detection of trans-splicing in these molecules may indicate broad RNA-joining properties, regardless of the polymerase used for transcription.

  8. SeqBuster, a bioinformatic tool for the processing and analysis of small RNAs datasets, reveals ubiquitous miRNA modifications in human embryonic cells.

    Science.gov (United States)

    Pantano, Lorena; Estivill, Xavier; Martí, Eulàlia

    2010-03-01

    High-throughput sequencing technologies enable direct approaches to catalog and analyze snapshots of the total small RNA content of living cells. Characterization of high-throughput sequencing data requires bioinformatic tools offering a wide perspective of the small RNA transcriptome. Here we present SeqBuster, a highly versatile and reliable web-based toolkit to process and analyze large-scale small RNA datasets. The high flexibility of this tool is illustrated by the multiple choices offered in the pre-analysis for mapping purposes and in the different analysis modules for data manipulation. To overcome the storage capacity limitations of the web-based tool, SeqBuster offers a stand-alone version that permits the annotation against any custom database. SeqBuster integrates multiple analyses modules in a unique platform and constitutes the first bioinformatic tool offering a deep characterization of miRNA variants (isomiRs). The application of SeqBuster to small-RNA datasets of human embryonic stem cells revealed that most miRNAs present different types of isomiRs, some of them being associated to stem cell differentiation. The exhaustive description of the isomiRs provided by SeqBuster could help to identify miRNA-variants that are relevant in physiological and pathological processes. SeqBuster is available at http://estivill_lab.crg.es/seqbuster. PMID:20008100

  9. A Simple Protocol for High Efficiency Protein Isolation After RNA Isolation from Mouse Thyroid and Other Very Small Tissue Samples.

    Science.gov (United States)

    Ziros, Panos G; Chartoumpekis, Dionysios V; Sykiotis, Gerasimos P

    2016-01-01

    As a dedicated hormone-secreting organ, the thyroid gland possesses a complement of proteostatic systems, including antioxidant, unfolded protein, and autophagic responses. The vast majority of animal investigations of thyroid physiology and, more recently, proteostasis, have utilized as model the rat, rather than the mouse. This is due to the very small size of the thyroid gland in the latter, with a total weight of ~2 mg (~1 mg per thyroid lobe). However, this strategy has limited the utilization of genetic approaches, such as taking advantage of the various transgenic and knockout mouse models. Here, we describe a simple and highly efficient protocol for the simultaneous isolation of mRNA, micro-RNA and 150-200 μg of protein from as little as 1 mg of mouse thyroid tissue, the average weight of one of the two thyroid lobes, thus preserving the other lobe for immunohistochemical or other analyses. While our workflow is similar to other protocols published in the literature and/or proposed by commercial reagent providers, we have introduced a key modification that addresses efficiently the most challenging step of the protein isolation process: the solubilization of the protein pellet after RNA extraction and protein precipitation. We demonstrate the feasibility of our approach and its utility for downstream analyses (including Western blotting) that facilitate the comparative study of proteostatic pathways in the mouse thyroid. We have also successfully applied this protocol on samples from mouse liver, brown and white adipose tissue, as well as from rodent cell lines. PMID:27613051

  10. Vasopressin mRNA and neurophysin-related cell-surface antigen (NRSA) in small-cell carcinoma.

    Science.gov (United States)

    North, W G; Yu, X M

    1993-01-01

    Production by small-cell carcinoma (SCCL) of neurophysins (HNPs) and neurophysin-related cell-surface antigen (NRSA) was examined for two cell lines, for mouse xenografts, and for a resected human tumor, using polyclonal and monoclonal antibodies to vasopressin-associated human neurophysin (VP-HNP) and polyclonal antibodies to vasopressin (VP). The nature of the mRNA responsible for giving rise to these neurophysin-related products was investigated by performing Northern analysis on preparations of poly A+RNA and cDNA probes complimentary to portions of the exon A, exon B, and exon C regions of the human VP gene. SDS-electrophoresis and Western analysis revealed two prominent proteins of 42,000 and 20,000 Da in acid extracts from all SCCL sources when the monoclonal anti-HNP or one of the two polyclonal anti-HNP preparations were used. These antibodies also disclosed the presence of a minor component of 10,000 Da. A second polyclonal anti-HNP preparation reacted with one prominent protein of 30,000 Da and, for one cell line and mouse xenografts, another protein of 32,000 Da. Both of two anti-VP preparations reacted with proteins of 42,000, 30,000, 25,000, and 20,000 Da in extracts from all SCCL source material. The immunoreactive proteins of 42,000, 30,000, and 20,000 Da were all components of a membrane fraction from SCCL cells and tissues. In Northern analysis, a single RNA of about 900 bases hybridized with exon A and exon B probes, but not with the cDNA probe complimentary to exon C of the VP gene.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8387189

  11. Persistence of smoking-induced dysregulation of miRNA expression in the small airway epithelium despite smoking cessation.

    Directory of Open Access Journals (Sweden)

    Guoqing Wang

    Full Text Available Even after quitting smoking, the risk of the development of chronic obstructive pulmonary disease (COPD and lung cancer remains significantly higher compared to healthy nonsmokers. Based on the knowledge that COPD and most lung cancers start in the small airway epithelium (SAE, we hypothesized that smoking modulates miRNA expression in the SAE linked to the pathogenesis of smoking-induced airway disease, and that some of these changes persist after smoking cessation. SAE was collected from 10th to 12th order bronchi using fiberoptic bronchoscopy. Affymetrix miRNA 2.0 arrays were used to assess miRNA expression in the SAE from 9 healthy nonsmokers and 10 healthy smokers, before and after they quit smoking for 3 months. Smoking status was determined by urine nicotine and cotinine measurement. There were significant differences in the expression of 34 miRNAs between healthy smokers and healthy nonsmokers (p1.5, with functions associated with lung development, airway epithelium differentiation, inflammation and cancer. After quitting smoking for 3 months, 12 out of the 34 miRNAs did not return to normal levels, with Wnt/β-catenin signaling pathway being the top identified enriched pathway of the target genes of the persistent dysregulated miRNAs. In the context that many of these persistent smoking-dependent miRNAs are associated with differentiation, inflammatory diseases or lung cancer, it is likely that persistent smoking-related changes in SAE miRNAs play a role in the subsequent development of these disorders.

  12. Regulation of the chitin degradation and utilization system by the ChiX small RNA in Serratia marcescens 2170.

    Science.gov (United States)

    Suzuki, Kazushi; Shimizu, Mari; Sasaki, Naomi; Ogawa, Chisana; Minami, Haruka; Sugimoto, Hayuki; Watanabe, Takeshi

    2016-01-01

    Serratia marcescens 2170 produces three different types of chitinases and chitin-binding protein CBP21. We found that transposon insertion into the 5' untranslated region (5' UTR) of chiPQ-ctb led to defective chitinase and CBP21 production. ChiX small RNA possessed the complementary sequence of the 5' UTRs of the chiPQ-ctb and chiR and repressed the expression of chiP and chiR. ChiX was detected in a medium containing glucose, glycerol, GlcNAc, and (GlcNAc)2, but the expression of both chiP and chiR was only observed in a medium containing (GlcNAc)2. ∆chiX mutant produced chitinases, CBP21, and chitobiase without induction. chiP transcripts were more abundant than those of chiR or chiX in a medium containing (GlcNAc)2. These results suggest that the constitutively expressed ChiX binds to the highly abundant chiP 5' UTR, thereby leading to the induction of chiR mRNA translation and the subsequent expression of chitinases and CBP21.

  13. Insulin-like growth factor II mRNA, peptides, and receptors in a thoracopulmonary malignant small round cell tumor

    DEFF Research Database (Denmark)

    Nielsen, F C; Orskov, C; Haselbacher, G;

    1994-01-01

    Insulin-like growth factor-(IGF) II and IGF-I and IGF-II/mannose 6-phosphate receptors were expressed in a thoracopulmonary malignant small round cell tumor (MSRCT) from a 14-year-old boy. Northern analysis showed that the MSRCT expresses multiple IGF-II mRNA of 6.0, 4.8, 4.2, and 2.2 kilobase from....... By a combined reverse transcription-polymerase chain reaction analysis, the authors also show that the MSRCT expresses IGF-I and IGF-II/mannose 6-phosphate receptor mRNA. The plasma concentration of IGF-II was 600 ng/ml and within the normal range of serum IGF-II. IGF binding proteins (IGFBP) of 41.5, 38.5, 34...... amounts of 10 kDa IGF-II and 7.5 kDa IGF-II. IGF-II may stimulate the proliferation of MSRCT by interaction with IGF-I receptors on the cells....

  14. An analysis of the small RNA transcriptome of four developmental stages of the citrus red mite (Panonychus citri).

    Science.gov (United States)

    Liu, B; Dou, W; Ding, T-B; Zhong, R; Liao, C-Y; Xia, W-K; Wang, J-J

    2014-04-01

    The citrus red mite (Panonychus citri) can feed on more than 112 plant species around the world. Endogenous small RNAs (sRNAs) have proved to be important components of gene regulation in many eukaryotes. Recently, many sRNAs have been shown to be involved in various biological processes, such as development in many animals, including insects; however, to date, no sRNAs have been reported in the citrus red mite. Using Illumina sequencing, several categories of sRNAs were identified, including 594 known microRNAs (miRNAs) grouped into 206 families and 31 novel miRNAs in the four developmental stages of citrus red mite. In addition, according to bioinformatics analysis and S-Poly(T) miRNA assays, the expression level of many miRNAs varied among the developmental stages. Furthermore, the prediction of miRNAs target genes and their functional annotation indicated that miRNAs are involved in the regulation of multiple pathways in the citrus red mite. As the first report of the sRNA world in citrus red mite, the present study furthers our understanding of the roles played by sRNAs in the development of citrus red mite and the data may help to develop methods of controlling the pests in the field. PMID:24330037

  15. DNAzyme-mediated recovery of small recombinant RNAs from a 5S rRNA-derived chimera expressed in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Willson Richard C

    2010-12-01

    Full Text Available Abstract Background Manufacturing large quantities of recombinant RNAs by overexpression in a bacterial host is hampered by their instability in intracellular environment. To overcome this problem, an RNA of interest can be fused into a stable bacterial RNA for the resulting chimeric construct to accumulate in the cytoplasm to a sufficiently high level. Being supplemented with cost-effective procedures for isolation of the chimera from cells and recovery of the recombinant RNA from stabilizing scaffold, this strategy might become a viable alternative to the existing methods of chemical or enzymatic RNA synthesis. Results Sequence encoding a 71-nucleotide recombinant RNA was inserted into a plasmid-borne deletion mutant of the Vibrio proteolyticus 5S rRNA gene in place of helix III - loop C segment of the original 5S rRNA. After transformation into Escherichia coli, the chimeric RNA (3×pen aRNA was expressed constitutively from E. coli rrnB P1 and P2 promoters. The RNA chimera accumulated to levels that exceeded those of the host's 5S rRNA. A novel method relying on liquid-solid partitioning of cellular constituents was developed for isolation of total RNA from bacterial cells. This protocol avoids toxic chemicals, and is therefore more suitable for large scale RNA purification than traditional methods. A pair of biotinylated 8-17 DNAzymes was used to bring about the quantitative excision of the 71-nt recombinant RNA from the chimera. The recombinant RNA was isolated by sequence-specific capture on beads with immobilized complementary deoxyoligonucleotide, while DNAzymes were recovered by biotin affinity chromatography for reuse. Conclusions The feasibility of a fermentation-based approach for manufacturing large quantities of small RNAs in vivo using a "5S rRNA scaffold" strategy is demonstrated. The approach provides a route towards an economical method for the large-scale production of small RNAs including shRNAs, siRNAs and aptamers for use

  16. Nuclear Ribosomal RNA Small Subunit (18S rRNA) Nucleotide Sequen Nuclear Ribosomal RNA Small Subunit (18S rRNA) Nucleotide Sequen cing and Characterization of Sailonggu(Whole Bone of Myospalax baileyi Thomas)cing%塞隆骨原动物高原鼢鼠核基因18S rRNA序列测定与分析

    Institute of Scientific and Technical Information of China (English)

    曹晖; 刘玉萍; 张绍来; 周开亚

    2001-01-01

    目的:测定仓鼠科动物高原鼢鼠Myospalax b aileyi的核rDNA基因序列,为塞隆骨正品基原检定提供分子依据。方法:采用PCR直接测序技术测定高原鼢鼠18S rRNA基因核苷酸序列并作序列特征分析。[ HT5”H〗结果:高原鼢鼠的18S rRNA序列长度为1 851 bp。根据排序比较,高原鼢鼠与2种鼠科动物间的DNA序列同源性 为72.04%~72.18%。结论:通过基因序列分析,DNA测序技术可成为 塞隆骨正品基原检定的准确有效手段。%Objective: Sequencing the nuclear ribosomal RNA small subunit (18S r RNA) gene of Myospalax baileyi (Cricetidae) to develop an ultimate and defi nitive means for origin identification of genuine Sailonggu. Methods: The total DNA wa s prepared from dried tail tissues. The nuclear 18S rRNA gene region was amplifi ed by PCR using a consensus primer set and its nucleotide sequence was determine d by PCR direct sequencing. The characteristic analysis of 18S rRNA sequences wa s generated usin software program Genetyx-SV/R Version 10.1. Results: The entire 18S rRNA gene region of M. baileyi spanned 1851 bp in length. Althou gh m ultiple alignment of sequence indicates that there are only lower homology (72.0 4%~72.18%)comparing with its two alias Mus musculus (GenBank Accession numb er X 00686)and Rattus norvegicus (M11188)(Muridae), their highly conservative dom ain i s located in 1020~1509 nt. There are many variable sites from upstream of 5'-e nd , which coud provide a novel information for molecular recognition of Sailonggu. Conclusion:DNA sequencing could be a useful and reliable tool in the origin identification of genuine Sailonggu.

  17. Topical Anti-Nuclear Factor-Kappa B Small Interfering RNA with Functional Peptides Containing Sericin-Based Hydrogel for Atopic Dermatitis

    OpenAIRE

    Takanori Kanazawa; Yuki Shizawa; Mayu Takeuchi; Kuniko Tamano; Hisako Ibaraki; Yasuo Seta; Yuki Takashima; Hiroaki Okada

    2015-01-01

    The small interfering RNA (siRNA) is suggested to offer a novel means of treating atopic dermatitis (AD) because it allows the specific silencing of genes related to AD pathogenesis. In our previous study, we found that siRNA targeted against RelA, an important nuclear factor-kappa B (NF-κB) subdomain, with functional peptides, showed therapeutic effects in a mouse model of AD. In the present study, to develop a topical skin application against AD, we prepared a hydrogel containing anti-RelA...

  18. In silico genome wide mining of conserved and novel miRNAs in the brain and pineal gland of Danio rerio using small RNA sequencing data

    OpenAIRE

    Suyash Agarwal; Naresh Sahebrao Nagpure; Prachi Srivastava; Basdeo Kushwaha; Ravindra Kumar; Manmohan Pandey; Shreya Srivastava

    2016-01-01

    MicroRNAs (miRNAs) are small, non-coding RNA molecules that bind to the mRNA of the target genes and regulate the expression of the gene at the post-transcriptional level. Zebrafish is an economically important freshwater fish species globally considered as a good predictive model for studying human diseases and development. The present study focused on uncovering known as well as novel miRNAs, target prediction of the novel miRNAs and the differential expression of the known miRNA using the ...

  19. Ultra-high throughput sequencing-based small RNA discovery and discrete statistical biomarker analysis in a collection of cervical tumours and matched controls

    Directory of Open Access Journals (Sweden)

    Gu Sam

    2010-05-01

    Full Text Available Abstract Background Ultra-high throughput sequencing technologies provide opportunities both for discovery of novel molecular species and for detailed comparisons of gene expression patterns. Small RNA populations are particularly well suited to this analysis, as many different small RNAs can be completely sequenced in a single instrument run. Results We prepared small RNA libraries from 29 tumour/normal pairs of human cervical tissue samples. Analysis of the resulting sequences (42 million in total defined 64 new human microRNA (miRNA genes. Both arms of the hairpin precursor were observed in twenty-three of the newly identified miRNA candidates. We tested several computational approaches for the analysis of class differences between high throughput sequencing datasets and describe a novel application of a log linear model that has provided the most effective analysis for this data. This method resulted in the identification of 67 miRNAs that were differentially-expressed between the tumour and normal samples at a false discovery rate less than 0.001. Conclusions This approach can potentially be applied to any kind of RNA sequencing data for analysing differential sequence representation between biological sample sets.

  20. Design and validation of small interfering RNA on respiratory syncytial virus M2-2 gene: A potential approach in RNA interference on viral replication.

    Science.gov (United States)

    Chin, V K; Atika Aziz, Nur A; Hudu, Shuaibu A; Harmal, Nabil S; Syahrilnizam, A; Jalilian, Farid A; Zamberi, S

    2016-10-01

    Human respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract infection in infants and young children globally and is a significant pathogen of the elderly and immunocompromised. The M2-2 protein of respiratory syncytial virus (RSV) is particularly important in regulation of viral RNA transcription and replication that could be a potential anti-viral candidate against RSV infection. In this study, we designed and validated siRNAs that specifically target the RSV M2-2 gene. Four siRNAs targeting different regions of the M2-2 gene were designed using web tool. In-vitro evaluation of silencing effect was performed by using RSV infected Vero cell line. Viral M2-2 linked GFP recombinant plasmid was co-transfected with non-targeted siRNA, Pooled siRNA, siRNA 1, siRNA 2, siRNA 3 and siRNA 4 using synthetic cationic polymer. The silencing effect of M2-2 gene at the protein level was measured both qualitatively and quantitatively by using fluorescence microscopy and flow cytometry. Meanwhile, the silencing effect at the mRNA level was assessed by using RT-qPCR. This study showed that all four designed siRNAs can effectively and efficiently silence M2-2 gene. siRNA 2 showed the highest (98%) silencing effect on protein level and siRNA 4 with 83.1% at the mRNA level. The viral assay showed no significant cytopathic effects observed after 6days post-infection with siRNAs. In conclusion, this study showed the effectiveness of siRNA in silencing M2-2 gene both at the protein and mRNA level which could potentially be used as a novel therapeutic agent in the treatment of RSV infection. However, further study is warranted to investigate the silencing effect of M2-2 protein and inhibition of RSV infection. PMID:27432115

  1. Small RNA profiling reveals phosphorus deficiency as a contributing factor in symptom expression for citrus huanglongbing disease.

    Science.gov (United States)

    Zhao, Hongwei; Sun, Ruobai; Albrecht, Ute; Padmanabhan, Chellappan; Wang, Airong; Coffey, Michael D; Girke, Thomas; Wang, Zonghua; Close, Timothy J; Roose, Mikeal; Yokomi, Raymond K; Folimonova, Svetlana; Vidalakis, Georgios; Rouse, Robert; Bowman, Kim D; Jin, Hailing

    2013-03-01

    Huanglongbing (HLB) is a devastating citrus disease that is associated with bacteria of the genus 'Candidatus Liberibacter' (Ca. L.). Powerful diagnostic tools and management strategies are desired to control HLB. Host small RNAs (sRNA) play a vital role in regulating host responses to pathogen infection and are used as early diagnostic markers for many human diseases, including cancers. To determine whether citrus sRNAs regulate host responses to HLB, sRNAs were profiled from Citrus sinensis 10 and 14 weeks post grafting with Ca. L. asiaticus (Las)-positive or healthy tissue. Ten new microRNAs (miRNAs), 76 conserved miRNAs, and many small interfering RNAs (siRNAs) were discovered. Several miRNAs and siRNAs were highly induced by Las infection, and can be potentially developed into early diagnosis markers of HLB. miR399, which is induced by phosphorus starvation in other plant species, was induced specifically by infection of Las but not Spiroplasma citri that causes citrus stubborn-a disease with symptoms similar to HLB. We found a 35% reduction of phosphorus in Las-positive citrus trees compared to healthy trees. Applying phosphorus oxyanion solutions to HLB-positive sweet orange trees reduced HLB symptom severity and significantly improved fruit production during a 3-year field trial in south-west Florida. Our molecular, physiological, and field data suggest that phosphorus deficiency is linked to HLB disease symptomology.

  2. The potential of circulating extracellular small RNAs (smexRNA) in veterinary diagnostics-Identifying biomarker signatures by multivariate data analysis.

    Science.gov (United States)

    Melanie, Spornraft; Benedikt, Kirchner; Pfaffl, Michael W; Irmgard, Riedmaier

    2015-09-01

    Worldwide growth and performance-enhancing substances are used in cattle husbandry to increase productivity. In certain countries however e.g., in the EU, these practices are forbidden to prevent the consumers from potential health risks of substance residues in food. To maximize economic profit, 'black sheep' among farmers might circumvent the detection methods used in routine controls, which highlights the need for an innovative and reliable detection method. Transcriptomics is a promising new approach in the discovery of veterinary medicine biomarkers and also a missing puzzle piece, as up to date, metabolomics and proteomics are paramount. Due to increased stability and easy sampling, circulating extracellular small RNAs (smexRNAs) in bovine plasma were small RNA-sequenced and their potential to serve as biomarker candidates was evaluated using multivariate data analysis tools. After running the data evaluation pipeline, the proportion of miRNAs (microRNAs) and piRNAs (PIWI-interacting small non-coding RNAs) on the total sequenced reads was calculated. Additionally, top 10 signatures were compared which revealed that the readcount data sets were highly affected by the most abundant miRNA and piRNA profiles. To evaluate the discriminative power of multivariate data analyses to identify animals after veterinary drug application on the basis of smexRNAs, OPLS-DA was performed. In summary, the quality of miRNA models using all mapped reads for both treatment groups (animals treated with steroid hormones or the β-agonist clenbuterol) is predominant to those generated with combined data sets or piRNAs alone. Using multivariate projection methodologies like OPLS-DA have proven the best potential to generate discriminative miRNA models, supported by small RNA-Seq data. Based on the presented comparative OPLS-DA, miRNAs are the favorable smexRNA biomarker candidates in the research field of veterinary drug abuse.

  3. In silico genome wide mining of conserved and novel miRNAs in the brain and pineal gland of Danio rerio using small RNA sequencing data.

    Science.gov (United States)

    Agarwal, Suyash; Nagpure, Naresh Sahebrao; Srivastava, Prachi; Kushwaha, Basdeo; Kumar, Ravindra; Pandey, Manmohan; Srivastava, Shreya

    2016-03-01

    MicroRNAs (miRNAs) are small, non-coding RNA molecules that bind to the mRNA of the target genes and regulate the expression of the gene at the post-transcriptional level. Zebrafish is an economically important freshwater fish species globally considered as a good predictive model for studying human diseases and development. The present study focused on uncovering known as well as novel miRNAs, target prediction of the novel miRNAs and the differential expression of the known miRNA using the small RNA sequencing data of the brain and pineal gland (dark and light treatments) obtained from NCBI SRA. A total of 165, 151 and 145 known zebrafish miRNAs were found in the brain, pineal gland (dark treatment) and pineal gland (light treatment), respectively. Chromosomes 4 and 5 of zebrafish reference assembly GRCz10 were found to contain maximum number of miR genes. The miR-181a and miR-182 were found to be highly expressed in terms of number of reads in the brain and pineal gland, respectively. Other ncRNAs, such as tRNA, rRNA and snoRNA, were curated against Rfam. Using GRCz10 as reference, the subsequent bioinformatic analyses identified 25, 19 and 9 novel miRNAs from the brain, pineal gland (dark treatment) and pineal gland (light treatment), respectively. Targets of the novel miRNAs were identified, based on sequence complementarity between miRNAs and mRNA, by searching for antisense hits in the 3'-UTR of reference RNA sequences of the zebrafish. The discovery of novel miRNAs and their targets in the zebrafish genome can be a valuable scientific resource for further functional studies not only in zebrafish but also in other economically important fishes. PMID:26981358

  4. The potential of circulating extracellular small RNAs (smexRNA in veterinary diagnostics—Identifying biomarker signatures by multivariate data analysis

    Directory of Open Access Journals (Sweden)

    Spornraft Melanie

    2015-09-01

    After running the data evaluation pipeline, the proportion of miRNAs (microRNAs and piRNAs (PIWI-interacting small non-coding RNAs on the total sequenced reads was calculated. Additionally, top 10 signatures were compared which revealed that the readcount data sets were highly affected by the most abundant miRNA and piRNA profiles. To evaluate the discriminative power of multivariate data analyses to identify animals after veterinary drug application on the basis of smexRNAs, OPLS-DA was performed. In summary, the quality of miRNA models using all mapped reads for both treatment groups (animals treated with steroid hormones or the β-agonist clenbuterol is predominant to those generated with combined data sets or piRNAs alone. Using multivariate projection methodologies like OPLS-DA have proven the best potential to generate discriminative miRNA models, supported by small RNA-Seq data. Based on the presented comparative OPLS-DA, miRNAs are the favorable smexRNA biomarker candidates in the research field of veterinary drug abuse.

  5. Inhibition of Hepatitis C Virus in Mice by a Small Interfering RNA Targeting a Highly Conserved Sequence in Viral IRES Pseudoknot.

    Directory of Open Access Journals (Sweden)

    Jae-Su Moon

    Full Text Available The hepatitis C virus (HCV internal ribosome entry site (IRES that directs cap-independent viral translation is a primary target for small interfering RNA (siRNA-based HCV antiviral therapy. However, identification of potent siRNAs against HCV IRES by bioinformatics-based siRNA design is a challenging task given the complexity of HCV IRES secondary and tertiary structures and association with multiple proteins, which can also dynamically change the structure of this cis-acting RNA element. In this work, we utilized siRNA tiling approach whereby siRNAs were tiled with overlapping sequences that were shifted by one or two nucleotides over the HCV IRES stem-loop structures III and IV spanning nucleotides (nts 277-343. Based on their antiviral activity, we mapped a druggable region (nts 313-343 where the targets of potent siRNAs were enriched. siIE22, which showed the greatest anti-HCV potency, targeted a highly conserved sequence across diverse HCV genotypes, locating within the IRES subdomain IIIf involved in pseudoknot formation. Stepwise target shifting toward the 5' or 3' direction by 1 or 2 nucleotides reduced the antiviral potency of siIE22, demonstrating the importance of siRNA accessibility to this highly structured and sequence-conserved region of HCV IRES for RNA interference. Nanoparticle-mediated systemic delivery of the stability-improved siIE22 derivative gs_PS1 siIE22, which contains a single phosphorothioate linkage on the guide strand, reduced the serum HCV genome titer by more than 4 log10 in a xenograft mouse model for HCV replication without generation of resistant variants. Our results provide a strategy for identifying potent siRNA species against a highly structured RNA target and offer a potential pan-HCV genotypic siRNA therapy that might be beneficial for patients resistant to current treatment regimens.

  6. Small interfering RNA targeting HIF-1{alpha} reduces hypoxia-dependent transcription and radiosensitizes hypoxic HT 1080 human fibrosarcoma cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Staab, Adrian [Wuerzburg Univ. (Germany). Dept. of Radiation Oncology; Paul Scherrer Institute (PSI), Villigen (Switzerland); Fleischer, Markus [Wuerzburg Univ. (Germany). Dept. of Radiation Oncology; Wuerzburg Univ. (Germany). Medical Clinic II; Loeffler, Juergen; Einsele, Herrmann [Wuerzburg Univ. (Germany). Medical Clinic II; Said, Harun M.; Katzer, Astrid; Flentje, Michael [Wuerzburg Univ. (Germany). Dept. of Radiation Oncology; Plathow, Christian [Freiburg Univ. (Germany). Dept. of Nuclear Medicine; Vordermark, Dirk [Wuerzburg Univ. (Germany). Dept. of Radiation Oncology; Halle-Wittenberg Univ. (Germany). Dept. of Radiation Oncology

    2011-04-15

    Background: Hypoxia inducible factor-1 has been identified as a potential target to overcome hypoxia-induced radioresistance The aim of the present study was to investigate whether selective HIF-1 inhibition via small interfering RNA (siRNA) targeting hypoxia-inducible factor 1{alpha} (HIF-1{alpha}) affects hypoxia-induced radioresistance in HT 1080 human fibrosarcoma cells. Material and Methods: HIF-1{alpha} expression in HT 1080 human fibrosarcoma cells in vitro was silenced using HIF-1{alpha} siRNA sequence primers. Quantitative real-time polymerase chain reaction assay was performed to quantify the mRNA expression of HIF-1{alpha}. HIF-1{alpha} protein levels were studied by Western blotting at 20% (air) or after 12 hours at 0.1% O{sub 2} (hypoxia). Cells were assayed for clonogenic survival after irradiation with 2, 5, or 10 Gy, under normoxic or hypoxic conditions in the presence of HIF-1{alpha}-targeted or control siRNA sequences. A modified oxygen enhancement ratio (OER') was calculated as the ratio of the doses to achieve the same survival at 0.1% O{sub 2} as at ambient oxygen tensions. OER' was obtained at cell survival levels of 50%, 37%, and 10%. Results: HIF-1{alpha}-targeted siRNA enhanced radiation treatment efficacy under severely hypoxic conditions compared to tumor cells treated with scrambled control siRNA. OER was reduced on all survival levels after treatment with HIF-1{alpha}-targeted siRNA, suggesting that inhibition of HIF-1 activation by using HIF-1{alpha}-targeted siRNA increases radiosensitivity of hypoxic tumor cells in vitro. Conclusion: Inhibition of HIF-1 activation by using HIF-1{alpha}-targeted siRNA clearly acts synergistically with radiotherapy and increase radiosensitivity of hypoxic cells in vitro. (orig.)

  7. Release of Small RNA-containing Exosome-like Vesicles from the Human Filarial Parasite Brugia malayi.

    Directory of Open Access Journals (Sweden)

    Mostafa Zamanian

    Full Text Available Lymphatic filariasis (LF is a socio-economically devastating mosquito-borne Neglected Tropical Disease caused by parasitic filarial nematodes. The interaction between the parasite and host, both mosquito and human, during infection, development and persistence is dynamic and delicately balanced. Manipulation of this interface to the detriment of the parasite is a promising potential avenue to develop disease therapies but is prevented by our very limited understanding of the host-parasite relationship. Exosomes are bioactive small vesicles (30-120 nm secreted by a wide range of cell types and involved in a wide range of physiological processes. Here, we report the identification and partial characterization of exosome-like vesicles (ELVs released from the infective L3 stage of the human filarial parasite Brugia malayi. Exosome-like vesicles were isolated from parasites in culture media and electron microscopy and nanoparticle tracking analysis were used to confirm that vesicles produced by juvenile B. malayi are exosome-like based on size and morphology. We show that loss of parasite viability correlates with a time-dependent decay in vesicle size specificity and rate of release. The protein cargo of these vesicles is shown to include common exosomal protein markers and putative effector proteins. These Brugia-derived vesicles contain small RNA species that include microRNAs with host homology, suggesting a potential role in host manipulation. Confocal microscopy shows J774A.1, a murine macrophage cell line, internalize purified ELVs, and we demonstrate that these ELVs effectively stimulate a classically activated macrophage phenotype in J774A.1. To our knowledge, this is the first report of exosome-like vesicle release by a human parasitic nematode and our data suggest a novel mechanism by which human parasitic nematodes may actively direct the host responses to infection. Further interrogation of the makeup and function of these bioactive

  8. MicroRNA-133a suppresses multiple oncogenic membrane receptors and cell invasion in non-small cell lung carcinoma.

    Directory of Open Access Journals (Sweden)

    Lu-Kai Wang

    Full Text Available Non-small cell lung cancers (NSCLCs cause high mortality worldwide, and the cancer progression can be activated by several genetic events causing receptor dysregulation, including mutation or amplification. MicroRNAs are a group of small non-coding RNA molecules that function in gene silencing and have emerged as the fine-tuning regulators during cancer progression. MiR-133a is known as a key regulator in skeletal and cardiac myogenesis, and it acts as a tumor suppressor in various cancers. This study demonstrates that miR-133a expression negatively correlates with cell invasiveness in both transformed normal bronchial epithelial cells and lung cancer cell lines. The oncogenic receptors in lung cancer cells, including insulin-like growth factor 1 receptor (IGF-1R, TGF-beta receptor type-1 (TGFBR1, and epidermal growth factor receptor (EGFR, are direct targets of miR-133a. MiR-133a can inhibit cell invasiveness and cell growth through suppressing the expressions of IGF-1R, TGFBR1 and EGFR, which then influences the downstream signaling in lung cancer cell lines. The cell invasive ability is suppressed in IGF-1R- and TGFBR1-repressed cells and this phenomenon is mediated through AKT signaling in highly invasive cell lines. In addition, by using the in vivo animal model, we find that ectopically-expressing miR-133a dramatically suppresses the metastatic ability of lung cancer cells. Accordingly, patients with NSCLCs who have higher expression levels of miR-133a have longer survival rates compared with those who have lower miR-133a expression levels. In summary, we identified the tumor suppressor role of miR-133a in lung cancer outcome prognosis, and we demonstrated that it targets several membrane receptors, which generally produce an activating signaling network during the progression of lung cancer.

  9. Small RNA Profiling Reveals Phosphorus Deficiency as a Contributing Factor in Symptom Expression for Citrus Huanglongbing Disease

    Institute of Scientific and Technical Information of China (English)

    Hongwei Zhao; Ruobai Sun; Ute Albrecht; Chellappan Padmanabhan; Airong Wang; Michael D.Coffey; Thomas Girke

    2013-01-01

    Huanglongbing (HLB) is a devastating citrus disease that is associated with bacteria of the genus "Candidatus Liberibacter' (Ca.L.).Powerful diagnostic tools and management strategies are desired to control HLB.Host small RNAs (sRNA) play a vital role in regulating host responses to pathogen infection and are used as early diagnostic markers for many human diseases,including cancers.To determine whether citrus sRNAs regulate host responses to HLB,sRNAs were profiled from Citrus sinensis 10 and 14 weeks post grafting with Ca.L.asiaticus (Las)-positive or healthy tissue.Ten new microRNAs (miRNAs),76 conserved miRNAs,and many small interfering RNAs (siRNAs) were discovered.Several miRNAs and siRNAs were highly induced by Las infection,and can be potentially developed into early diagnosis markers of HLB.miR399,which is induced by phosphorus starvation in other plant species,was induced specifically by infection of Las but not Spiroplasma citri that causes citrus stubborn--a disease with symptoms similar to HLB.We found a 35% reduction of phosphorus in Las-positive citrus trees compared to healthy trees.Applying phosphorus oxyanion solutions to HLB-positive sweet orange trees reduced HLB symptom severity and significantly improved fruit production during a 3-year field trial in south-west Florida.Our molecular,physiological,and field data suggest that phosphorus deficiency is linked to HLB disease symptomology.

  10. Topical Anti-Nuclear Factor-Kappa B Small Interfering RNA with Functional Peptides Containing Sericin-Based Hydrogel for Atopic Dermatitis

    Directory of Open Access Journals (Sweden)

    Takanori Kanazawa

    2015-09-01

    Full Text Available The small interfering RNA (siRNA is suggested to offer a novel means of treating atopic dermatitis (AD because it allows the specific silencing of genes related to AD pathogenesis. In our previous study, we found that siRNA targeted against RelA, an important nuclear factor-kappa B (NF-κB subdomain, with functional peptides, showed therapeutic effects in a mouse model of AD. In the present study, to develop a topical skin application against AD, we prepared a hydrogel containing anti-RelA siRNA and functional peptides and determined the intradermal permeation and the anti-AD effects in an AD mouse model. We selected the silk protein, sericin (SC, which is a versatile biocompatible biomaterial to prepare hydrogel as an aqueous gel base. We found that the siRNA was more widely delivered to the site of application in AD-induced ear skin of mice after topical application via the hydrogel containing functional peptides than via the preparation without functional peptides. In addition, the ear thickness and clinical skin severity of the AD-induced mice treated with hydrogel containing anti-RelA siRNA with functional peptides improved more than that of mice treated with the preparation formulated with negative siRNA.

  11. Topical Anti-Nuclear Factor-Kappa B Small Interfering RNA with Functional Peptides Containing Sericin-Based Hydrogel for Atopic Dermatitis.

    Science.gov (United States)

    Kanazawa, Takanori; Shizawa, Yuki; Takeuchi, Mayu; Tamano, Kuniko; Ibaraki, Hisako; Seta, Yasuo; Takashima, Yuki; Okada, Hiroaki

    2015-01-01

    The small interfering RNA (siRNA) is suggested to offer a novel means of treating atopic dermatitis (AD) because it allows the specific silencing of genes related to AD pathogenesis. In our previous study, we found that siRNA targeted against RelA, an important nuclear factor-kappa B (NF-κB) subdomain, with functional peptides, showed therapeutic effects in a mouse model of AD. In the present study, to develop a topical skin application against AD, we prepared a hydrogel containing anti-RelA siRNA and functional peptides and determined the intradermal permeation and the anti-AD effects in an AD mouse model. We selected the silk protein, sericin (SC), which is a versatile biocompatible biomaterial to prepare hydrogel as an aqueous gel base. We found that the siRNA was more widely delivered to the site of application in AD-induced ear skin of mice after topical application via the hydrogel containing functional peptides than via the preparation without functional peptides. In addition, the ear thickness and clinical skin severity of the AD-induced mice treated with hydrogel containing anti-RelA siRNA with functional peptides improved more than that of mice treated with the preparation formulated with negative siRNA. PMID:26371030

  12. Expression and clinical significance of genes frequently mutated in small cell lung cancers defined by whole exome/RNA sequencing.

    Science.gov (United States)

    Iwakawa, Reika; Kohno, Takashi; Totoki, Yasushi; Shibata, Tatsuhiro; Tsuchihara, Katsuya; Mimaki, Sachiyo; Tsuta, Koji; Narita, Yoshitaka; Nishikawa, Ryo; Noguchi, Masayuki; Harris, Curtis C; Robles, Ana I; Yamaguchi, Rui; Imoto, Seiya; Miyano, Satoru; Totsuka, Hirohiko; Yoshida, Teruhiko; Yokota, Jun

    2015-06-01

    Small cell lung cancer (SCLC) is the most aggressive type of lung cancer. Only 15% of SCLC patients survive beyond 2 years after diagnosis. Therefore, for the improvement of patients' outcome in this disease, it is necessary to identify genetic alterations applicable as therapeutic targets in SCLC cells. The purpose of this study is the identification of genes frequently mutated and expressed in SCLCs that will be targetable for therapy of SCLC patients. Exome sequencing was performed in 28 primary tumors and 16 metastatic tumors from 38 patients with SCLCs. Expression of mutant alleles was verified in 19 cases by RNA sequencing. TP53, RB1 and PTEN were identified as being significantly mutated genes. Additional 36 genes were identified as being frequently (≥10%) mutated in SCLCs by combining the results of this study and two recent studies. Mutated alleles were expressed in 8 of the 36 genes, TMEM132D, SPTA1, VPS13B, CSMD2, ANK2, ASTN1, ASPM and FBN3. In particular, the TMEM132D, SPTA1 and VPS13B genes were commonly mutated in both early and late stage tumors, primary tumors and metastases, and tumors before and after chemotherapy, as in the case of the TP53 and RB1 genes. Therefore, in addition to TP53, RB1 and PTEN, TMEM132D, SPTA1 and VPS13B could be also involved in SCLC development, with the products from their mutated alleles being potential therapeutic targets in SCLC patients. PMID:25863124

  13. Prognostic significance of RNA-dependent protein kinase (PKR) on non-small cell lung cancer patients

    Science.gov (United States)

    Pataer, Abujiang; Raso, Maria Gabriela; Correa, Arlene M; Behrens, Carmen; Tsuta, Koji; Solis, Luisa; Fang, Bingliang; Roth, Jack A.; Wistuba, Ignacio I.; Swisher, Stephen G.

    2011-01-01

    Purpose The role of RNA-dependent protein kinase (PKR) in antiviral defence mechanisms and in cellular differentiation, growth, and apoptosis is well known, but the role of PKR in human lung cancer remains poorly understood. To explore the role of PKR in human lung cancer, we evaluated PKR’s expression in tissue microarray specimens from both non-small cell lung cancer (NSCLC) and normal human bronchial epithelium tissue. Experimental Design Tissue microarray samples (TMA-1) from 231 lung cancers were stained with PKR antibody and validated on TMA-2 from 224 lung cancers. Immunohistochemical expression score was quantified by three pathologists independently. Survival probability was computed by the Kaplan-Meier method. Results The NSCLC cells showed lower levels of PKR expression than normal bronchial epithelium cells did. We also found a significant association between lower levels of PKR expression and lymph node metastasis. We found that loss of PKR expression is correlated with a more aggressive behavior, and that a high PKR expression predicts a subgroup of patients with a favorable outcome. Univariate and multivariate Cox proportional hazards regression models showed that a lower level of PKR expression was significantly associated with shorter survival in NSCLC patients. We further validated and confirmed that PKR to be a powerful prognostic factor in TMA-2 lung cancer (HR=0.22, P<0.0001). Conclusions Our findings first indicate that PKR expression is an independent prognostic variable in NSCLC patients. PMID:20930042

  14. Evaluating hypotheses of basal animal phylogeny using complete sequences of large and small subunit rRNA

    Energy Technology Data Exchange (ETDEWEB)

    Medina, Monica; Collins, Allen G.; Silberman, Jeffrey; Sogin, Mitchell L.

    2001-06-21

    We studied the evolutionary relationships among basal metazoan lineages by using complete large subunit (LSU) and small subunit (SSU) ribosomal RNA sequences for 23 taxa. After identifying competing hypotheses, we performed maximum likelihood searches for trees conforming to each hypothesis. Kishino-Hasegawa tests were used to determine whether the data (LSU, SSU, and combined) reject any of the competing hypotheses. We also conducted unconstrained tree searches, compared the resulting topologies, and calculated bootstrap indices. Shimodaira-Hasegawa tests were applied to determine whether the data reject any of the topologies resulting from the constrained and unconstrained tree searches. LSU, SSU, and the combined data strongly contradict two assertions pertaining to sponge phylogeny. Hexactinellid sponges are not likely to be the basal lineage of amonophyletic Porifera or the sister group to all other animals. Instead, Hexactinellida and Demospongia form a well-supported clade of siliceous sponges, Silicea. It remains unclear, on the basis of these data alone, whether the calcarean sponges are more closely related to Silicea or to nonsponge animals. The SSU and combined data reject the hypothesis that Bilateria is more closely related to Ctenophora than it is to Cnidaria, whereas LSU data alone do not refute either hypothesis. LSU and SSU data agree in supporting the monophyly of Bilateria, Cnidaria, Ctenophora, and Metazoa. LSU sequence data reveal phylogenetic structure in a data set with limited taxon sampling. Continued accumulation of LSU sequences should increase our understanding of animal phylogeny.

  15. RNA targeting by small molecule alkaloids: Studies on the binding of berberine and palmatine to polyribonucleotides and comparison to ethidium

    Science.gov (United States)

    Islam, Md. Maidul; Suresh Kumar, Gopinatha

    2008-03-01

    The binding affinity, energetics and conformational aspects of the interaction of isoquinoline alkaloids berberine and palmatine to four single stranded polyribonucleotides polyguanylic acid [poly(G)], polyinosinic acid [poly(I)], polycytidylic acid [poly(C)] and polyuridylic acid [poly(U)] were studied by absorption, fluorescence, isothermal titration calorimetry and circular dichroism spectroscopy and compared with ethidium. Berberine, palmatine and ethidium binds strongly with poly(G) and poly(I) with affinity in the order 10 5 M -1 while their binding to poly(C) and poly(U) were very weak or practically nil. The same conclusions have also emerged from isothermal titration calorimetric studies. The binding of all the three compounds to poly(C) and poly(I) was exothermic and favored by both negative enthalpy change and positive entropy change. Conformational change in the polymer associated with the binding was observed in poly(I) with all the three molecules and poly(U) with ethidium but not in poly(G) and poly(C) revealing differences in the orientation of the bound molecules in the hitherto different helical organization of these polymers. These fundamental results may be useful and serve as database for the development of futuristic RNA based small molecule therapeutics.

  16. A small RNA controls expression of the chitinase ChiA in Listeria monocytogenes

    DEFF Research Database (Denmark)

    Nielsen, Jesper S; Larsen, Marianne Halberg; Lillebæk, Eva Maria Sternkopf;

    2011-01-01

    In recent years, more than 60 small RNAs (sRNAs) have been identified in the gram-positive human pathogen Listeria monocytogenes, but their putative roles and mechanisms of action remain largely unknown. The sRNA LhrA was recently shown to be a post-transcriptional regulator of a single gene, lmo...... role of LhrA in L. monocytogenes. To this end, we determined the effects of LhrA on global-wide gene expression. We observed that nearly 300 genes in L. monocytogenes are either positively or negatively affected by LhrA. Among these genes, we identified lmo0302 and chiA as direct targets of LhrA, thus...... establishing LhrA as a multiple target regulator. Lmo0302 encodes a hypothetical protein with no known function, whereas chiA encodes one of two chitinases present in L. monocytogenes. We show here that LhrA acts as a post-transcriptional regulator of lmo0302 and chiA by interfering with ribosome recruitment...

  17. Phylogenetics of the brachyuran crabs (Crustacea: Decapoda): the status of Podotremata based on small subunit nuclear ribosomal RNA.

    Science.gov (United States)

    Ahyong, Shane T; Lai, Joelle C Y; Sharkey, Deirdre; Colgan, Donald J; Ng, Peter K L

    2007-11-01

    The true crabs, the Brachyura, are generally divided into two major groups: Eubrachyura or 'advanced' crabs, and Podotremata or 'primitive' crabs. The status of Podotremata is one of the most controversial issues in brachyuran systematics. The podotreme crabs, best recognised by the possession of gonopores on the coxae of the pereopods, have variously been regarded as mono-, para- or polyphyletic, or even as non-brachyuran. For the first time, the phylogenetic positions of the podotreme crabs were studied by cladistic analysis of small subunit nuclear ribosomal RNA sequences. Eight of 10 podotreme families were represented along with representatives of 17 eubrachyuran families. Under both maximum parsimony and Bayesian Inference, Podotremata was found to be significantly paraphyletic, comprising three major clades: Dromiacea, Raninoida, and Cyclodorippoida. The most 'basal' is Dromiacea, followed by Raninoida and Cylodorippoida. Notably, Cyclodorippoida was identified as the sister group of the Eubrachyura. Previous hypotheses that the dromiid crab, Hypoconcha, is an anomuran were unsupported, though Dromiidae as presently composed could be paraphyletic. Topologies constrained for podotreme monophyly were found to be significantly worse (P < 0.04) than unconstrained topologies under Templeton and S-H tests. The clear pattern of podotreme paraphyly and robustness of topologies recovered indicates that Podotremata as a formal concept is untenable. Relationships among the eubrachyurans were generally equivocal, though results indicate the majoids or dorippoids were the least derived of the Eubrachyura. A new high level classification of the Brachyura is proposed.

  18. MicroRNA-193b modulates proliferation, migration, and invasion of non-small cell lung cancer cells

    Institute of Scientific and Technical Information of China (English)

    Huajun Hu; Shangao Li; Jun Liu; Bin Ni

    2012-01-01

    MicroRNAs have been reported to be closely related to the development of human lung cancers.However,the functions of microRNAs in non-small cell lung cancer (NSCLC) remain largely undefined.Here,we investigated the role of microRNA-193b (miR-193b) in NSCLC.Our data showed that miR-193b was markedly down-regulated in NSCLC cancer tissues compared with adjacent normal tissues.The NSCLC cell line (A549) transfected with the miR-193b exhibited significantly decreased proliferation,migration,and invasion capacities when compared with the control cells.In contrast,inhibition of miR-193bincreased the proliferation,migration,and invasion of A549 cells.Moreover,miR-193b repressed the expressions of cyclin D1 and urokinase-type plasminogen activator in A549 cells.These data suggest that miR-193b is a tumor suppressor in NSCLC.

  19. Serum level of microRNA-147 as diagnostic biomarker in human non-small cell lung cancer.

    Science.gov (United States)

    Chu, Guangmin; Zhang, Jianbo; Chen, Xiaobing

    2016-08-01

    Objectives In this study, we intended to examine the gene expression level and the clinical significance of microRNA-147 (miR-147) in cancer tissues and sera of patients with non-small cell lung cancer (NSCLC). Methods Quantitative real-time PCR (qRT-PCR) was used to investigate the expression levels of miR-147 in 32 paired NSCLC tissues and their adjacent normal lung tissues, sera of 122 control and 87 NSCLC patients. The correlation of serum miR-147 expression level with clinicopathological characteristics, and the prognosis of NSCLC patients was statistically evaluated. Results MiR-147 was significantly down-regulated in NSCLC tissues than in paired adjacent normal tissues, and in sera of NSCLC patients than in sera of control patients. In addition, serum miR-147 was markedly down-regulated in advanced NSCLC patients and the patients with lymph node metastasis (LNM). Low serum miR-147 expression level was found to be significantly correlated with tumor, lymph node, metastasis stage, LNM, and tumor size. Statistical analysis showed that patients with low serum miR-147 had much worse overall survival, and low serum miR-147 expression level was an independent prognostic factor for poor prognosis for NSCLC. Conclusion Low serum miR-147 expression level may be a useful biomarker for patients with NSCLC. PMID:26581116

  20. Quantitative and qualitative analysis of small RNAs in human endothelial cells and exosomes provides insights into localized RNA processing, degradation and sorting

    NARCIS (Netherlands)

    van Balkom, Bas W M; Eisele, Almut S; Pegtel, D Michiel; Bervoets, Sander; Verhaar, Marianne C

    2015-01-01

    Exosomes are small vesicles that mediate cell-cell communication. They contain proteins, lipids and RNA, and evidence is accumulating that these molecules are specifically sorted for release via exosomes. We recently showed that endothelial-cell-produced exosomes promote angiogenesis in vivo in a sm

  1. Small RNAs derived from natural antisense transcripts%天然反义转录物(NAT)来源的小分子RNA

    Institute of Scientific and Technical Information of China (English)

    谢兆辉

    2009-01-01

    天然反义转录物(natural antisense transcript,NAT)通常指自然情况下生物体内生成的内源RNA,它们可以与其他RNA部分或完全互补.NAT在生物中非常普遍,并且可以产生多种具有调节作用的小RNA,如天然反义转录干扰小RNA、天然反义微RNA、长的十扰短RNA、扫描RNA和与Piwi相互作用的RNA等.这些小RNA或许是NAT调节基因表达的重要物质分子之一.本文就NAT来源的小RNA及其功能作一慨述.%Natural antisense transcripts (NATs) are endogenous RNA molecules that exhibit partial or complete complementarity to other RNAs. They are common in prokaryotes and eukaryotes, and can produce some small regulatory RNAs. Such as nat-small interfering RNAs (nat-siRNAs), nat-microRNAs (nat-miRNAs), long short interfering RNAs (lsiRNAs), scan RNA (scnRNAs) and PIWl-interacting RNAs (piRNAs). These small RNAs may be involved in the gene regulation by NATs, In this paper, small RNAs derived from NATs and their mechanisms are discussed.

  2. Mono-arginine Cholesterol-based Small Lipid Nanoparticles as a Systemic siRNA Delivery Platform for Effective Cancer Therapy.

    Science.gov (United States)

    Lee, Jinju; Saw, Phei Er; Gujrati, Vipul; Lee, Yonghyun; Kim, Hyungjun; Kang, Sukmo; Choi, Minsuk; Kim, Jae-Il; Jon, Sangyong

    2016-01-01

    Although efforts have been made to develop a platform carrier for the delivery of RNAi therapeutics, systemic delivery of siRNA has shown only limited success in cancer therapy. Cationic lipid-based nanoparticles have been widely used for this purpose, but their toxicity and undesired liver uptake after systemic injection owing to their cationic surfaces have hampered further clinical translation. This study describes the development of neutral, small lipid nanoparticles (SLNPs) made of a nontoxic cationic cholesterol derivative, as a suitable carrier of systemic siRNA to treat cancers. The cationic cholesterol derivative, mono arginine-cholesterol (MA-Chol), was synthesized by directly attaching an arginine moiety to cholesterol via a cleavable ester bond. siRNA-loaded SLNPs (siRNA@SLNPs) were prepared using MA-Chol and a neutral helper lipid, dioleoyl phosphatidylethanolamine (DOPE), as major components and a small amount of PEGylated phospholipid mixed with siRNA. The resulting nanoparticles were less than ~50 nm in diameter with neutral zeta potential and much lower toxicity than typical cationic cholesterol (DC-Chol)-based lipid nanoparticles. SLNPs loaded with siRNA against kinesin spindle protein (siKSP@SLNPs) exhibited a high level of target gene knockdown in various cancer cell lines, as shown by measurement of KSP mRNA and cell death assays. Furthermore, systemic injection of siKSP@SLNPs into prostate tumor-bearing mice resulted in preferential accumulation of the delivered siRNA at the tumor site and significant inhibition of tumor growth, with little apparent toxicity, as shown by body weight measurements. These results suggest that these SLNPs may provide a systemic delivery platform for RNAi-based cancer therapy.

  3. 番茄叶片小RNA提取方法的优化%Majorization of small RNA extraction methods in tomato leaf

    Institute of Scientific and Technical Information of China (English)

    陈华; 昌伟; 杨瑞; 王绍辉

    2012-01-01

    This experiment used tomato leaf to extract small RNA.UNIQ-10 Column Trizol Extraction Kit+LiCl method,a general Trizol+ LiClmethod and the improved Trizol + LiCl method were compared on the extraction effect.The results showed that the improved Trizol+LiCl method could extract high-quality small RNA from tomato leaves,Agarose gel electro-phoresis of total RNA showed clear and complete strips,nucleic acid protein detector measured the 1 026.8 ng/μL in concentration,2.00 for A260/A280 and 2.31 for A260/A230.15% polyacrylamide - 7 M urea gel electrophoresis showed clear small RNA strips area.The high-quality small RNA from tomato leaves by improved Trizol+LiCl method met the criterion for small RNA Northern blot and other molecular biology experiments.%以番茄叶片为材料提取小RNA,比较分析UNlQ-10柱式Trizol总RNA抽提试剂盒+LiC1法,常规Tr-izol+LiC1法和改进的Trizol法+LiC1法的提取效果.结果表明,改进的Trizol+ LiCl法能从番茄叶片中提取高质量的小RNA,总RNA琼脂糖凝胶电泳条带完整清晰,核酸蛋白测定仪显示RNA的浓度为1 026.8 ng/μL,A260/A280比值为2.00;A260/A230比值为2.31.15%聚丙烯酰胺—7 mol/L尿素凝胶电泳显示小RNA带型区域清晰.用该方法提取的高质量的番茄叶片小RNA符合RT-PCR,Northern blot等分子生物学试验的标准.

  4. Study on FSHR and LHR mRNA Levels of Different BMPRIB Genotypes of Small Tail Han Sheep During the Oestrum

    Institute of Scientific and Technical Information of China (English)

    JIA Cun-ling; LI Ning; WEI Ze-hui; ZHU Xiao-ping; LIU Hai-ying; JIA Zhi-hai

    2007-01-01

    The relationship between different BMPRIB genotypes of Small Tail Han sheep and FSHR and LHR mRNA levels during the oestrum was studied using semiquantitative PCR. The results indicated that FSHR mRNA extracted from the right ovary of BB (1.14 ± 0.11) ewes showed higher levels compared with AA (0.44 ± 0.11) and AB (0.36±0.08) ewes (P < 0.01), and LHR mRNA extracted from the right ovary of BB (0.42±0.02) ewes showed significantly higher levels compared with AA (0.23 ±0.02) and AB (0.25 ±0.04) ewes (P<0.01); however, the mRNA extracted from the left ovary showed no significant difference in levels among the genotypes during the oestrum. It indicated that the fecundity induced by a mutation of BMPRIB in Small Tail Han sheep may be related to the changes of the mRNA expression of LHR and FSHR in ovary.

  5. A mathematical model and quantitative comparison of the small RNA circuit in the Vibrio harveyi and Vibrio cholerae quorum sensing systems

    International Nuclear Information System (INIS)

    Quorum sensing is the process by which bacteria regulate their gene expression based on the local cell-population density. The quorum sensing systems of Vibrio harveyi and Vibrio cholerae are comprised of a phosphorelay cascade coupled to a small RNA (sRNA) circuit. The sRNA circuit contains multiple quorum regulated small RNA (Qrr) that regulate expression of the homologous master transcriptional regulators LuxR (in V. harveyi) and HapR (in V. cholerae). Their quorum sensing systems are topologically similar and homologous thereby making it difficult to understand why repression of HapR is more robust than LuxR to changes in Qrr. In this work we formulate and parameterize a novel mathematical model of the V. harveyi and V. cholerae sRNA circuit. We parameterize the model by fitting it to a variety of empirical data from both species. We show that we can distinguish all of the parameters and that the parameterizations (one for each species) are robust to errors in the data. We then use our model to propose some experiments to identify and explain kinetic differences between the species. We find that V. cholerae Qrr are more abundant and more sensitive to changes in LuxO than V. harveyi Qrr and argue that this is why expression of HapR is more robust than LuxR to changes in Qrr. (paper)

  6. A mathematical model and quantitative comparison of the small RNA circuit in the Vibrio harveyi and Vibrio cholerae quorum sensing systems

    Science.gov (United States)

    Hunter, G. A. M.; Guevara Vasquez, F.; Keener, J. P.

    2013-08-01

    Quorum sensing is the process by which bacteria regulate their gene expression based on the local cell-population density. The quorum sensing systems of Vibrio harveyi and Vibrio cholerae are comprised of a phosphorelay cascade coupled to a small RNA (sRNA) circuit. The sRNA circuit contains multiple quorum regulated small RNA (Qrr) that regulate expression of the homologous master transcriptional regulators LuxR (in V. harveyi) and HapR (in V. cholerae). Their quorum sensing systems are topologically similar and homologous thereby making it difficult to understand why repression of HapR is more robust than LuxR to changes in Qrr. In this work we formulate and parameterize a novel mathematical model of the V. harveyi and V. cholerae sRNA circuit. We parameterize the model by fitting it to a variety of empirical data from both species. We show that we can distinguish all of the parameters and that the parameterizations (one for each species) are robust to errors in the data. We then use our model to propose some experiments to identify and explain kinetic differences between the species. We find that V. cholerae Qrr are more abundant and more sensitive to changes in LuxO than V. harveyi Qrr and argue that this is why expression of HapR is more robust than LuxR to changes in Qrr.

  7. Small RNA Derived from the Virulence Modulating Region of the Potato spindle tuber viroid Silences callose synthase Genes of Tomato Plants.

    Science.gov (United States)

    Adkar-Purushothama, Charith Raj; Brosseau, Chantal; Giguère, Tamara; Sano, Teruo; Moffett, Peter; Perreault, Jean-Pierre

    2015-08-01

    The tomato (Solanum lycopersicum) callose synthase genes CalS11-like and CalS12-like encode proteins that are essential for the formation of callose, a major component of pollen mother cell walls; these enzymes also function in callose formation during pathogen infection. This article describes the targeting of these callose synthase mRNAs by a small RNA derived from the virulence modulating region of two Potato spindle tuber viroid variants. More specifically, viroid infection of tomato plants resulted in the suppression of the target mRNAs up to 1.5-fold, depending on the viroid variant used and the gene targeted. The targeting of these mRNAs by RNA silencing was validated by artificial microRNA experiments in a transient expression system and by RNA ligase-mediated rapid amplification of cDNA ends. Viroid mutants incapable of targeting callose synthase mRNAs failed to induce typical infection phenotypes, whereas a chimeric viroid obtained by swapping the virulence modulating regions of a mild and a severe variant of Potato spindle tuber viroid greatly affected the accumulation of viroids and the severity of disease symptoms. These data provide evidence of the silencing of multiple genes by a single small RNA derived from a viroid. PMID:26290537

  8. Evaluation of the mRNA and Protein Expressions of Nutritional Biomarkers in the Gastrointestinal Mucosa of Patients with Small Intestinal Disorders.

    Science.gov (United States)

    Nakamura, Masanao; Hirooka, Yoshiki; Watanabe, Osamu; Yamamura, Takeshi; Funasaka, Kohei; Ohno, Eizaburo; Miyahara, Ryoji; Kawashima, Hiroki; Shimoyama, Yoshie; Goto, Hidemi

    2016-01-01

    Objective The objectives of this study were to investigate the mRNA and protein expression of biomarkers related to absorption in the small intestinal mucosa of humans and determine the relationships between small intestinal diseases and nutrition. Methods The study subjects consisted of patients scheduled to undergo double-balloon endoscopy (DBE) or total colonoscopy for suspected gastrointestinal disorder in a clinical practice. Biopsies were taken from apparently normal mucosa in the visible areas of 6 parts of the intestines from the duodenum to the colon. The mRNA expression of specific biomarkers (SGLT1, SGLT5, GIP, GLP, LAT1, LAT2, and NPC1L1) in the mucosa was compared among three patient groups: Inflammation, Tumor, and Control. Results Sixty-six patients participated in this study. Both routes of DBE were performed in 20 patients, in whom biopsy samples were obtained from the mucosa for all sections. There were no remarkable differences in the mRNA expression levels among the 3 groups. However, SGLT1, GIP, GLP, and NPC1L1 exhibited specific distribution patterns. The expression levels of GIP and NPC1L1 were highest in the upper jejunum, but were extremely low in the terminal ileum and colon. A comparison of the mRNA expression profile in each intestinal section revealed that the SGLT1 mRNA expression in the Tumor group and the GIP mRNA expression in the Inflammation group were significantly higher than the corresponding levels in the Control group in the upper jejunum. Conclusion The gastrointestinal mucosa of patients with small bowel diseases can maintain proper nutrient absorption, except in the upper jejunum. PMID:27522989

  9. Analysis of U3 snoRNA and small subunit processome components in the parasitic protist Entamoeba histolytica.

    Science.gov (United States)

    Srivastava, Ankita; Ahamad, Jamaluddin; Ray, Ashwini Kumar; Kaur, Devinder; Bhattacharya, Alok; Bhattacharya, Sudha

    2014-02-01

    In the early branching parasitic protist Entamoeba histolytica, pre-rRNA synthesis continues when cells are subjected to growth stress, but processing slows down and unprocessed pre-rRNA accumulates. To gain insight into the regulatory mechanisms leading to accumulation, it is necessary to define the pre-rRNA processing machinery in E. histolytica. We searched the E. histolytica genome sequence for homologs of the SSU processome, which contains the U3snoRNA, and 72 proteins in yeast. We could identify 57 of the proteins with high confidence. Of the rest, 6 were absent in human, and 4 were non-essential in yeast. The remaining 5 were absent in other parasite genomes as well. Analysis of U3snoRNA showed that the E. histolytica U3snoRNA adopted the same conserved secondary structure as seen in yeast and human. The predicted structure was verified by chemical modification followed by primer extension (SHAPE). Further we showed that the predicted interactions of Eh_U3snoRNA boxes A and A' with pre-18S rRNA were highly conserved both in position and sequence. The predicted interactions of 5'-hinge and 3'-hinge sequences of Eh_U3 snoRNA with the 5'-ETS sequences were conserved in position but not in sequence. Transcription of selected genes of SSU processome was tested by northern analysis, and transcripts of predicted sizes were obtained. During serum starvation, when unprocessed pre-RNA accumulated, the transcript levels of some of these genes declined. This is the first report on pre-rRNA processing machinery in E. histolytica, and shows that the components are well conserved with respect to yeast and human.

  10. Differential mRNA Accumulation upon Early Arabidopsis thaliana Infection with ORMV and TMV-Cg Is Associated with Distinct Endogenous Small RNAs Level.

    Directory of Open Access Journals (Sweden)

    Diego Zavallo

    Full Text Available Small RNAs (sRNAs play important roles in plant development and host-pathogen interactions. Several studies have highlighted the relationship between viral infections, endogenous sRNA accumulation and transcriptional changes associated with symptoms. However, few studies have described a global analysis of endogenous sRNAs by comparing related viruses at early stages of infection, especially before viral accumulation reaches systemic tissues. An sRNA high-throughput sequencing of Arabidopsis thaliana leaf samples infected either with Oilseed rape mosaic virus (ORMV or crucifer-infecting Tobacco mosaic virus (TMV-Cg with slightly different symptomatology at two early stages of infection (2 and 4 dpi was performed. At early stages, both viral infections strongly alter the patterns of several types of endogenous sRNA species in distal tissues with no virus accumulation suggesting a systemic signaling process foregoing to virus spread. A correlation between sRNAs derived from protein coding genes and the associated mRNA transcripts was also detected, indicating that an unknown recursive mechanism is involved in a regulatory circuit encompassing this sRNA/mRNA equilibrium. This work represents the initial step in uncovering how differential accumulation of endogenous sRNAs contributes to explain the massive alteration of the transcriptome associated with plant-virus interactions.

  11. Phylogenetic positions of two marine ciliates, Metanophrys similis and Pseudocohnilembus hargisi (Protozoa, Ciliophora, Scuticociliatia), inferred from complete small subunit rRNA gene sequences

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    The small subunit rRNA (SSrRNA) gene was sequenced for two marine scuticociliates Metanophrys similis and Pseudocohnilembus hargisi. The results show that this gene comprises 1763 and 1753 nucleotides in the two marine ciliates respectively.Metanophrys similis is phylogenetically closely related to the clade containing Mesanophrys carcini and Anophyroides haemophila, which branches basally to other species within the order Philasterida. Pseudocohnilembus hargisi groups with its congener, P. marinus, with strong bootstrap support. Paranophrys magna groups with the clade including Cohnilembus and Uronema, representing a sister clade to that containing the two Pseudocohnilembus species.

  12. Characterization of a small auxin-up RNA (SAUR-like gene involved in Arabidopsis thaliana development.

    Directory of Open Access Journals (Sweden)

    Marios Nektarios Markakis

    Full Text Available The root of Arabidopsis thaliana is used as a model system to unravel the molecular nature of cell elongation and its arrest. From a micro-array performed on roots that were treated with aminocyclopropane-1-carboxylic acid (ACC, the precursor of ethylene, a Small auxin-up RNA (SAUR-like gene was found to be up regulated. As it appeared as the 76th gene in the family, it was named SAUR76. Root and leaf growth of overexpression lines ectopically expressing SAUR76 indicated the possible involvement of the gene in the division process. Using promoter::GUS and GFP lines strong expression was seen in endodermal and pericycle cells at the end of the elongation zone and during several stages of lateral root primordia development. ACC and IAA/NAA were able to induce a strong up regulation of the gene and changed the expression towards cortical and even epidermal cells at the beginning of the elongation zone. Confirmation of this up regulation of expression was delivered using qPCR, which also indicated that the expression quickly returned to normal levels when the inducing IAA-stimulus was removed, a behaviour also seen in other SAUR genes. Furthermore, confocal analysis of protein-GFP fusions localized the protein in the nucleus, cytoplasm and plasma membrane. SAUR76 expression was quantified in several mutants in ethylene and auxin-related pathways, which led to the conclusion that the expression of SAUR76 is mainly regulated by the increase in auxin that results from the addition of ACC, rather than by ACC itself.

  13. AB094. High-throughput sequencing of small RNA component of penile in a post-radical prostatectomy model of erectile dysfunction

    Science.gov (United States)

    Ruan, Yajun; Luan, Yang; Zhang, Yan; Li, Hao; Li, Rui; Cui, Kai; Jiang, Hongyang; Li, Mingchao; Wang, Tao; Liu, Jihong

    2016-01-01

    Objective The introduction of nerve-sparing radical prostatectomy represents a milestone in the treatment of prostate cancer. However, a certain percentage of cancer survivors still suffer from erectile dysfunction. Recent research has stated that using PDE 5-inhibitors after radical prostatectomy may lead to biochemical recurrence. This study was performed to identify the expression profile of small RNA in rats with neurogenic erectile dysfunction, and to investigate possible genes and signaling pathways involving in the disease. Methods Neurogenic erectile dysfunction (ED) was induced in male rats by bilateral cavernous nerve crushing injury (BCNI). After 28 days, erectile function was evaluated by cavernous nerve electrostimulation. Masson’s trichrome staining was performed to assess histologic changes. RNA was isolated from the corpus cavernosum (CC) of both control rats and neurogenic ED rats. Small RNA sequencing was conducted using an Illumina Hiseq 2,500/2,000 platform. Candidate small RNAs were validated by real-time polymerase chain reaction. Results Intracavernous pressure (ICP) was significantly decreased in BCNI group compared with SHAM group. Corporal tissue in the neurogenic ED rats showed a significantly lower smooth muscle/collagen ratio compared with tissue in the SHAM controls. Real time PCR validated that miR-9a-5p, miR-203a-5p, miR-378a-3p and miR-3557-5p were upregulated, and meanwhile miR-3084a-3p was downregulated. Conclusions Small RNA, including microRNA, may play an important role in the regulation of genes in CC and some certain miRs may participate in post-prostatectomy ED. Further studies will be designed to investigate the specific mechanisms of these changes.

  14. Transfer RNA Derived Small RNAs Targeting Defense Responsive Genes Are Induced during Phytophthora capsici Infection in Black Pepper (Piper nigrum L.).

    Science.gov (United States)

    Asha, Srinivasan; Soniya, Eppurath V

    2016-01-01

    Small RNAs derived from transfer RNAs were recently assigned as potential gene regulatory candidates for various stress responses in eukaryotes. In this study, we report on the cloning and identification of tRNA derived small RNAs from black pepper plants in response to the infection of the quick wilt pathogen, Phytophthora capsici. 5'tRFs cloned from black pepper were validated as highly expressed during P. capsici infection. A high-throughput systematic analysis of the small RNAome (sRNAome) revealed the predominance of 5'tRFs in the infected leaf and root. The abundance of 5'tRFs in the sRNAome and the defense responsive genes as their potential targets indicated their regulatory role during stress response in black pepper. The 5'Ala(CGC) tRF mediated cleavage was experimentally mapped at the tRF binding sites on the mRNA targets of Non-expresser of pathogenesis related protein (NPR1), which was down-regulated during pathogen infection. Comparative sRNAome further demonstrated sequence conservation of 5'Ala tRFs across the angiosperm plant groups, and many important genes in the defense response were identified in silico as their potential targets. Our findings uncovered the diversity, differential expression and stress responsive functional role of tRNA-derived small RNAs during Phytophthora infection in black pepper. PMID:27313593

  15. Transfer RNA Derived Small RNAs Targeting Defense Responsive Genes Are Induced during Phytophthora capsici Infection in Black Pepper (Piper nigrum L.).

    Science.gov (United States)

    Asha, Srinivasan; Soniya, Eppurath V

    2016-01-01

    Small RNAs derived from transfer RNAs were recently assigned as potential gene regulatory candidates for various stress responses in eukaryotes. In this study, we report on the cloning and identification of tRNA derived small RNAs from black pepper plants in response to the infection of the quick wilt pathogen, Phytophthora capsici. 5'tRFs cloned from black pepper were validated as highly expressed during P. capsici infection. A high-throughput systematic analysis of the small RNAome (sRNAome) revealed the predominance of 5'tRFs in the infected leaf and root. The abundance of 5'tRFs in the sRNAome and the defense responsive genes as their potential targets indicated their regulatory role during stress response in black pepper. The 5'Ala(CGC) tRF mediated cleavage was experimentally mapped at the tRF binding sites on the mRNA targets of Non-expresser of pathogenesis related protein (NPR1), which was down-regulated during pathogen infection. Comparative sRNAome further demonstrated sequence conservation of 5'Ala tRFs across the angiosperm plant groups, and many important genes in the defense response were identified in silico as their potential targets. Our findings uncovered the diversity, differential expression and stress responsive functional role of tRNA-derived small RNAs during Phytophthora infection in black pepper.

  16. Enhancement of Gene Silencing Effect and Membrane Permeability by Peptide-Conjugated 27-Nucleotide Small Interfering RNA

    Directory of Open Access Journals (Sweden)

    Toshio Seyama

    2012-09-01

    Full Text Available Two different sizes of siRNAs, of which one type was 21-nucleotide (nt siRNA containing 2-nt dangling ends and the other type was 27-nt siRNA with blunt ends, were conjugated with a nuclear export signal peptide of HIV-1 Rev at the 5′-sense end. Processing by Dicer enzyme, cell membrane permeability, and RNAi efficiency of the peptide-conjugated siRNAs were examined. Dicer cleaved the peptide-conjugated 27-nt siRNA leading to the release of 21-nt siRNA, whereas the peptide-conjugated 21-nt siRNA was not cleaved. High membrane permeability and cytoplasmic localization was found in the conjugates. Moreover, the peptide-conjugated 27-nt siRNA showed increased potency of RNAi in comparison with the nonmodified 21-nt and 27-nt siRNAs, whereas the peptide-conjugated 21-nt siRNA showed decreased RNAi efficacy. This potent RNAi efficacy is probably owing to acceleration of RISC through recognition by Dicer, as well as to the improvement of cell membrane permeability and intracellular accumulation.

  17. Establishment of a rapid, inexpensive protocol for extraction of high quality RNA from small amounts of strawberry plant tissues and other recalcitrant fruit crops.

    Science.gov (United States)

    Christou, Anastasis; Georgiadou, Egli C; Filippou, Panagiota; Manganaris, George A; Fotopoulos, Vasileios

    2014-03-01

    Strawberry plant tissues and particularly fruit material are rich in polysaccharides and polyphenolic compounds, thus rendering the isolation of nucleic acids a difficult task. This work describes the successful modification of a total RNA extraction protocol, which enables the isolation of high quantity and quality of total RNA from small amounts of strawberry leaf, root and fruit tissues. Reverse-transcription polymerase chain reaction (RT-PCR) amplification of GAPDH housekeeping gene from isolated RNA further supports the proposed protocol efficiency and its use for downstream molecular applications. This novel procedure was also successfully followed using other fruit tissues, such as olive and kiwifruit. In addition, optional treatment with RNase A following initial nucleic acid extraction can provide sufficient quality and quality of genomic DNA for subsequent PCR analyses, as evidenced from PCR amplification of housekeeping genes using extracted genomic DNA as template. Overall, this optimized protocol allows easy, rapid and economic isolation of high quality RNA from small amounts of an important fruit crop, such as strawberry, with extended applicability to other recalcitrant fruit crops.

  18. Small interfering RNAs targeting peste des petits ruminants virus M mRNA increase virus-mediated fusogenicity and inhibit viral replication in vitro.

    Science.gov (United States)

    Liu, Fuxiao; Wu, Xiaodong; Zou, Yanli; Li, Lin; Liu, Shan; Chi, Tianying; Wang, Zhiliang

    2015-11-01

    Peste des petits ruminants (PPR), caused by peste des petits ruminants virus (PPRV), is an acute or subacute, highly contagious and economically important disease of small ruminants. The PPRV is classified into the genus Morbillivirus in the family Paramyxoviridae. The PPRV matrix (M) protein possesses an intrinsic ability to bind to lipid membranes, and plays a crucial role in viral assembly and further budding. In this study, three different small interfering RNAs (siRNA) were designed on the basis of translated region for PPRV Nigeria 75/1M mRNA, and were subsequently synthesized for their transfection into Vero-SLAM cells, followed by infection with PPRVs. The results showed that two out of three siRNAs robustly induced cell-to-cell fusion as early as 36h post-infection with PPRVs, effectively suppressed expression of the M protein by interference for the M mRNA, and eventually inhibited viral replication in vitro. These findings led us to speculate that siRNA-mediated knockdown of the M protein would alter its interaction with viral glycoproteins, thus exacerbating intercellular fusion but hampering virus release. PMID:26318517

  19. A motif-based search in bacterial genomes identifies the ortholog of the small RNA Yfr1 in all lineages of cyanobacteria

    Directory of Open Access Journals (Sweden)

    Axmann Ilka M

    2007-10-01

    Full Text Available Abstract Background Non-coding RNAs (ncRNA are regulators of gene expression in all domains of life. They control growth and differentiation, virulence, motility and various stress responses. The identification of ncRNAs can be a tedious process due to the heterogeneous nature of this molecule class and the missing sequence similarity of orthologs, even among closely related species. The small ncRNA Yfr1 has previously been found in the Prochlorococcus/Synechococcus group of marine cyanobacteria. Results Here we show that screening available genome sequences based on an RNA motif and followed by experimental analysis works successfully in detecting this RNA in all lineages of cyanobacteria. Yfr1 is an abundant ncRNA between 54 and 69 nt in size that is ubiquitous for cyanobacteria except for two low light-adapted strains of Prochlorococcus, MIT 9211 and SS120, in which it must have been lost secondarily. Yfr1 consists of two predicted stem-loop elements separated by an unpaired sequence of 16–20 nucleotides containing the ultraconserved undecanucleotide 5'-ACUCCUCACAC-3'. Conclusion Starting with an ncRNA previously found in a narrow group of cyanobacteria only, we show here the highly specific and sensitive identification of its homologs within all lineages of cyanobacteria, whereas it was not detected within the genome sequences of E. coli and of 7 other eubacteria belonging to the alpha-proteobacteria, chlorobiaceae and spirochaete. The integration of RNA motif prediction into computational pipelines for the detection of ncRNAs in bacteria appears as a promising step to improve the quality of such predictions.

  20. In vivo study of Dicer-2-mediated immune response of the small interfering RNA pathway upon systemic infections of virulent and avirulent viruses in Bombus terrestris.

    Science.gov (United States)

    Niu, Jinzhi; Smagghe, Guy; De Coninck, Dieter I M; Van Nieuwerburgh, Filip; Deforce, Dieter; Meeus, Ivan

    2016-03-01

    Recent studies suggest a potent role of the small interfering RNA (siRNA) pathway in the control of bee viruses and its usefulness to tackle these viral diseases. However, the involvement of the siRNA pathway in the defense against different bee viruses is still poorly understood. Therefore, in this report, we comprehensively analyzed the response of the siRNA pathway in bumblebees of Bombus terrestris to systemic infections of the virulent Israeli acute paralysis virus (IAPV) and the avirulent slow bee paralysis virus (SBPV). Our results showed that IAPV and SBPV infections induced the expression of Dicer-2. IAPV infections also triggered the production of predominantly 22 nt-long virus-derived siRNAs (vsiRNAs). Intriguingly, these 22 nt-long vsiRNAs showed a high proportion of antigenomic IAPV sequences. Conversely, these predominantly 22 nt-long vsiRNAs of SBPV were not detected in SBPV infected bees. Furthermore, an "RNAi-of-RNAi" experiment on Dicer-2 did not result in altered genome copy numbers of IAPV (n = 17-18) and also not of SBPV (n = 11-12). Based on these results, we can speculate about the importance of the siRNA pathway in bumblebees for the antiviral response. During infection of IAPV, this pathway is probably recruited but it might be insufficient to control viral infection in our experimental setup. The host can control SBPV infection, but aside from the induction of Dicer-2 expression, no further evidence of the antiviral activity of the siRNA pathway was observed. This report may also enhance the current understanding of the siRNA pathway in the innate immunity of non-model insects upon different viral infections. PMID:26711439

  1. Rapid, single-tube method for quantitative preparation and analysis of RNA and DNA in samples as small as one cell

    Directory of Open Access Journals (Sweden)

    Anshelevich Aleksandra

    2005-01-01

    Full Text Available Abstract Background Current methods for accurate quantification of nucleic acids typically begin with a template preparation step in which DNA and/or RNA are freed of bound proteins and are then purified. Isolation of RNA is particularly challenging because this molecule is sensitive to elevated temperatures and is degraded by RNases, which therefore have to be immediately inactivated upon cell lysis. Many protocols for nucleic acids purification, reverse transcription of RNA and/or amplification of DNA require repeated transfers from tube to tube and other manipulations during which materials may be lost. Results This paper introduces a novel and highly reliable single-tube method for rapid cell lysis, followed by quantitative preparation and analysis of both RNA and/or DNA molecules in small samples. In contrast to previous approaches, this procedure allows all steps to be carried out by sequential dilution in a single tube, without chemical extraction or binding to a matrix. We demonstrate the utility of this method by quantification of four genes, Xist, Sry and the two heat-inducible hsp70i (hsp70.1 and hsp70.3, as well as their RNA transcripts in single mouse embryos and in isolated blastomeres. Conclusion This method virtually eliminates losses of nucleic acids and is sensitive and accurate down to single molecules.

  2. The role and potential mechanisms of LncRNA-TATDN1 on metastasis and invasion of non-small cell lung cancer.

    Science.gov (United States)

    Zequn, Niu; Xuemei, Zhang; Wei, Li; Zongjuan, Ming; Yujie, Zhong; Yanli, Hou; Yuping, Zhang; Xia, Meng; Wei, Wang; Wenjing, Deng; Na, Fan; Shuanying, Yang

    2016-04-01

    The invasion and metastasis of malignant tumor cells lead to normal tissue destruction and are major prognostic factors for many malignant cancers. Long non-coding RNA (LncRNA) is associated with occurrence, development and prognoses of non-small cell lung cancer (NSCLC), but its mechanisms of action involved in tumor invasion and metastasis are not clear. In this study, we screened and detected the expression of LncRNA in two NSCLC lines 95D and 95C by using high throughput LncRNA chip. We found that TATDN1 (Homo sapiens TatD DNase domain containing 1, TATDN1), one of LncRNAs, was highly expressed in 95D cells and NSCLC tumor tissues compared to 95C cells. Knockdown of TATDN1-1 by shRNA significantly inhibited cell proliferation, adhesion, migration and invasion in 95D cells. Further mechanism study showed that TATDN1 knockdown suppressed the expression of E-cadherin, HER2, β-catenin and Ezrin. Moreover, knockdown TATDN1 also inhibited tumor growth and metastasis in a 95D mouse model in vivo by inhibiting β-catenin and Ezrin. These data indicate that TATDN1 expression is associated with 95D cells' higher potential of invasion and metastasis, and suggest that TATDN1 may be a potential prognostic factor and therapeutic target for NSCLCs. PMID:26943769

  3. Genome-wide small RNA analysis of soybean reveals auxin-responsive microRNAs that are differentially expressed in response to salt stress in root apex

    Directory of Open Access Journals (Sweden)

    Zhengxi eSun

    2016-01-01

    Full Text Available Root growth and the architecture of the root system in Arabidopsis are largely determined by root meristematic activity. Legume roots show strong developmental plasticity in response to both abiotic and biotic stimuli, including symbiotic rhizobia. However, a global analysis of gene regulation in the root meristem of soybean plants is lacking. In this study, we performed a global analysis of the small RNA transcriptome of root tips from soybean seedlings grown under normal and salt stress conditions. In total, 71 miRNA candidates, including known and novel variants of 59 miRNA families, were identified. We found 66 salt-responsive miRNAs in the soybean root meristem; among them, 22 are novel miRNAs. Interestingly, we found auxin-responsive cis-elements in the promoters of many salt-responsive miRNAs, implying that these miRNAs may be regulated by auxin and auxin signaling plays a key role in regulating the plasticity of the miRNAome and root development in soybean. A functional analysis of miR399, a salt-responsive miRNA in the root meristem, indicates the crucial role of this miRNA in modulating soybean root developmental plasticity. Our data provide novel insight into the miRNAome-mediated regulatory mechanism in soybean root growth under salt stress.

  4. Genome-Wide Small RNA Analysis of Soybean Reveals Auxin-Responsive microRNAs that are Differentially Expressed in Response to Salt Stress in Root Apex

    Science.gov (United States)

    Sun, Zhengxi; Wang, Youning; Mou, Fupeng; Tian, Yinping; Chen, Liang; Zhang, Senlei; Jiang, Qiong; Li, Xia

    2016-01-01

    Root growth and the architecture of the root system in Arabidopsis are largely determined by root meristematic activity. Legume roots show strong developmental plasticity in response to both abiotic and biotic stimuli, including symbiotic rhizobia. However, a global analysis of gene regulation in the root meristem of soybean plants is lacking. In this study, we performed a global analysis of the small RNA transcriptome of root tips from soybean seedlings grown under normal and salt stress conditions. In total, 71 miRNA candidates, including known and novel variants of 59 miRNA families, were identified. We found 66 salt-responsive miRNAs in the soybean root meristem; among them, 22 are novel miRNAs. Interestingly, we found auxin-responsive cis-elements in the promoters of many salt-responsive miRNAs, implying that these miRNAs may be regulated by auxin and auxin signaling plays a key role in regulating the plasticity of the miRNAome and root development in soybean. A functional analysis of miR399, a salt-responsive miRNA in the root meristem, indicates the crucial role of this miRNA in modulating soybean root developmental plasticity. Our data provide novel insight into the miRNAome-mediated regulatory mechanism in soybean root growth under salt stress. PMID:26834773

  5. MicroRNA-196a promotes non-small cell lung cancer cell proliferation and invasion through targeting HOXA5

    International Nuclear Information System (INIS)

    MicroRNAs (miRNAs) are short, non-coding RNAs (~22 nt) that play important roles in the pathogenesis of human diseases by negatively regulating gene expression. Although miR-196a has been implicated in several other cancers, its role in non-small cell lung cancer (NSCLC) is unknown. The aim of the present study was to examine the expression pattern of miR-196a in NSCLC and its clinical significance, as well as its biological role in tumor progression. Expression of miR-196a was analyzed in 34 NSCLC tissues and five NSCLC cell lines by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The effect of DNA methylation on miR-196a expression was investigated by 5-aza-2-deoxy-cytidine treatment and bisulfite sequencing. The effect of miR-196a on proliferation was evaluated by MTT and colony formation assays, and cell migration and invasion were evaluated by transwell assays. Analysis of target protein expression was determined by western blotting. Luciferase reporter plasmids were constructed to confirm the action of miR-196a on downstream target genes, including HOXA5. Differences between the results were tested for significance using Student’s t-test (two-tailed). miR-196a was highly expressed both in NSCLC samples and cell lines compared with their corresponding normal counterparts, and the expression of miR-196a may be affected by DNA demethylation. Higher expression of miR-196a in NSCLC tissues was associated with a higher clinical stage, and also correlated with NSCLC lymph-node metastasis. In vitro functional assays demonstrated that modulation of miR-196a expression affected NSCLC cell proliferation, migration and invasion. Our analysis showed that miR-196a suppressed the expression of HOXA5 both at the mRNA and protein levels, and luciferase assays confirmed that miR-196a directly bound to the 3’untranslated region of HOXA5. Knockdown of HOXA5 expression in A549 cells using RNAi was shown to promote NSCLC cell proliferation, migration

  6. Congruent Phylogenies of Most Common Small-Subunit rRNA and Dissimilatory Sulfite Reductase Gene Sequences Retrieved from Estuarine Sediments

    OpenAIRE

    Joulian, Catherine; Ramsing, Niels B.; Ingvorsen, Kjeld

    2001-01-01

    The diversity of sulfate-reducing bacteria (SRB) in brackish sediment was investigated using small-subunit rRNA and dissimilatory sulfite reductase (DSR) gene clone libraries and cultivation. The phylogenetic affiliation of the most commonly retrieved clones for both genes was strikingly similar and produced Desulfosarcina variabilis-like sequences from the inoculum but Desulfomicrobium baculatum-like sequences from a high dilution in natural media. Related organisms were subsequently cultiva...

  7. Sensitive PCR diagnosis of Infections by Enterocytozoon bieneusi (microsporidia) using primers based on the region coding for small-subunit rRNA.

    OpenAIRE

    DA SILVA, A. J.; Schwartz, D A; Visvesvara, G S; De Moura, H; Slemenda, S B; Pieniazek, N J

    1996-01-01

    Enterocytozoon bieneusi is the most common microsporidian infecting patients with AIDS. We have developed a PCR primer pair, named EBIEF1/EBIER1, based on the small-subunit rRNA sequence of this microsporidian. Compared with other PCR-based methods, this primer pair shows a higher efficiency of detection in diagnostic applications than does another previously described primer pair, V1/EB450.

  8. Small-RNA deep sequencing reveals Arctium tomentosum as a natural host of Alstroemeria virus X and a new putative Emaravirus.

    Directory of Open Access Journals (Sweden)

    Yaqi Bi

    Full Text Available BACKGROUND: Arctium species (Asteraceae are distributed worldwide and are used as food and rich sources of secondary metabolites for the pharmaceutical industry, e.g., against avian influenza virus. RNA silencing is an antiviral defense mechanism that detects and destroys virus-derived double-stranded RNA, resulting in accumulation of virus-derived small RNAs (21-24 nucleotides that can be used for generic detection of viruses by small-RNA deep sequencing (SRDS. METHODOLOGY/PRINCIPAL FINDINGS: SRDS was used to detect viruses in the biennial wild plant species Arctium tomentosum (woolly burdock; family Asteraceae displaying virus-like symptoms of vein yellowing and leaf mosaic in southern Finland. Assembly of the small-RNA reads resulted in contigs homologous to Alstroemeria virus X (AlsVX, a positive/single-stranded RNA virus of genus Potexvirus (family Alphaflexiviridae, or related to negative/single-stranded RNA viruses of the genus Emaravirus. The coat protein gene of AlsVX was 81% and 89% identical to the two AlsVX isolates from Japan and Norway, respectively. The deduced, partial nucleocapsid protein amino acid sequence of the emara-like virus was only 78% or less identical to reported emaraviruses and showed no variability among the virus isolates characterized. This virus--tentatively named as Woolly burdock yellow vein virus--was exclusively associated with yellow vein and leaf mosaic symptoms in woolly burdock, whereas AlsVX was detected in only one of the 52 plants tested. CONCLUSIONS/SIGNIFICANCE: These results provide novel information about natural virus infections in Acrtium species and reveal woolly burdock as the first natural host of AlsVX besides Alstroemeria (family Alstroemeriaceae. Results also revealed a new virus related to the recently emerged Emaravirus genus and demonstrated applicability of SRDS to detect negative-strand RNA viruses. SRDS potentiates virus surveys of wild plants, a research area underrepresented in

  9. Genetic characterization and phylogenetic relationships based on 18S rRNA and ITS1 region of small form of canine Babesia spp. from India.

    Science.gov (United States)

    Mandal, M; Banerjee, P S; Garg, Rajat; Ram, Hira; Kundu, K; Kumar, Saroj; Kumar, G V P P S Ravi

    2014-10-01

    Canine babesiosis is a vector borne disease caused by intra-erythrocytic apicomplexan parasites Babesia canis (large form) and Babesia gibsoni (small form), throughout the globe. Apart from few sporadic reports on the occurrence of B. gibsoni infection in dogs, no attempt has been made to characterize Babesia spp. of dogs in India. Fifteen canine blood samples, positive for small form of Babesia, collected from northern to eastern parts of India, were used for amplification of 18S rRNA gene (∼1665bp) of Babesia sp. and partial ITS1 region (∼254bp) of B. gibsoni Asian genotype. Cloning and sequencing of the amplified products of each sample was performed separately. Based on sequences and phylogenetic analysis of 18S rRNA and ITS1 sequences, 13 were considered to be B. gibsoni. These thirteen isolates shared high sequence identity with each other and with B. gibsoni Asian genotype. The other two isolates could not be assigned to any particular species because of the difference(s) in 18S rRNA sequence with B. gibsoni and closer identity with Babesiaoccultans and Babesiaorientalis. In the phylogenetic tree, all the isolates of B. gibsoni Asian genotype formed a separate major clade named as Babesia spp. sensu stricto clade with high bootstrap support. The two unnamed Babesia sp. (Malbazar and Ludhiana isolates) clustered close together with B. orientalis, Babesia sp. (Kashi 1 isolate) and B. occultans of bovines. It can be inferred from this study that 18S rRNA gene and ITS1 region are highly conserved among 13 B. gibsoni isolates from India. It is the maiden attempt of genetic characterization by sequencing of 18S rRNA gene and ITS1 region of B. gibsoni from India and is also the first record on the occurrence of an unknown Babesia sp. of dogs from south and south-east Asia.

  10. Nuclear Factor-κB Signaling Pathway Constitutively Activated in Esophageal Squamous Cell Carcinoma Cell Lines and Inhibition of Growth of Cells by Small Interfering RNA

    Institute of Scientific and Technical Information of China (English)

    Fang TIAN; Wei-Dong ZANG; Wei-Hong HOU; Hong-Tao LIU; Le-Xun XUE

    2006-01-01

    Although constitutive nuclear factor (NF)-κB activation has been reported in many human tumors, the role of the NF-κB pathway in esophageal squamous cell carcinoma (ESCC) has not been known.In this study, NF-κB pathway in two ESCC cell lines was investigated using immunocytochemistry, Western blot and reverse transcription-polymerase chain reaction. The activation of NF-κB DNA binding was determined by electrophoretic mobility-shift assay. RNA interference was used to specifically inhibit the expression of p65. Growth of cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.The results showed that p50, p65, Iκ Bα, p-Iκ Bα and Iκ B kinase β were expressed and mainly localized in the cytoplasm. Reverse transcription-polymerase chain reaction results showed the constitutive expressions of p50, p65 and Iκ Bα mRNA in the two ESCC cell lines. Furthermore, the nuclear extracts revealed that p50 and p65 translocated to the nucleus had DNA-binding activity. Finally, small interfering RNA of p65 decreased the expression of p65, and the viability of cells transfected with p65 small interfering RNA was significantly suppressed at the same concentration of 5-fluorouracil (P<0.05) compared to untransfected cells. The results of this study showed that there was the constitutively activated NF-κB signaling pathway in the ESCC cell lines. RNA interference targeting at p65 increased the sensitivity of the ESCC cell lines to 5-fluorouracil,suggesting that NF-κB might be a good target for cancer treatment.

  11. Diagnostic utility of LunX mRNA in peripheral blood and pleural fluid in patients with primary non-small cell lung cancer

    Directory of Open Access Journals (Sweden)

    Tian Zhigang

    2008-05-01

    Full Text Available Abstract Background Progress in lung cancer is hampered by the lack of clinically useful diagnostic markers. The goal of this study was to provide a detailed evaluation of lung cancer tumor markers indicative of molecular abnormalities and to assess their diagnostic utility in non-small cell lung cancer (NSCLC patients. Methods Quantitative real-time RT-PCR was used to determine LunX, CK19, CEA, VEGF-C and hnRNP A2/B1 mRNA levels in peripheral blood and pleural fluid from NSCLC patients, compared with those from patients with other epithelial cancer (esophagus cancer and breast cancer, benign lung disease (pneumonia and tuberculo pleurisy and from healthy volunteers. Results In peripheral blood LunX mRNA was detectable in 75.0% (33/44 of patients with NSCLC, but not in patients with other epithelial cancer (0/28, benign lung disease (0/10 or in healthy volunteers (0/15. In contrast, all other genetic markers were detected in patients with either NSCLC, other epithelia cancer or benign lung disease, and in healthy volunteers. The expression level and positive rate of LunX mRNA in peripheral blood correlated with the pathologic stage of NSCLC (P LunX mRNA was detected in 92.9% (13/14 of malignant pleural fluid samples and was the only marker whose expression level was significantly different between malignant and benign pleural fluid (P LunX mRNA in the peripheral blood of NSCLC patients decreased shortly after clinical treatment (P = 0.005. Conclusion Of several commonly used genetic markers, LunX mRNA is the most specific gene marker for lung cancer and has potential diagnostic utility when measured in the peripheral blood and pleural fluid of NSCLC patients.

  12. Suppression of non-small cell lung tumor development by the let-7 microRNA family

    NARCIS (Netherlands)

    M.S. Kumar (Madhu); S.J. Erkeland (Stefan); R.E. Pester (Ryan); C.Y. Chen (Cindy); M.S. Ebert (Margaret); P.A. Sharp (Phillip); T. Jacks (Tyler)

    2008-01-01

    textabstractMany microRNAs (miRNAs) target mRNAs involved in processes aberrant in tumorigenesis, such as proliferation, survival, and differentiation. In particular, the let-7 miRNA family has been proposed to function in tumor suppression, because reduced expression of let-7 family members is comm

  13. A versatile method to design stem-loop primer-based quantitative PCR assays for detecting small regulatory RNA molecules.

    Directory of Open Access Journals (Sweden)

    Zsolt Czimmerer

    Full Text Available Short regulatory RNA-s have been identified as key regulators of gene expression in eukaryotes. They have been involved in the regulation of both physiological and pathological processes such as embryonal development, immunoregulation and cancer. One of their relevant characteristics is their high stability, which makes them excellent candidates for use as biomarkers. Their number is constantly increasing as next generation sequencing methods reveal more and more details of their synthesis. These novel findings aim for new detection methods for the individual short regulatory RNA-s in order to be able to confirm the primary data and characterize newly identified subtypes in different biological conditions. We have developed a flexible method to design RT-qPCR assays that are very sensitive and robust. The newly designed assays were tested extensively in samples from plant, mouse and even human formalin fixed paraffin embedded tissues. Moreover, we have shown that these assays are able to quantify endogenously generated shRNA molecules. The assay design method is freely available for anyone who wishes to use a robust and flexible system for the quantitative analysis of matured regulatory RNA-s.

  14. Highly Efficient Transfection of Small Interfering RNA into Murine Primary Chondrocytes via Optimized Electroporation%电穿孔法介导 siRNA 高效转染小鼠原代软骨细胞

    Institute of Scientific and Technical Information of China (English)

    刘安军; 麻献华; 杨瑞; 高琳; 陈李斌佶; 章卫平; 谢志芳

    2015-01-01

    目的:建立小鼠原代软骨细胞高效电转siRNA的方法。方法常规分离得到的小鼠原代软骨细胞继续用链丝菌蛋白酶消化3h,然后应用高效电转缓冲液和优化的电转参数转染pCMV-EGFP表达质粒或siRNA,用台盼蓝检测细胞存活率;转染后48h分析转染效率和siRNA靶分子的mRNA和蛋白表达水平。结果电穿孔后原代软骨细胞的存活率>80%,质粒的转染效率达到37.3%±5.2%;siRNA靶分子的mRNA和蛋白表达水平分别下调了75%和66%。结论成功建立了通过电穿孔介导siRNA转染小鼠原代软骨细胞的方法,达到了很好的基因沉默效果且保持了较高的细胞存活率。%Objective To develop an efficient and reliable method to transfect murine primary chondrocytes with small interfering RNA ( siRNA) by electroporation.Methods Murine primary chondrocytes were isolated and treated with pronase for 3 hours.The cells were then electroporated with either pCMV-EGFP plasmid or siRNA using a high performance electroporation buffer and optimized condi-tions.Cell viability was determined by trypan blue.The transfection efficiency and expression levels of siRNA-targeted gene were evalua-ted 48 hours post-electroporation.Results By using proper electroporation condition, 37.3%±5.2%of cells were transfected by the plasmid with high cellular viability (>80%) .Transfection of siRNA using the same electroporation resulted in effective down-regulation of its targeted gene expression at both mRNA and protein levels (75% and 66% decrease, respectively).Conclusion Transfection of murine primary chondrocytes with siRNA in this optimized electroporation condition was successful and resulted in effective gene silencing and high cellular viability.

  15. A Small Indel Mutant Mouse Model of Epidermolytic Palmoplantar Keratoderma and Its Application to Mutant-specific shRNA Therapy.

    Science.gov (United States)

    Lyu, Ya-Su; Shi, Pei-Liang; Chen, Xiao-Ling; Tang, Yue-Xiao; Wang, Yan-Fang; Liu, Rong-Rong; Luan, Xiao-Rui; Fang, Yu; Mei, Ru-Huan; Du, Zhen-Fang; Ke, Hai-Ping; Matro, Erik; Li, Ling-En; Lin, Zhao-Yu; Zhao, Jing; Gao, Xiang; Zhang, Xian-Ning

    2016-03-22

    Epidermolytic palmoplantar keratoderma (EPPK) is a relatively common autosomal-dominant skin disorder caused by mutations in the keratin 9 gene (KRT9), with few therapeutic options for the affected so far. Here, we report a knock-in transgenic mouse model that carried a small insertion-deletion (indel) mutant of Krt9, c.434delAinsGGCT (p.Tyr144delinsTrpLeu), corresponding to the human mutation KRT9/c.500delAinsGGCT (p.Tyr167delinsTrpLeu), which resulted in a human EPPK-like phenotype in the weight-stress areas of the fore- and hind-paws of both Krt9(+/mut) and Krt9(mut/mut) mice. The phenotype confirmed that EPPK is a dominant-negative condition, such that mice heterozygotic for the K9-mutant allele (Krt9(+/mut)) showed a clear EPPK-like phenotype. Then, we developed a mutant-specific short hairpin RNA (shRNA) therapy for EPPK mice. Mutant-specific shRNAs were systematically identified in vitro using a luciferase reporter gene assay and delivered into Krt9(+/mut) mice. shRNA-mediated knockdown of mutant protein resulted in almost normal morphology and functions of the skin, whereas the same shRNA had a negligible effect in wild-type K9 mice. Our results suggest that EPPK can be treated by gene therapy, and this has significant implications for future clinical application.

  16. Identification of a long non-coding RNA gene, growth hormone secretagogue receptor opposite strand, which stimulates cell migration in non-small cell lung cancer cell lines.

    Science.gov (United States)

    Whiteside, Eliza J; Seim, Inge; Pauli, Jana P; O'Keeffe, Angela J; Thomas, Patrick B; Carter, Shea L; Walpole, Carina M; Fung, Jenny N T; Josh, Peter; Herington, Adrian C; Chopin, Lisa K

    2013-08-01

    The molecular mechanisms involved in non‑small cell lung cancer tumourigenesis are largely unknown; however, recent studies have suggested that long non-coding RNAs (lncRNAs) are likely to play a role. In this study, we used public databases to identify an mRNA-like, candidate long non-coding RNA, GHSROS (GHSR opposite strand), transcribed from the antisense strand of the ghrelin receptor gene, growth hormone secretagogue receptor (GHSR). Quantitative real-time RT-PCR revealed higher expression of GHSROS in lung cancer tissue compared to adjacent, non-tumour lung tissue. In common with many long non-coding RNAs, GHSROS is 5' capped and 3' polyadenylated (mRNA-like), lacks an extensive open reading frame and harbours a transposable element. Engineered overexpression of GHSROS stimulated cell migration in the A549 and NCI-H1299 non-small cell lung cancer cell lines, but suppressed cell migration in the Beas-2B normal lung-derived bronchoepithelial cell line. This suggests that GHSROS function may be dependent on the oncogenic context. The identification of GHSROS, which is expressed in lung cancer and stimulates cell migration in lung cancer cell lines, contributes to the growing number of non-coding RNAs that play a role in the regulation of tumourigenesis and metastatic cancer progression.

  17. A local role for the small ribosomal subunit primary binder rpS5 in final 18S rRNA processing in yeast.

    Directory of Open Access Journals (Sweden)

    Andreas Neueder

    Full Text Available In vivo depletion of the yeast small ribosomal subunit (SSU protein S5 (rpS5 leads to nuclear degradation of nascent SSUs and to a perturbed global assembly state of the SSU head domain. Here, we report that rpS5 plays an additional local role at the head/platform interface in efficient SSU maturation. We find that yeast small ribosomal subunits which incorporated an rpS5 variant lacking the seven C-terminal amino acids have a largely assembled head domain and are exported to the cytoplasm. On the other hand, 3' processing of 18S rRNA precursors is inhibited in these ribosomal particles, although they associate with the putative endonuclease Nob1p and other late acting 40S biogenesis factors. We suggest that the SSU head component rpS5 and platform components as rpS14 are crucial constituents of a highly defined spatial arrangement in the head-platform interface of nascent SSUs, which is required for efficient processing of the therein predicted SSU rRNA 3' end. Positioning of rpS5 in nascent SSUs, including its relative orientation towards platform components in the head-platform cleft, will depend on the general assembly and folding state of the head domain. Therefore, the suggested model can explain 18S precursor rRNA 3' processing phenotypes observed in many eukaryotic SSU head assembly mutants.

  18. Small interfering RNA-mediated suppression of Ccl2 in Müller cells attenuates microglial recruitment and photoreceptor death following retinal degeneration

    Directory of Open Access Journals (Sweden)

    Rutar Matt

    2012-09-01

    Full Text Available Abstract Background The recruitment and activation of inflammatory cells is thought to exacerbate photoreceptor death in retinal degenerative conditions such as age-related macular degeneration (AMD. We investigated the role of Müller cell-derived chemokine (C-C motif ligand (Ccl2 expression on monocyte/microglia infiltration and photoreceptor death in light-mediated retinal degeneration, using targeted small interfering (siRNA. Methods Adult Sprague–Dawley rats were injected intravitreally with 1 μg of either Ccl2 siRNA or scrambled siRNA, and were then exposed to 1000 lux of light for a period of 24 hours. The mice were given an overdose of barbiturate, and the retinas harvested and evaluated for the effects of bright-light exposure. Ccl2 expression was assessed by quantitative PCR, immunohistochemistry, and in situ hybridization. Monocytes/microglia were counted on retinal cryostat sections immunolabeled with the markers ED1 and ionized calcium binding adaptor (IBA1, and photoreceptor apoptosis was assessed using terminal dUTP nick end labeling. Results Intravitreal injection of Ccl2 siRNA significantly reduced the expression of Ccl2 following light damage to 29% compared with controls. In retinas injected with Ccl2 siRNA, in situ hybridization and immunohistochemistry on retinal cryostat sections showed a substantial decrease in Ccl2 within Müller cells. Cell counts showed significantly fewer ED1-positive and IBA1-positive cells in the retinal vasculature and outer nuclear layer of Ccl2 siRNA-injected retinas, compared with controls. Moreover, there was significantly less photoreceptor apoptosis in Ccl2 siRNA-injected retinas compared with controls. Conclusions Our data indicate that Ccl2 expression by Müller cells promotes the infiltration of monocytes/microglia, thereby contributing to the neuroinflammatory response and photoreceptor death following retinal injury. Modulation of exaggerated chemokine responses using siRNA may have

  19. 微小RNA与卵巢癌的发病机制研究进展%Small RNA research progress and the pathogenesis of ovarian cancer

    Institute of Scientific and Technical Information of China (English)

    汪春年; 王红卫; 李征

    2015-01-01

    Small RNA (miRNAs) as a short chain, non-coding RNA transcription is the important regulatory factors in the regulation of network. Studies have shown that miRNAs play important roles in the formation and progress of ovarian cancer, may become the new target for ovarian cancer treatment. This paper summarize the miRNAs in its role in the development of ovarian cancer.%微小RNA(miRNAs)作为一种短链非编码RNA,是转录后调控网络中重要的调控因子。研究表明miRNAs在卵巢癌形成与进展中发挥着重要作用,可能成为卵巢癌治疗的新靶点。本文就近年来miRNAs在卵巢癌发生发展中的作用做一综述。

  20. A genome-wide small interfering RNA (siRNA) screen reveals nuclear factor-κB (NF-κB)-independent regulators of NOD2-induced interleukin-8 (IL-8) secretion.

    Science.gov (United States)

    Warner, Neil; Burberry, Aaron; Pliakas, Maria; McDonald, Christine; Núñez, Gabriel

    2014-10-10

    NOD2 encodes an intracellular multidomain pattern recognition receptor that is the strongest known genetic risk factor in the pathogenesis of Crohn disease (CD), a chronic relapsing inflammatory disorder of the intestinal tract. NOD2 functions as a sensor for bacterial cell wall components and activates proinflammatory and antimicrobial signaling pathways. Here, using a genome-wide small interfering RNA (siRNA) screen, we identify numerous genes that regulate secretion of the proinflammatory cytokine IL-8 in response to NOD2 activation. Moreover, many of the identified IL-8 regulators are linked by protein-protein interactions, revealing subnetworks of highly connected IL-8 regulators implicated in processes such as vesicle formation, mRNA stability, and protein ubiquitination and trafficking. A TNFα counterscreen to induce IL-8 secretion in an NOD2-independent manner reveals that the majority of the identified regulators affect IL-8 secretion irrespective of the initiating stimuli. Using immortalized macrophages, we validate the ubiquitin protease, USP8, and the endosomal sorting protein, VPS28, as negative regulators of NOD2-induced cytokine secretion. Interestingly, several genes that affect NOD2-induced IL-8 secretion are present in loci associated with CD risk by genome-wide association studies, supporting a role for the NOD2/IL-8 pathway, and not just NOD2, in the pathogenesis of CD. Overall, this screen provides a valuable resource in the advancement of our understanding of the genes that regulate the secretion of IL-8. PMID:25170077

  1. A Genome-wide Small Interfering RNA (siRNA) Screen Reveals Nuclear Factor-κB (NF-κB)-independent Regulators of NOD2-induced Interleukin-8 (IL-8) Secretion*

    Science.gov (United States)

    Warner, Neil; Burberry, Aaron; Pliakas, Maria; McDonald, Christine; Núñez, Gabriel

    2014-01-01

    NOD2 encodes an intracellular multidomain pattern recognition receptor that is the strongest known genetic risk factor in the pathogenesis of Crohn disease (CD), a chronic relapsing inflammatory disorder of the intestinal tract. NOD2 functions as a sensor for bacterial cell wall components and activates proinflammatory and antimicrobial signaling pathways. Here, using a genome-wide small interfering RNA (siRNA) screen, we identify numerous genes that regulate secretion of the proinflammatory cytokine IL-8 in response to NOD2 activation. Moreover, many of the identified IL-8 regulators are linked by protein-protein interactions, revealing subnetworks of highly connected IL-8 regulators implicated in processes such as vesicle formation, mRNA stability, and protein ubiquitination and trafficking. A TNFα counterscreen to induce IL-8 secretion in an NOD2-independent manner reveals that the majority of the identified regulators affect IL-8 secretion irrespective of the initiating stimuli. Using immortalized macrophages, we validate the ubiquitin protease, USP8, and the endosomal sorting protein, VPS28, as negative regulators of NOD2-induced cytokine secretion. Interestingly, several genes that affect NOD2-induced IL-8 secretion are present in loci associated with CD risk by genome-wide association studies, supporting a role for the NOD2/IL-8 pathway, and not just NOD2, in the pathogenesis of CD. Overall, this screen provides a valuable resource in the advancement of our understanding of the genes that regulate the secretion of IL-8. PMID:25170077

  2. Regulation of Heterochromatin Assembly on Unpaired Chromosomes during Caenorhabditis elegans Meiosis by Components of a Small RNA-Mediated Pathway

    OpenAIRE

    Xingyu She; Xia Xu; Alexander Fedotov; Kelly, William G; Maine, Eleanor M.

    2009-01-01

    Many organisms have a mechanism for down regulating the expression of non-synapsed chromosomes and chromosomal regions during meiosis. This phenomenon is thought to function in genome defense. During early meiosis in Caenorhabditis elegans, unpaired chromosomes (e.g., the male X chromosome) become enriched for a modification associated with heterochromatin and transcriptional repression, dimethylation of histone H3 on lysine 9 (H3K9me2). This enrichment requires activity of the cellular RNA-d...

  3. Small RNA sequencing-microarray analyses in Parkinson leukocytes reveal deep brain stimulation-induced and splicing changes that classify brain region transcriptomes

    Directory of Open Access Journals (Sweden)

    Lilach eSoreq

    2013-05-01

    Full Text Available MicroRNAs (miRNAs are key post transcriptional regulators of their multiple target genes. However, the detailed profile of miRNA expression in Parkinson's disease, the second most common neurodegenerative disease worldwide and the first motor disorder has not been charted yet. Here, we report comprehensive miRNA profiling by next-generation small-RNA sequencing, combined with targets inspection by splice-junction and exon arrays interrogating leukocyte RNA in Parkinson’s disease patients before and after deep brain stimulation (DBS treatment and of matched healthy control volunteers (HC. RNA-Seq analysis identified 254 miRNAs and 79 passenger strand forms as expressed in blood leukocytes, 16 of which were modified in patients pre treatment as compared to HC. 11 miRNAs were modified following brain stimulation, 5 of which were changed inversely to the disease induced changes. Stimulation cessation further induced changes in 11 miRNAs. Transcript isoform abundance analysis yielded 332 changed isoforms in patients compared to HC, which classified brain transcriptomes of 47 PD and control independent microarrays. Functional enrichment analysis highlighted mitochondrion organization. DBS induced 155 splice changes, enriched in ubiquitin homeostasis. Cellular composition analysis revealed immune cell activity pre and post treatment. Overall, 217 disease and 74 treatment alternative isoforms were predictably targeted by modified miRNAs within both 3’ and 5’ untranslated ends and coding sequence sites. The stimulation-induced network sustained 4 miRNAs and 7 transcripts of the disease network. We believe that the presented dynamic networks provide a novel avenue for identifying disease and treatment-related therapeutic targets. Furthermore, the identification of these networks is a major step forward in the road for understanding the molecular basis for neurological and neurodegenerative diseases and assessment of the impact of brain stimulation

  4. Combining NMR and small angle X-ray and neutron scattering in the structural analysis of a ternary protein-RNA complex

    Energy Technology Data Exchange (ETDEWEB)

    Hennig, Janosch; Wang, Iren; Sonntag, Miriam [Institute of Structural Biology, Helmholtz Zentrum Muenchen (Germany); Gabel, Frank [Extremophiles and Large Molecular Assemblies Group (ELMA), Institut de Biologie Structurale (IBS) CEA-CNRS-UJF (France); Sattler, Michael, E-mail: sattler@helmholtz-muenchen.de [Institute of Structural Biology, Helmholtz Zentrum Muenchen (Germany)

    2013-05-15

    Many processes in the regulation of gene expression and signaling involve the formation of protein complexes involving multi-domain proteins. Individual domains that mediate protein-protein and protein-nucleic acid interactions are typically connected by flexible linkers, which contribute to conformational dynamics and enable the formation of complexes with distinct binding partners. Solution techniques are therefore required for structural analysis and to characterize potential conformational dynamics. Nuclear magnetic resonance spectroscopy (NMR) provides such information but often only sparse data are obtained with increasing molecular weight of the complexes. It is therefore beneficial to combine NMR data with additional structural restraints from complementary solution techniques. Small angle X-ray/neutron scattering (SAXS/SANS) data can be efficiently combined with NMR-derived information, either for validation or by providing additional restraints for structural analysis. Here, we show that the combination of SAXS and SANS data can help to refine structural models obtained from data-driven docking using HADDOCK based on sparse NMR data. The approach is demonstrated with the ternary protein-protein-RNA complex involving two RNA recognition motif (RRM) domains of Sex-lethal, the N-terminal cold shock domain of Upstream-to-N-Ras, and msl-2 mRNA. Based on chemical shift perturbations we have mapped protein-protein and protein-RNA interfaces and complemented this NMR-derived information with SAXS data, as well as SANS measurements on subunit-selectively deuterated samples of the ternary complex. Our results show that, while the use of SAXS data is beneficial, the additional combination with contrast variation in SANS data resolves remaining ambiguities and improves the docking based on chemical shift perturbations of the ternary protein-RNA complex.

  5. MicroRNA-429 induces tumorigenesis of human non-small cell lung cancer cells and targets multiple tumor suppressor genes

    Energy Technology Data Exchange (ETDEWEB)

    Lang, Yaoguo; Xu, Shidong; Ma, Jianqun; Wu, Jun [Department of Thoracic Surgery, Harbin Medical University Cancer Hospital, 150 Haping Road, Harbin, Heilongjiang 150081 (China); Jin, Shi; Cao, Shoubo [Department of Medical Oncology, Harbin Medical University Cancer Hospital, 150 Haping Road, Harbin, Heilongjiang 150081 (China); Yu, Yan, E-mail: yuyan@hrbmu.edu.cn [Department of Medical Oncology, Harbin Medical University Cancer Hospital, 150 Haping Road, Harbin, Heilongjiang 150081 (China)

    2014-07-18

    Highlights: • MiR-429 expression is upregulated in non-small cell lung cancer (NSCLC). • MiR-429 inhibits PTEN, RASSF8 and TIMP2 expression. • MiR-429 promotes metastasis and proliferation. • We report important regulatory mechanisms involved in NSCLC progression. • MiR-429 is a potential therapeutic target and diagnostic marker. - Abstract: Lung cancer is the major cause of cancer death globally. MicroRNAs are evolutionally conserved small noncoding RNAs that are critical for the regulation of gene expression. Aberrant expression of microRNA (miRNA) has been implicated in cancer initiation and progression. In this study, we demonstrated that the expression of miR-429 are often upregulated in non-small cell lung cancer (NSCLC) compared with normal lung tissues, and its expression level is also increased in NSCLC cell lines compared with normal lung cells. Overexpression of miR-429 in A549 NSCLC cells significantly promoted cell proliferation, migration and invasion, whereas inhibition of miR-429 inhibits these effects. Furthermore, we demonstrated that miR-429 down-regulates PTEN, RASSF8 and TIMP2 expression by directly targeting the 3′-untranslated region of these target genes. Taken together, our results suggest that miR-429 plays an important role in promoting the proliferation and metastasis of NSCLC cells and is a potential target for NSCLC therapy.

  6. MicroRNA-429 induces tumorigenesis of human non-small cell lung cancer cells and targets multiple tumor suppressor genes

    International Nuclear Information System (INIS)

    Highlights: • MiR-429 expression is upregulated in non-small cell lung cancer (NSCLC). • MiR-429 inhibits PTEN, RASSF8 and TIMP2 expression. • MiR-429 promotes metastasis and proliferation. • We report important regulatory mechanisms involved in NSCLC progression. • MiR-429 is a potential therapeutic target and diagnostic marker. - Abstract: Lung cancer is the major cause of cancer death globally. MicroRNAs are evolutionally conserved small noncoding RNAs that are critical for the regulation of gene expression. Aberrant expression of microRNA (miRNA) has been implicated in cancer initiation and progression. In this study, we demonstrated that the expression of miR-429 are often upregulated in non-small cell lung cancer (NSCLC) compared with normal lung tissues, and its expression level is also increased in NSCLC cell lines compared with normal lung cells. Overexpression of miR-429 in A549 NSCLC cells significantly promoted cell proliferation, migration and invasion, whereas inhibition of miR-429 inhibits these effects. Furthermore, we demonstrated that miR-429 down-regulates PTEN, RASSF8 and TIMP2 expression by directly targeting the 3′-untranslated region of these target genes. Taken together, our results suggest that miR-429 plays an important role in promoting the proliferation and metastasis of NSCLC cells and is a potential target for NSCLC therapy

  7. Small RNA Sequencing Uncovers New miRNAs and moRNAs Differentially Expressed in Normal and Primary Myelofibrosis CD34+ Cells.

    Directory of Open Access Journals (Sweden)

    Paola Guglielmelli

    Full Text Available Myeloproliferative neoplasms (MPN are chronic myeloid cancers thought to arise at the level of CD34+ hematopoietic stem/progenitor cells. They include essential thrombocythemia (ET, polycythemia vera (PV and primary myelofibrosis (PMF. All can progress to acute leukemia, but PMF carries the worst prognosis. Increasing evidences indicate that deregulation of microRNAs (miRNAs might plays an important role in hematologic malignancies, including MPN. To attain deeper knowledge of short RNAs (sRNAs expression pattern in CD34+ cells and of their possible role in mediating post-transcriptional regulation in PMF, we sequenced with Illumina HiSeq2000 technology CD34+ cells from healthy subjects and PMF patients. We detected the expression of 784 known miRNAs, with a prevalence of miRNA up-regulation in PMF samples, and discovered 34 new miRNAs and 99 new miRNA-offset RNAs (moRNAs, in CD34+ cells. Thirty-seven small RNAs were differentially expressed in PMF patients compared with healthy subjects, according to microRNA sequencing data. Five miRNAs (miR-10b-5p, miR-19b-3p, miR-29a-3p, miR-379-5p, and miR-543 were deregulated also in PMF granulocytes. Moreover, 3'-moR-128-2 resulted consistently downregulated in PMF according to RNA-seq and qRT-PCR data both in CD34+ cells and granulocytes. Target predictions of these validated small RNAs de-regulated in PMF and functional enrichment analyses highlighted many interesting pathways involved in tumor development and progression, such as signaling by FGFR and DAP12 and Oncogene Induced Senescence. As a whole, data obtained in this study deepened the knowledge of miRNAs and moRNAs altered expression in PMF CD34+ cells and allowed to identify and validate a specific small RNA profile that distinguishes PMF granulocytes from those of normal subjects. We thus provided new information regarding the possible role of miRNAs and, specifically, of new moRNAs in this disease.

  8. Small molecule-mediated up-regulation of microRNA targeting a key cell death modulator BNIP3 improves cardiac function following ischemic injury.

    Science.gov (United States)

    Lee, Se-Yeon; Lee, Seahyoung; Choi, Eunhyun; Ham, Onju; Lee, Chang Youn; Lee, Jiyun; Seo, Hyang-Hee; Cha, Min-Ji; Mun, Bohyun; Lee, Yunmi; Yoon, Cheesoon; Hwang, Ki-Chul

    2016-01-01

    Genetic ablation of BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), an essential regulator of cardiac cell death, is an effective way to prevent cardiac cell death triggered by pathologic conditions. However, currently there exists no known means, such as inhibitors, to down-regulate BNIP3 in mature heart. Here, we report that a small molecule inducer of microRNA-182 (miR-182) suppressed ischemia/reperfusion (I/R)-induced cardiac cell death by down-regulating BNIP3. We first selected miR-182 as a potent BNIP3-targeting miRNA based on miRNA-target prediction databases and empirical data. The subsequent screening of small molecules for inducing miR-182 expression identified Kenpaullone as a hit compound. Both exogenous miR-182 and Kenpaullone significantly suppressed hypoxia-induced cardiomyocyte death in vitro. To investigate the effect of changing substituents of Kenpaullone on miR-182 expression, we synthesized 9 derivatives of Kenpaullone. Among these derivatives, compound 5 showed significantly improved ability to induce miR-182 expression. The results of the in vivo study showed that compound 5 significantly improved heart function following I/R-injury in rats. Our study provides strong evidence that the small molecule-mediated up-regulation of miRNAs is a viable strategy to down-regulate target proteins with no known chemical inhibitor and that compound 5 may have potential to prevent I/R-inflicted cardiac cell death. PMID:27008992

  9. Impact of CHK2-small interfering RNA on CpG ODN7909-enhanced radiosensitivity in lung cancer A549 cells

    Directory of Open Access Journals (Sweden)

    Chen W

    2012-12-01

    Full Text Available Wei Chen,* Xiaoqun Liu,* Tiankui Qiao, Sujuan Yuan Department of Oncology, Jinshan Hospital, Fudan University, Shanghai, People's Republic of China*These authors contributed equally to this workObjective: To investigate the impact of checkpoint kinase 2 (CHK2-small interfering RNA (CHK2-siRNA on the enhancement of radiosensitivity by CpG oligodeoxynucleotide (ODN 7909 in lung cancer A549 cells.Methods: The A549 cells were randomly divided into five groups: control, CpG, X-ray, CpG + X-ray, and CHK2-siRNA + CpG + X-ray. Cell colonization was observed using inverted microscopy. Cell cycle and apoptosis were analyzed by flow cytometry. CHK2 expression was detected by Western blot. CHK2-siRNA was adopted to silence the expression of CHK2.Results: The level of CHK2 phosphorylation was higher in the CpG + X-ray group than in the X-ray group. Increases in G2/mitotic (M phase arrest and apoptosis and a decrease of cell survival rate in the CpG + X-ray group were statistically significant (P < 0.05 when compared with the CHK2-siRNA + CpG + X-ray group in which the expression of CHK2 was obviously inhibited. The combination of CpG ODN7909 and X-ray irradiation was found to enhance the mitotic death of A549 cells. The sensitization enhancement ratio of mean death dose (D0 was 1.42 in the CpG + X-ray group, which was higher than that of the CHK2-siRNA + CpG + X-ray group, in which D0 was 1.05.Conclusion: To a certain extent, the impact of a combination of CpG ODN7909 and X-ray on G2/M phase arrest, apoptosis, and rate of cell survival was attenuated by CHK2-siRNA in human lung adenocarcinoma A549 cells, indicating that increased phosphorylation of CHK2 might be a radiosensitive pathway.Keywords: oligodeoxynucleotide, checkpoint kinase 2, mitotic death, apoptosis, X-ray

  10. MicroRNA-663a is downregulated in non-small cell lung cancer and inhibits proliferation and invasion by targeting JunD

    OpenAIRE

    Zhang, Yi; Xu, Xiaoman; Zhang, Meng; wang, xin; Bai, Xue; Li, Hui; KAN, LIANG; Zhou, Yong; Niu, Huiyan; He, Ping

    2016-01-01

    Background MicroRNA-663a expression is downregulated in several tumors. However, its functions and mechanisms in human non-small cell lung (NSCLC) cancer remain obscure. The present study aimed to identify the expression pattern, biological roles and potential mechanisms by which miR-663a dysregulation is associated with NSCLC. Methods We examined expression level of miR-663a in 62 cases of NSCLC tissues and 5 NSCLC cell lines by reverse transcription PCR. In vitro, gain-of-function and loss-...

  11. P53-regulated long non-coding RNA TUG1 affects cell proliferation in human non-small cell lung cancer, partly through epigenetically regulating HOXB7 expression

    OpenAIRE

    Zhang, E-b; Yin, D-d; M. Sun; Kong, R.; Liu, X-h; You, L-h; Han, L.; Xia, R; Wang, K-m; Yang, J-s; De, W; Shu, Y-q; Wang, Z-X

    2014-01-01

    Recently, a novel class of transcripts, long non-coding RNAs (lncRNAs), is being identified at a rapid pace. These RNAs have critical roles in diverse biological processes, including tumorigenesis. Here we report that taurine-upregulated gene 1 (TUG1), a 7.1-kb lncRNA, recruiting and binding to polycomb repressive complex 2 (PRC2), is generally downregulated in non-small cell lung carcinoma (NSCLC) tissues. In a cohort of 192 NSCLC patients, the lower expression of TUG1 was associated with a ...

  12. Specific responses in rat small intestinal epithelial mRNA expression and protein levels during chemotherapeutic damage and regeneration

    NARCIS (Netherlands)

    M. Verburg (Melissa); I.B. Renes (Ingrid); D.J. van Nispen; S. Ferdinandusse; M. Jorritsma; H.A. Büller (Hans); A.W.C. Einerhand (Sandra); J. Dekker (Jan)

    2002-01-01

    textabstractThe rapidly dividing small intestinal epithelium is very sensitive to the cytostatic drug methotrexate. We investigated the regulation of epithelial gene expression in rat jejunum during methotrexate-induced damage and regeneration. Ten differentiation markers were loca

  13. Identification of Zinc Deficiency-Responsive MicroRNAs in Brassica juncea Roots by Small RNA Sequencing

    Institute of Scientific and Technical Information of China (English)

    SHI Dong-qing; ZHANG Yuan; MA Jin-hu; LI Yu-long; and XU Jin

    2013-01-01

    The importance of zinc (Zn) as a micronutrient essential for plant growth and development is becoming increasingly apparent. Much of the world’s soil is Zn-deficient, and soil-based Zn deficiency is often accompanied by Zn deficiency in human populations. MicroRNAs (miRNAs) play important roles in the regulation of plant gene expression at the level of translation. Many miRNAs involved in the modulation of heavy metal toxicity responses in plants have been identiifed;however, the role of miRNAs in the plant Zn deifciency response is almost completely unknown. Using high-throughput Solexa sequencing, we identiifed several miRNAs that respond to Zn deifciency in Brassica juncea roots. At least 21 conserved candidate miRNA families, and 101 individual members within those families, were identiifed in both the control and the Zn-deifcient B. juncea roots. Among this, 15 miRNAs from 9 miRNA families were differentially expressed in the control and Zn-deifcient plants. Of the 15 differentially expressed miRNAs, 13 were up-regulated in the Zn-deifcient B. juncea roots, and only two, miR399b and miR845a, were down-regulated. Bioinformatics analysis indicated that these miRNAs were involved in modulating phytohormone response, plant growth and development, and abiotic stress responses in B. juncea roots. These data help to lay the foundation for further understanding of miRNA function in the regulation of the plant Zn deifciency response and its impact on plant growth and development.

  14. RNA-binding protein HuR promotes growth of small intestinal mucosa by activating the Wnt signaling pathway

    OpenAIRE

    Liu, Lan; Christodoulou-Vafeiadou, Eleni; Rao, Jaladanki N.; Zou, Tongtong; Xiao, Lan; Kyoung Chung, Hee; Yang, Hong; Gorospe, Myriam; Kontoyiannis, Dimitris; Wang, Jian-Ying

    2014-01-01

    Inhibition of growth of the intestinal epithelium, a rapidly self-renewing tissue, is commonly found in various critical disorders. The RNA-binding protein HuR is highly expressed in the gut mucosa and modulates the stability and translation of target mRNAs, but its exact biological function in the intestinal epithelium remains unclear. Here, we investigated the role of HuR in intestinal homeostasis using a genetic model and further defined its target mRNAs. Targeted deletion of HuR in intest...

  15. Analysis of a cDNA clone expressing a human autoimmune antigen: full-length sequence of the U2 small nuclear RNA-associated B antigen

    International Nuclear Information System (INIS)

    A U2 small nuclear RNA-associated protein, designated B'', was recently identified as the target antigen for autoimmune sera from certain patients with systemic lupus erythematosus and other rheumatic diseases. Such antibodies enabled them to isolate cDNA clone λHB''-1 from a phage λgt11 expression library. This clone appeared to code for the B'' protein as established by in vitro translation of hybrid-selected mRNA. The identity of clone λHB''-1 was further confirmed by partial peptide mapping and analysis of the reactivity of the recombinant antigen with monospecific and monoclonal antibodies. Analysis of the nucleotide sequence of the 1015-base-pair cDNA insert of clone λHB''-1 revealed a large open reading frame of 800 nucleotides containing the coding sequence for a polypeptide of 25,457 daltons. In vitro transcription of the λHB''-1 cDNA insert and subsequent translation resulted in a protein product with the molecular size of the B'' protein. These data demonstrate that clone λHB''-1 contains the complete coding sequence of this antigen. The deduced polypeptide sequence contains three very hydrophilic regions that might constitute RNA binding sites and/or antigenic determinants. These findings might have implications both for the understanding of the pathogenesis of rheumatic diseases as well as for the elucidation of the biological function of autoimmune antigens

  16. Cross-talk between a regulatory small RNA, cyclic-di-GMP signalling and flagellar regulator FlhDC for virulence and bacterial behaviours.

    Science.gov (United States)

    Yuan, Xiaochen; Khokhani, Devanshi; Wu, Xiaogang; Yang, Fenghuan; Biener, Gabriel; Koestler, Benjamin J; Raicu, Valerica; He, Chenyang; Waters, Christopher M; Sundin, George W; Tian, Fang; Yang, Ching-Hong

    2015-11-01

    Dickeya dadantii is a globally dispersed phytopathogen which causes diseases on a wide range of host plants. This pathogen utilizes the type III secretion system (T3SS) to suppress host defense responses, and secretes pectate lyase (Pel) to degrade the plant cell wall. Although the regulatory small RNA (sRNA) RsmB, cyclic diguanylate monophosphate (c-di-GMP) and flagellar regulator have been reported to affect the regulation of these two virulence factors or multiple cell behaviours such as motility and biofilm formation, the linkage between these regulatory components that coordinate the cell behaviours remain unclear. Here, we revealed a sophisticated regulatory network that connects the sRNA, c-di-GMP signalling and flagellar master regulator FlhDC. We propose multi-tiered regulatory mechanisms that link the FlhDC to the T3SS through three distinct pathways including the FlhDC-FliA-YcgR3937 pathway; the FlhDC-EcpC-RpoN-HrpL pathway; and the FlhDC-rsmB-RsmA-HrpL pathway. Among these, EcpC is the most dominant factor for FlhDC to positively regulate T3SS expression.

  17. Development and application of small-subunit rRNA probes for assessment of selected Thiobacillus species and members of the genus Acidiphilium.

    Science.gov (United States)

    Peccia, J; Marchand, E A; Silverstein, J; Hernandez, M

    2000-07-01

    Culture-dependent studies have implicated sulfur-oxidizing bacteria as the causative agents of acid mine drainage and concrete corrosion in sewers. Thiobacillus species are considered the major representatives of the acid-producing bacteria in these environments. Small-subunit rRNA genes from all of the Thiobacillus and Acidiphilium species catalogued by the Ribosomal Database Project were identified and used to design oligonucleotide DNA probes. Two oligonucleotide probes were synthesized to complement variable regions of 16S rRNA in the following acidophilic bacteria: Thiobacillus ferrooxidans and T. thiooxidans (probe Thio820) and members of the genus Acidiphilium (probe Acdp821). Using (32)P radiolabels, probe specificity was characterized by hybridization dissociation temperature (T(d)) with membrane-immobilized RNA extracted from a suite of 21 strains representing three groups of bacteria. Fluorochrome-conjugated probes were evaluated for use with fluorescent in situ hybridization (FISH) at the experimentally determined T(d)s. FISH was used to identify and enumerate bacteria in laboratory reactors and environmental samples. Probing of laboratory reactors inoculated with a mixed culture of acidophilic bacteria validated the ability of the oligonucleotide probes to track specific cell numbers with time. Additionally, probing of sediments from an active acid mine drainage site in Colorado demonstrated the ability to identify numbers of active bacteria in natural environments that contain high concentrations of metals, associated precipitates, and other mineral debris.

  18. The small subunit rRNA gene sequence of the chonotrich Chilodochona carcini Jankowski, 1973 confirms chonotrichs as a dysteriid-derived clade (Phyllopharyngea, Ciliophora).

    Science.gov (United States)

    Lynn, Denis H

    2016-08-01

    The chonotrichs are sessile ciliated protozoa that are ectosymbiotic on the body parts of a variety of crustaceans. They have long been considered a separate group because their sessile habit has resulted in the evolution of a very divergent body form and reproductive strategy compared to free-living ciliates. In the mid-20th Century, the free-living dysteriid cyrtophorian ciliates were proposed as a potential sister clade because the chonotrich bud or daughter cell showed similarities during division morphogenesis (i.e. ontogeny) to these free-living dysteriids. A single small subunit (SSU) rRNA gene sequence is available for the chonotrich Isochona sp. However, its authenticity has recently been questioned, and the placement of this sequence within the dysteriid clade has added to this controversy. In this report, the SSUrRNA gene sequence of the chonotrich Chilodochona carcini, ectosymbiotic on the green crab Carcinus maenas, is provided. Topology testing of the SSUrRNA gene phylogeny, constructed by Bayesian Inference, robustly supports the sister-group relationship of Isochona sp. and Chilodochona carcini, the monophyly of these two chonotrichs, and the divergence of the chonotrich clade within the dysteriid clade. PMID:27151876

  19. Down-regulation of STAT3 expression by vector-based small interfering RNA inhibits pancreatic cancer growth

    Institute of Scientific and Technical Information of China (English)

    Chen Huang; Guang Yang; Tao Jiang; Jun Cao; Ke-Jian Huang; Zheng-Jun Qiu

    2011-01-01

    AIM: To evaluate the effect of RNA interference (RNAi) mediated silence of signal transduction and activation of transcription (STAT)3 on the growth of human pancreatic cancer cells both in vitro and in vivo . METHODS: STAT3 specific shRNA was used to silence the expression of STAT3 in pancreatic cancer cell line SW1990. The anti-growth effects of RNAi against STAT3 were studied in vitro and in experimental cancer xenografts in nude mice. The potential pathways involved in STAT3 signaling were detected using reverse transcription polymerase chain reaction and western blotting. RESULTS: The expression of the STAT3 was inhibited using RNAi in SW1990 cells. RNAi against STAT3 inhibited cell proliferation, induced cell apoptosis and significantly reduced the levels of CyclinD1 and Bcl-xL when compared with parental and control vector-transfected cells. In vivo experiments showed that RNAi against STAT3 inhibited the tumorigenicity of SW1990 cells and significantly suppressed tumor growth when it was directly injected into tumors. CONCLUSION: STAT3 signaling pathway plays an important role in the progression of pancreatic cancer, and silence of STAT3 gene using RNAi technique may be a novel therapeutic option for treatment of pancreatic cancer.

  20. Small interfering RNA against transcription factor STAT6 leads to increased cholesterol synthesis in lung cancer cell lines.

    Directory of Open Access Journals (Sweden)

    Richa Dubey

    Full Text Available STAT6 transcription factor has become a potential molecule for therapeutic intervention because it regulates broad range of cellular processes in a large variety of cell types. Although some target genes and interacting partners of STAT6 have been identified, its exact mechanism of action needs to be elucidated. In this study, we sought to further characterize the molecular interactions, networks, and functions of STAT6 by profiling the mRNA expression of STAT6 silenced human lung cells (NCI-H460 using microarrays. Our analysis revealed 273 differentially expressed genes after STAT6 silencing. Analysis of the gene expression data with Ingenuity Pathway Analysis (IPA software revealed Gene expression, Cell death, Lipid metabolism as the functions associated with highest rated network. Cholesterol biosynthesis was among the most enriched pathways in IPA as well as in PANTHER analysis. These results have been validated by real-time PCR and cholesterol assay using scrambled siRNA as a negative control. Similar findings were also observed with human type II pulmonary alveolar epithelial cells, A549. In the present study we have, for the first time, shown the inverse relationship of STAT6 with the cholesterol biosynthesis in lung cancer cells. The present findings are potentially significant to advance the understanding and design of therapeutics for the pathological conditions where both STAT6 and cholesterol biosynthesis are implicated viz. asthma, atherosclerosis etc.

  1. High Throughput Sequencing of Entamoeba 27nt Small RNA Population Reveals Role in Permanent Gene Silencing But No Effect on Regulating Gene Expression Changes during Stage Conversion, Oxidative, or Heat Shock Stress.

    Science.gov (United States)

    Zhang, Hanbang; Ehrenkaufer, Gretchen M; Manna, Dipak; Hall, Neil; Singh, Upinder

    2015-01-01

    The human parasite Entamoeba histolytica has an active RNA interference (RNAi) pathway with an extensive repertoire of 27nt small RNAs that silence genes. However the role of this pathway in regulating amebic biology remains unknown. In this study, we address whether silencing via 27nt small RNAs may be a mechanism for controlling gene expression changes during conversion between the trophozoite and cyst stages of the parasite. We sequenced small RNA libraries generated from trophozoites, early cysts, mature cysts, and excysting cells and mapped them to the E. invadens genome. Our results show that, as in E. histolytica, small RNAs in E. invadens are largely ~27nt in length, have an unusual 5'-polyphosphate structure and mediate gene silencing. However, when comparing the libraries from each developmental time-point we found few changes in the composition of the small RNA populations. Furthermore, genes targeted by small RNAs were permanently silenced with no changes in transcript abundance during development. Thus, the E. invadens 27nt small RNA population does not mediate gene expression changes during development. In order to assess the generalizability of our observations, we examined whether small RNAs may be regulating gene expression changes during stress response in E. histolytica. Comparison of the 27nt small RNA populations from E. histolytica trophozoites from basal conditions, or after heat shock or exposure to oxidative stress showed few differences. Similar to data in E. invadens development, genes targeted by small RNAs were consistently silenced and did not change expression under tested stress conditions. Thus, the biological roles of the 27nt small RNA population in Entamoeba remain elusive. However, as the first characterization of the RNAi pathway in E. invadens these data serve as a useful resource for the study of Entamoeba development and open the door to the development of RNAi-based gene silencing tools in E. invadens.

  2. Small interfering RNA of cyclooxygenase-2 induces growth inhibition and apoptosis independently of Bcl-2 in human myeloma RPMI8226 cells

    Institute of Scientific and Technical Information of China (English)

    Qiu-bai LI; Zhi-chao CHEN; Yong YOU; Ping ZOU

    2007-01-01

    Aim: To investigate the effects of small interfering RNA of cyclooxygenase-2 (COX-2) on the proliferation and apoptosis of human multiple myeloma RPMI8226 cells and its relation with the Bcl-2 family in vitro. Methods: Transcription and expression of COX-2 in human myeloma RPMI8226 cells were checked by RT-PCR and Western blot analysis, respectively. The COX-2 siRNA fragment targeting exon 5 of COX-2 gene was transfected into the cells with the Amaxa nucleofection technique. The inhibition of cell growth was detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) assay. Apoptosis was estimated by Annexin-V/propidium iodide double-labeled cytometry and confirmed by termi-nal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay. Bcl-2 and Bax expression was evaluated by Western blot analysis. Results: The COX-2 siRNA fragment could be successfully transfected into RPMI8226 cells, which resulted in the significant inhibition of transcription and expression of COX-2 in the myeloma cells. Proliferation of the transfected cells was inhibited and apoptosis was induced (6.52%±0.32%, 12.53%±2.52%, 24.39%±3.51% and 36.48%±4.96% for 0, 12, 24, and 48 h, respectively) in a time-dependent manner (P<0.01), However, the expression of Bcl-2 and Bax in the RPMI8226 cells had no significant changes after nucleofection. Conclusion: COX-2 siRNA transfection can suppress COX-2 expression in human myeloma RPMI8226 cells, which leads to growth inhibition and apoptosis independent of Bcl-2.

  3. Comparison of PCR-based detection of chromogranin A mRNA with traditional histological lymph node staging of small intestinal neuroendocrine neoplasia

    Directory of Open Access Journals (Sweden)

    Lawrence Ben

    2012-06-01

    Full Text Available Abstract Background Accurate neuroendocrine neoplasia (NEN staging is vital for determining prognosis and therapeutic strategy. The great majority of NENs express chromogranin A (CgA which can be detected at a protein or transcript level. The current standards for lymph node metastasis detection are histological examination after Hematoxylin and Eosin (H&E and CgA immunohistochemical (IHC staining. We hypothesized that detection of CgA mRNA transcripts would be a more sensitive method of detecting these metastases. Findings We compared these traditional methods with PCR for CgA mRNA extracted from formalin fixed paraffin embedded slides of lymph nodes (n = 196 from small intestinal NENs, other gastrointestinal cancers and benign gastrointestinal disease. CgA PCR detected significantly more NEN lymph nodes (75% than H&E (53% or CgA IHC (57% (p = 0.02. PCR detected CgA mRNA in 50% (14 of the 28 of SI-NEN lymph nodes previously considered negative. The false positive rate for detection of CgA mRNA was 19% in non-neuroendocrine cancers, and appeared to be due to occult neuroendocrine differentiation or contamination by normal epithelium during histological processing. Conclusions Molecular pathological analysis demonstrates the limitations of observer-dependent histopathology. CgA PCR analysis detected the presence of CgA transcripts in lymph nodes without histological evidence of tumor metastasis. Molecular node positivity (stage molN1 of SI-NEN lymph nodes could confer greater staging accuracy and facilitate early and accurate therapeutic intervention. This technique warrants investigation using clinically annotated tumor samples with follow-up data.

  4. Intrageneric relationships of Enterococci as determined by reverse transcriptase sequencing of small-subunit rRNA.

    Science.gov (United States)

    Williams, A M; Rodrigues, U M; Collins, M D

    1991-01-01

    The 16S ribosomal ribonucleic acid (rRNA) sequences of eleven Enterococcus species were determined by reverse transcription in an attempt to clarify their intrageneric relationships. Comparative analysis of the sequence data revealed the presence of several species groups within the genus. The species E. avium, E. malodoratus, E. pseudoavium and E. raffinosus formed a distinct group as did E. durans, E. faecium, E. hirae and E. mundtii and the pair of species E. casseliflavus and E. gallinarum. Of the remaining species, E. cecorum, E. columbae, E. faecalis and E. saccharolyticus formed distinct lines of descent within the genus, whereas E. solitarius displayed a closer affinity with Tetragenococcus halophilus than with other enterococcal species. PMID:1712504

  5. Small regulatory RNAs of the RNA interference (RNAi) pathway as a prophylactic treatment against fish pathogenic viruses

    DEFF Research Database (Denmark)

    Schyth, Brian Dall; Hajiabadi, Seyed Amir Hossein Jalali; Kristensen, Lasse Bøgelund Juel;

    2011-01-01

    . The mechanism can be programmed with several types of small double stranded RNAs - the type of which defines the destiny of the target. One such class of regulatory RNAs called microRNAs are upregulated due to various physiological responses of the cell and they suppress many genes simultaneously believed...

  6. Small interfering RNA targeting mcl-1 enhances proteasome inhibitor-induced apoptosis in various solid malignant tumors

    Directory of Open Access Journals (Sweden)

    Zhou Wei

    2011-11-01

    Full Text Available Abstract Background Targeting the ubiquitin-proteasome pathway is a promising approach for anticancer strategies. Recently, we found Bik accumulation in cancer cell lines after they were treated with bortezomib. However, recent evidence indicates that proteasome inhibitors may also induce the accumulation of anti-apoptotic Bcl-2 family members. The current study was designed to analyze the levels of several anti-apoptotic members of Bcl-2 family in different human cancer cell lines after they were treated with proteasome inhibitors. Methods Different human cancer cell lines were treated with proteasome inhibitors. Western blot were used to investigate the expression of Mcl-1 and activation of mitochondrial apoptotic signaling. Cell viability was investigated using SRB assay, and induction of apoptosis was measured using flow cytometry. Results We found elevated Mcl-1 level in human colon cancer cell lines DLD1, LOVO, SW620, and HCT116; human ovarian cancer cell line SKOV3; and human lung cancer cell line H1299, but not in human breast cancer cell line MCF7 after they were treated with bortezomib. This dramatic Mcl-1 accumulation was also observed when cells were treated with other two proteasome inhibitors, MG132 and calpain inhibitor I (ALLN. Moreover, our results showed Mcl-1 accumulation was caused by stabilization of the protein against degradation. Reducing Mcl-1 accumulation by Mcl-1 siRNA reduced Mcl-1 accumulation and enhanced proteasome inhibitor-induced cell death and apoptosis, as evidenced by the increased cleavage of caspase-9, caspase-3, and poly (ADP-ribose polymerase. Conclusions Our results showed that it was not only Bik but also Mcl-1 accumulation during the treatment of proteasome inhibitors, and combining proteasome inhibitors with Mcl-1 siRNA would enhance the ultimate anticancer effect suggesting this combination might be a more effective strategy for cancer therapy.

  7. Shock-induced poration, cholesterol flip-flop and small interfering RNA transfection in a phospholipid membrane: Multimillion atom, microsecond molecular dynamics simulations

    Science.gov (United States)

    Choubey, Amit

    performing a 15 mus all-atom MD simulation of a DPPC-CHOL bilayer. We find that the CHOL flip-flop rates are on the sub microsecond timescale. These results are verified by performing various independent parallel replica (PR) simulations. Our PR simulations provide significant boost in sampling of the flip-flop events. We observe that the CHOL flip-flop can induce membrane order, regulate membrane-bending energy, and facilitate membrane relaxation. The rapid flip-flop rates reported here have important implications for the role of CHOL in mechanical properties of cell membranes, formation of domains, and maintaining CHOL concentration asymmetry in plasma membrane. Our PR approach can reach submillisecond time scales and bridge the gap between MD simulations and Nuclear Magnetic Resonance (NMR) experiments on CHOL flip-flop dynamics in membranes. The last project deals with transfection barriers encountered by a bare small interfering RNA (siRNA) in a phospholipid bilayer. SiRNA molecules play a pivotal role in therapeutic applications. A key limitation to the widespread implementation of siRNA-based therapeutics is the difficulty of delivering siRNA-based drugs to cells. We have examined structural and mechanical barriers to siRNA passage across a phospholipid bilayer using all-atom MD simulations. We find that the electrostatic interaction between the anionic siRNA and head groups of phospholipid molecules induces a phase transformation from the liquid crystalline to ripple phase. Steered MD simulations reveal that the siRNA transfection through the ripple phase requires a force of ˜ 1.5 nN.

  8. Small-molecule inhibition of HIV pre-mRNA splicing as a novel antiretroviral therapy to overcome drug resistance.

    Directory of Open Access Journals (Sweden)

    Nadia Bakkour

    2007-10-01

    Full Text Available The development of multidrug-resistant viruses compromises antiretroviral therapy efficacy and limits therapeutic options. Therefore, it is an ongoing task to identify new targets for antiretroviral therapy and to develop new drugs. Here, we show that an indole derivative (IDC16 that interferes with exonic splicing enhancer activity of the SR protein splicing factor SF2/ASF suppresses the production of key viral proteins, thereby compromising subsequent synthesis of full-length HIV-1 pre-mRNA and assembly of infectious particles. IDC16 inhibits replication of macrophage- and T cell-tropic laboratory strains, clinical isolates, and strains with high-level resistance to inhibitors of viral protease and reverse transcriptase. Importantly, drug treatment of primary blood cells did not alter splicing profiles of endogenous genes involved in cell cycle transition and apoptosis. Thus, human splicing factors represent novel and promising drug targets for the development of antiretroviral therapies, particularly for the inhibition of multidrug-resistant viruses.

  9. Study of genetic diversity of eukaryotic picoplankton in different oceanic regions by small-subunit rRNA gene cloning and sequencing.

    Science.gov (United States)

    Díez, B; Pedrós-Alió, C; Massana, R

    2001-07-01

    Very small eukaryotic organisms (picoeukaryotes) are fundamental components of marine planktonic systems, often accounting for a significant fraction of the biomass and activity in a system. Their identity, however, has remained elusive, since the small cells lack morphological features for identification. We determined the diversity of marine picoeukaryotes by sequencing cloned 18S rRNA genes in five genetic libraries from North Atlantic, Southern Ocean, and Mediterranean Sea surface waters. Picoplankton were obtained by filter size fractionation, a step that excluded most large eukaryotes and recovered most picoeukaryotes. Genetic libraries of eukaryotic ribosomal DNA were screened by restriction fragment length polymorphism analysis, and at least one clone of each operational taxonomic unit (OTU) was partially sequenced. In general, the phylogenetic diversity in each library was rather great, and each library included many different OTUs and members of very distantly related phylogenetic groups. Of 225 eukaryotic clones, 126 were affiliated with algal classes, especially the Prasinophyceae, the Prymnesiophyceae, the Bacillariophyceae, and the Dinophyceae. A minor fraction (27 clones) was affiliated with clearly heterotrophic organisms, such as ciliates, the chrysomonad Paraphysomonas, cercomonads, and fungi. There were two relatively abundant novel lineages, novel stramenopiles (53 clones) and novel alveolates (19 clones). These lineages are very different from any organism that has been isolated, suggesting that there are previously unknown picoeukaryotes. Prasinophytes and novel stramenopile clones were very abundant in all of the libraries analyzed. These findings underscore the importance of attempts to grow the small eukaryotic plankton in pure culture.

  10. Differential gene expression in liver and small intestine from lactating rats compared to age-matched virgin controls detects increased mRNA of cholesterol biosynthetic genes

    Directory of Open Access Journals (Sweden)

    Jungsuwadee Paiboon

    2011-02-01

    Full Text Available Abstract Background Lactation increases energy demands four- to five-fold, leading to a two- to three-fold increase in food consumption, requiring a proportional adjustment in the ability of the lactating dam to absorb nutrients and to synthesize critical biomolecules, such as cholesterol, to meet the dietary needs of both the offspring and the dam. The size and hydrophobicity of the bile acid pool increases during lactation, implying an increased absorption and disposition of lipids, sterols, nutrients, and xenobiotics. In order to investigate changes at the transcriptomics level, we utilized an exon array and calculated expression levels to investigate changes in gene expression in the liver, duodenum, jejunum, and ileum of lactating dams when compared against age-matched virgin controls. Results A two-way mixed models ANOVA was applied to detect differentially expressed genes. Significance calls were defined as a p Cyp7a1, which catalyzes the rate limiting step in the bile acid biosynthetic pathway, was also significantly increased in liver. In addition, decreased levels of mRNA associated with T-cell signaling were found in the jejunum and ileum. Several members of the Solute Carrier (SLC and Adenosine Triphosphate Binding Cassette (ABC superfamilies of membrane transporters were found to be differentially expressed; these genes may play a role in differences in nutrient and xenobiotic absorption and disposition. mRNA expression of SLC39a4_predicted, a zinc transporter, was increased in all tissues, suggesting that it is involved in increased zinc uptake during lactation. Microarray data are available through GEO under GSE19175. Conclusions We detected differential expression of mRNA from several pathways in lactating dams, including upregulation of the cholesterol biosynthetic pathway in liver and intestine, consistent with Srebp activation. Differential T-Cell signaling in the two most distal regions of the small intestine (ileum and

  11. Small ncRNA Expression-Profiling of Blood from Hemophilia A Patients Identifies miR-1246 as a Potential Regulator of Factor 8 Gene.

    Directory of Open Access Journals (Sweden)

    Tewarit Sarachana

    Full Text Available Hemophilia A (HA is a bleeding disorder caused by deficiency of functional plasma clotting factor VIII (FVIII. Genetic mutations in the gene encoding FVIII (F8 have been extensively studied. Over a thousand different mutations have been reported in the F8 gene. These span a diverse range of mutation types, namely, missense, splice-site, deletions of single and multiple exons, inversions, etc. There is nonetheless evidence that other molecular mechanisms, in addition to mutations in the gene encoding the FVIII protein, may be involved in the pathobiology of HA. In this study, global small ncRNA expression profiling analysis of whole blood from HA patients, and controls, was performed using high-throughput ncRNA microarrays. Patients were further sub-divided into those that developed neutralizing-anti-FVIII antibodies (inhibitors and those that did not. Selected differentially expressed ncRNAs were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR analysis. We identified several ncRNAs, and among them hsa-miR-1246 was significantly up-regulated in HA patients. In addition, miR-1246 showed a six-fold higher expression in HA patients without inhibitors. We have identified an miR-1246 target site in the noncoding region of F8 mRNA and were able to confirm the suppressory role of hsa-miR-1246 on F8 expression in a stable lymphoblastoid cell line expressing FVIII. These findings suggest several testable hypotheses vis-à-vis the role of nc-RNAs in the regulation of F8 expression. These hypotheses have not been exhaustively tested in this study as they require carefully curated clinical samples.

  12. Analysis of a cDNA clone expressing a human autoimmune antigen: full-length sequence of the U2 small nuclear RNA-associated B antigen

    Energy Technology Data Exchange (ETDEWEB)

    Habets, W.J.; Sillekens, P.T.G.; Hoet, M.H.; Schalken, J.A.; Roebroek, A.J.M.; Leunissen, J.A.M.; Van de Ven, W.J.M.; Van Venrooij, W.J.

    1987-04-01

    A U2 small nuclear RNA-associated protein, designated B'', was recently identified as the target antigen for autoimmune sera from certain patients with systemic lupus erythematosus and other rheumatic diseases. Such antibodies enabled them to isolate cDNA clone lambdaHB''-1 from a phage lambdagt11 expression library. This clone appeared to code for the B'' protein as established by in vitro translation of hybrid-selected mRNA. The identity of clone lambdaHB''-1 was further confirmed by partial peptide mapping and analysis of the reactivity of the recombinant antigen with monospecific and monoclonal antibodies. Analysis of the nucleotide sequence of the 1015-base-pair cDNA insert of clone lambdaHB''-1 revealed a large open reading frame of 800 nucleotides containing the coding sequence for a polypeptide of 25,457 daltons. In vitro transcription of the lambdaHB''-1 cDNA insert and subsequent translation resulted in a protein product with the molecular size of the B'' protein. These data demonstrate that clone lambdaHB''-1 contains the complete coding sequence of this antigen. The deduced polypeptide sequence contains three very hydrophilic regions that might constitute RNA binding sites and/or antigenic determinants. These findings might have implications both for the understanding of the pathogenesis of rheumatic diseases as well as for the elucidation of the biological function of autoimmune antigens.

  13. Identification of small auxin-up RNA (SAUR) genes in Urticales plants: mulberry (Morus notabilis), hemp (Cannabis sativa) and ramie (Boehmeria nivea).

    Science.gov (United States)

    Huang, Xing; Bao, Yaning; Wang, B O; Liu, Lijun; Chen, Jie; Dai, Lunjin; Baloch, Sana Ullah; Peng, Dingxiang

    2016-03-01

    Small auxin-up RNA (SAUR) genes are important gene families in auxin signalling transduction and are commonly used as early auxin responsive markers. Till date, no SAUR gene is identified in Urticales plants despite of the published bioinformation of mulberry, hemp and ramie. In this study, we used Arabidopsis sequences as query to search against mulberry, hemp genomes and ramie transcriptome database. In total, we obtained 62, 56 and 71 SAUR genes in mulberry, hemp and ramie, respectively. Phylogenetic analysis revealed the Urticales specific expansion of SAUR genes. Expression analysis showed 15 randomly selected ramie SAUR genes that were diversely functioned in ramie tissues and revealed a series of IAA-responsive, drought-responsive and high temperature-responsive genes. Moreover, comparison of qRT-PCR data and previous RNA-Seq data suggested the reliability of our work. In this study, we first report the identification of SAUR genes in Urticales plants. These results will provide a foundation for their function validation in Urticales plant growth and development.

  14. Silencing of hpv16 e6 and e7 oncogenic activities by small interference rna induces autophagy and apoptosis in human cervical cancer cells

    Directory of Open Access Journals (Sweden)

    Jonathan Salazar-León

    2011-08-01

    Full Text Available Cervical cancer is the second most common form of death by cancer in women worldwide and has special attention for the development of new treatment strategies. Human Papilloma Virus (HPV persistent infection is the main etiological agent of this neoplasia, and the main cellular transformation mechanism is by disruption of p53 and pRb function by interaction with HPV E6 and E7 oncoproteins. This generates alterations in cellular differentiation and cellular death inhibition. Thus, HPV E6 and E7 oncogenes represent suitable targets for the development of gene therapy strategies against cervical cancer. An attractive technology platform is developing for post-transcriptional selective silencing of gene expression, using small interference RNA. Therefore, in the present study, we used SiHa cells (HPV16+ transiently transfected with specific siRNA expression plasmids for HPV16 E6 and E7 oncogenes. In this model we detected repression of E6 and E7 oncogene and oncoprotein expression, an increase in p53 and hypophosphorylated pRb isoform protein expression, and autophagy and apoptosis morphology features. These findings suggest that selective silencing of HPV16 E6 and E7 oncogenes by siRNAs, has significant biological effects on the survival of human cancer cells and is a potential gene therapy strategy against cervical cancer.

  15. Identification of small auxin-up RNA (SAUR) genes in Urticales plants: mulberry (Morus notabilis), hemp (Cannabis sativa) and ramie (Boehmeria nivea)

    Indian Academy of Sciences (India)

    Xing Huang; Yaning Bao; Bo Wang; Lijun Liu; Jie Chen; Lunjin Dai; Sana Ullah Baloch; Dingxiang Peng

    2016-03-01

    Small auxin-up RNA (SAUR) genes are important gene families in auxin signalling transduction and are commonly used asearly auxin responsive markers. Till date, no SAUR gene is identified in Urticales plants despite of the published bioinformationof mulberry, hemp and ramie. In this study, we used Arabidopsis sequences as query to search against mulberry, hempgenomes and ramie transcriptome database. In total, we obtained 62, 56 and 71 SAUR genes in mulberry, hemp and ramie,respectively. Phylogenetic analysis revealed the Urticales specific expansion of SAUR genes. Expression analysis showed 15 randomly selected ramie SAUR genes that were diversely functioned in ramie tissues and revealed a series of IAA-responsive,drought-responsive and high temperature-responsive genes. Moreover, comparison of qRT-PCR data and previous RNA-Seqdata suggested the reliability of our work. In this study, we first report the identification of SAUR genes in Urticales plants.These results will provide a foundation for their function validation in Urticales plant growth and development.

  16. MicroRNA-related polymorphisms in apoptosis pathway genes are predictive of clinical outcome in patients with limited disease small cell lung cancer

    Science.gov (United States)

    Jiang, Wei; Bi, Nan; Zhang, Wen-Jue; Wu, Li-Hong; Liu, Li-Pin; Men, Yu; Wang, Jing-Bo; Liang, Jun; Hui, Zhou-Guang; Zhou, Zong-Mei; Wang, Lu-Hua

    2016-01-01

    We examined the impact of single nucleotide polymorphisms (SNPs) at miRNA binding sites in the 3′-UTRs of genes in the apoptosis pathway on the prognosis of patients with limited disease-small cell lung cancer (LD-SCLC). Twelve tagSNPs in seven genes were genotyped using blood samples from 146 LD-SCLC patients treated with chemoradiotherapy. Cox proportional hazard regression models and recursive partitioning analysis were performed to identify SNPs significantly associated with overall survival. Three SNPs, CASP8: rs1045494 (C > T), PIK3R1: rs3756668 (A > G) and CASP7: rs4353229 (T > C), were associated with longer overall survival in LD-SCLC patients after chemoradiotherapy. The adjusted hazard ratios (95% confidence intervals) were 0.480 (0.258–0.894), 0.405 (0.173–0.947) and 0.446 (0.247–0.802), respectively, and remained significant after multiple comparison correction. Moreover, subset analysis showed these SNPs were still predictive of overall survival in stage III patients. Recursive partitioning analysis enabled patients to be classified into three risk subgroups based on unfavorable genotype combinations of the rs1045494 and rs4353229 SNPs. These findings suggest miRNA-related polymorphisms in the apoptosis pathway may be useful biomarkers for selection of LD-SCLC patients likely to benefit from chemoradiotherapy. PMID:26988918

  17. LncRNA-UCA1 exerts oncogenic functions in non-small cell lung cancer by targeting miR-193a-3p.

    Science.gov (United States)

    Nie, Wei; Ge, Hui-juan; Yang, Xiao-qun; Sun, Xiangjie; Huang, Hai; Tao, Xia; Chen, Wan-sheng; Li, Bing

    2016-02-01

    Recently, the long non-coding RNA (lncRNA) urothelial carcinoma-associated 1 (UCA1) has been identified as an oncogenic gene in multiple human tumor entitles, and dysregulation of UCA1 was tightly linked to carcinogenesis and cancer progression. However, whether the aberrant expression of UCA1 in non-small cell lung cancer (NSCLC) is associated with malignancy, metastasis or prognosis has not been characterized. In this study, we found that UCA1 was upregulated in NSCLC tissues. Higher expression of UCA1 led to a significantly poorer survival time, and multivariate analysis revealed that UCA1 was an independent risk factor of prognosis. UCA1 overexpression enhanced, whereas UCA1 silencing impaired the proliferation and colony formation of NSCLC cells. Moreover, mechanistic investigations showed that UCA1 upregulated the expression of miR-193a-3p target gene ERBB4 through competitively 'spongeing' miR-193a-3p. Overall, we concluded that UCA1 functions as an oncogene in NSCLC, acting mechanistically by upregulating ERBB4 in part through 'spongeing' miR-193a-3p.

  18. The rnc Gene Promotes Exopolysaccharide Synthesis and Represses the vicRKX Gene Expressions via MicroRNA-Size Small RNAs in Streptococcus mutans

    Science.gov (United States)

    Mao, Meng-Ying; Yang, Ying-Ming; Li, Ke-Zeng; Lei, Lei; Li, Meng; Yang, Yan; Tao, Xiang; Yin, Jia-Xin; Zhang, Ru; Ma, Xin-Rong; Hu, Tao

    2016-01-01

    Dental caries is a biofilm-dependent disease that largely relies on the ability of Streptococcus mutans to synthesize exopolysaccharides. Although the rnc gene is suggested to be involved in virulence mechanisms in many other bacteria, the information regarding it in S. mutans is very limited. Here, using deletion or overexpression mutant assay, we demonstrated that rnc in S. mutans significantly positively regulated exopolysaccharide synthesis and further altered biofilm formation. Meanwhile, the cariogenecity of S. mutans was decreased by deletion of rnc in a specific pathogen-free (SPF) rat model. Interestingly, analyzing the expression at mRNA level, we found the downstream vic locus was repressed by rnc in S. mutans. Using deep sequencing and bioinformatics analysis, for the first time, three putative microRNA-size small RNAs (msRNAs) targeting vicRKX were predicted in S. mutans. The expression levels of these msRNAs were negatively correlated with vicRKX but positively correlated with rnc, indicating rnc probably repressed vicRKX expression through msRNAs at the post-transcriptional level. In all, the results present that rnc has a potential role in the regulation of exopolysaccharide synthesis and can affect vicRKX expressions via post-transcriptional repression in S. mutans. This study provides an alternative avenue for further research aimed at preventing caries. PMID:27242713

  19. Determination of SGK1 mRNA in non-small cell lung cancer samples underlines high expression in squamous cell carcinomas

    Directory of Open Access Journals (Sweden)

    Abbruzzese Claudia

    2012-01-01

    Full Text Available Abstract Background Lung cancer represents the most frequent cause of death for cancer. In non-small cell lung cancer (NSCLC, which accounts for the vast majority of this disease, only early detection and treatment, when possible, may significantly affect patient's prognosis. An important role in NSCLC malignancy is attributed to the signal transduction pathways involving PI3Kinase, with consequent activation of the AKT family factors. The serum and glucocorticoid kinase (SGK factors, which share high structural and functional homologies with the AKT factors, are a family of ubiquitously expressed serine/threonine kinases under the control of cellular stress and hormones. SGK1 is the most represented SGK member. Methods By means of immunohistochemistry and quantitative real-time PCR, we determined SGK1 protein and mRNA expression in a cohort of 66 formalin-fixed, paraffin-embedded NSCLC surgical samples. All samples belonged to patients with a well-documented clinical history. Results mRNA expression was significantly higher in squamous cell carcinomas, and correlated with several clinical prognostic indicators, being elevated in high-grade tumors and in tumors with bigger size and worse clinical stage. No correlation was found between SGK1 protein expression and these clinical parameters. Conclusions This explorative analysis of SGK1 expression in NSCLC samples highlights the potential role of this factor in NSCLC patients' prognosis. Moreover, the higher expression in the squamous cell carcinoma subtype opens new therapeutic possibilities in this NSCLC subtype by designing specific kinase inhibitors.

  20. Identification of small auxin-up RNA (SAUR) genes in Urticales plants: mulberry (Morus notabilis), hemp (Cannabis sativa) and ramie (Boehmeria nivea).

    Science.gov (United States)

    Huang, Xing; Bao, Yaning; Wang, B O; Liu, Lijun; Chen, Jie; Dai, Lunjin; Baloch, Sana Ullah; Peng, Dingxiang

    2016-03-01

    Small auxin-up RNA (SAUR) genes are important gene families in auxin signalling transduction and are commonly used as early auxin responsive markers. Till date, no SAUR gene is identified in Urticales plants despite of the published bioinformation of mulberry, hemp and ramie. In this study, we used Arabidopsis sequences as query to search against mulberry, hemp genomes and ramie transcriptome database. In total, we obtained 62, 56 and 71 SAUR genes in mulberry, hemp and ramie, respectively. Phylogenetic analysis revealed the Urticales specific expansion of SAUR genes. Expression analysis showed 15 randomly selected ramie SAUR genes that were diversely functioned in ramie tissues and revealed a series of IAA-responsive, drought-responsive and high temperature-responsive genes. Moreover, comparison of qRT-PCR data and previous RNA-Seq data suggested the reliability of our work. In this study, we first report the identification of SAUR genes in Urticales plants. These results will provide a foundation for their function validation in Urticales plant growth and development. PMID:27019439

  1. Identification of Circulating miRNAs in a Mouse Model of Nerve Allograft Transplantation under FK506 Immunosuppression by Illumina Small RNA Deep Sequencing

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    Shao-Chun Wu

    2015-01-01

    Full Text Available Background. This study aimed to establish the expression profile of circulating microRNAs (miRNAs during nerve allotransplantation in the presence and absence of FK506 immunosuppression. Methods. A 1 cm BALB/c donor sciatic nerve graft was transplanted into the sciatic nerve gaps created in recipient C57BL/6 mice with or without daily FK506 immunosuppression [1 mg/(kg·d]. At 3, 7, and 14 d after nerve allotransplantation, serum samples were collected for miRNA expression analysis by Illumina small RNA deep sequencing. Results. Sequence analysis showed that the dominant size of circulating small RNAs after nerve allotransplantation was 22 nucleotides, followed by 23-nucleotide sequences. Nine upregulated circulating miRNAs (let-7e-5p, miR-101a-3p, miR-151-5p, miR-181a-5p, miR-204-5p, miR-340-5p, miR-381-3p, miR-411-5p, miR-9-5p, and miR-219-2-3p were identified at 3 d, but none was identified at 7 or 14 d. Among them, miR-9-5p had the highest fold-change of >50-fold, followed by miR-340-5p with 38.8-fold. The presence of these nine miRNAs was not significant at 7 and 14 d after nerve allotransplantation with or without immunosuppression, showing that these miRNAs are not ideal biomarkers for monitoring rejection of deep-buried nerve allografts, a response usually observed later. Conclusions. We identified nine upregulated circulating miRNAs, which may have a biological function, particularly during the early stages after nerve allotransplantation under FK506 immunosuppression.

  2. Regulation function of non-coding small RNA in Salmonella%沙门菌非编码小RNA调控功能研究进展

    Institute of Scientific and Technical Information of China (English)

    孟霞; 王亨; 朱国强

    2011-01-01

    细菌非编码小RNA(sRNA)是原核生物中新发现的基因表达调控因子,可在转录后水平调节基因的表达。沙门菌是sRNA研究的模式菌,研究者利用生物信息学预测技术、全基因组分析技术和高通量RNA测序技术,至少发现70余种沙门菌sRNA。它们通过感应温度,pH值,渗透压或氧分压等环境信号,利用碱基互补方式与靶标mRNA结合,调控靶标mRNA的翻译、降解或稳定性。通常一种sRNA有多个靶基因或靶位点,可调节多种基因的表达,在沙门菌营养物质代谢、外膜蛋白合成、群体感应和毒力表达等诸多生命过程中发挥重要的调控作用。%Non-coding small RNA(sRNA) is a new factor to regulate gene expression at post-transcriptional level in prokaryote.As a model organism for sRNA research,at least 70 sRNAs have been identified by biocomputational prediction approaches,the whole genome analysis and high-throughout sequencing technology in Salmonella species.These sRNA can regulate translation,degradation and stability of target mRNAs by base pairing with them while sensing environmental signal such as temperature,pH,osmotic pressure and oxygen concentration.One sRNA can regulate multiple genes expression via targeting mRNA or gene sites.They play an important regulation role in substance metabolism,outer membrane proteins synthesis,quorum sensing and virulence gene expression.

  3. Identification of the base-pairing requirements for repression of hctA translation by the small RNA IhtA leads to the discovery of a new mRNA target in Chlamydia trachomatis.

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    Nicole A Grieshaber

    Full Text Available The non-coding small RNA, IhtA expressed by the obligate intracellular human pathogen Chlamydia trachomatis modulates the translation of HctA, a key protein involved in replicative to infectious cell type differentiation. Using a combination of bioinformatics and mutagenesis we sought to identify the base pairing requirement for functional repression of HctA protein expression, with an eye to applying our findings towards the identification of additional targets. IhtA is predicted to fold into a three stem:loop structure. We found that loop 1 occludes the initiation codon of hctA, while loop 2 and 3 are not required for function. This 7 nucleotide region forms G/C rich interactions surrounding the AUG of hctA. Two additional genes in the chlamydial genome, CTL0322 and CTL0097, contained some elements of the hctA:IhtA recognition sequence. The mRNA of both CTL0322and CTL0097 interacted with IhtA in vitro as measured by biolayer interferometry. However, using a CheZ reporter expression system, IhtA only inhibited the translation of CTL0322. The proposed IhtA recognition site in the CTL0322 message contains significant G/C base pairing on either side of the initiation codon while CTL0097 only contains G/C base pairing 3' to the AUG initiation codon. These data suggest that as the functional interacting region is only 6-7nt in length that full translation repression is dependent on the degree of G/C base pairing. Additionally our results indicate that IhtA may regulate multiple mRNAs involved in the chlamydial infectious cycle.

  4. Identification of the Base-Pairing Requirements for Repression of hctA Translation by the Small RNA IhtA Leads to the Discovery of a New mRNA Target in Chlamydia trachomatis

    Science.gov (United States)

    Grieshaber, Nicole A.; Tattersall, Jeremiah S.; Liguori, Johella; Lipat, Joseph N.; Runac, Justin; Grieshaber, Scott S.

    2015-01-01

    The non-coding small RNA, IhtA expressed by the obligate intracellular human pathogen Chlamydia trachomatis modulates the translation of HctA, a key protein involved in replicative to infectious cell type differentiation. Using a combination of bioinformatics and mutagenesis we sought to identify the base pairing requirement for functional repression of HctA protein expression, with an eye to applying our findings towards the identification of additional targets. IhtA is predicted to fold into a three stem:loop structure. We found that loop 1 occludes the initiation codon of hctA, while loop 2 and 3 are not required for function. This 7 nucleotide region forms G/C rich interactions surrounding the AUG of hctA. Two additional genes in the chlamydial genome, CTL0322 and CTL0097, contained some elements of the hctA:IhtA recognition sequence. The mRNA of both CTL0322and CTL0097 interacted with IhtA in vitro as measured by biolayer interferometry. However, using a CheZ reporter expression system, IhtA only inhibited the translation of CTL0322. The proposed IhtA recognition site in the CTL0322 message contains significant G/C base pairing on either side of the initiation codon while CTL0097 only contains G/C base pairing 3’ to the AUG initiation codon. These data suggest that as the functional interacting region is only 6-7nt in length that full translation repression is dependent on the degree of G/C base pairing. Additionally our results indicate that IhtA may regulate multiple mRNAs involved in the chlamydial infectious cycle. PMID:25756658

  5. Antitumor effect of RNA interference on non-small-cell lung cancer in vivo%RNA干扰技术抗非小细胞肺癌作用的体内实验研究

    Institute of Scientific and Technical Information of China (English)

    Min Zhang; Chunxue Bai; Xin Zhang; Ling Mao; Yuehong Wang

    2009-01-01

    Objective: Lung cancer has emerged as a leading cause of cancer death in the world. Current therapies are ineffective, thus new approaches are needed to improve the therapeutic ratio. RNA interference (RNAi) has shown promise in gene silencing in vitro, the potential of which in developing new methods for the therapy of non-small-cell lung cancer (NSCLC) needs to be further tested in vivo. In this study, chemically synthesized double-stranded RNA (dsRNA) targeting epidermal growth factor receptor (EGFR) was transfected into NSCLC cell line SPC-A1 cells and established the tumor bur-dened athymic nude mice model to investigate whether dsRNA could induce gene silencing in NSCLC cells in vivo. Methods:SPC-A1 was transfected with EGFR sequence-specific dsRNA formulated with Lipofectamine 2000. SPC-A1 cells (1 x 107>/mL) in 200 μL were injected s.c. Into the left flank area of the mice to establish the tumor burdened athymic nude mice model.Calculate the tumor growth inhibition rate by measuring the diameter and the weight of the tumor. Immunohistochemistry and Western blot were used to monitor the reduction in the production of the EGFR protein. Realtime RT-PCR was used to detect the silencing of the EGFR mRNA level. Results: It displayed that EGFR sequence specific dsRNA (dsRNA-EGFR)significantly inhibited the tumor growth in vivo. The tumor growth inhibition rate was 75.03%. The dsRNA-EGFR sequence specifically silenced EGFR with 53.6% of down-regulation of EGFR protein production and 32.3% of silencing of EGFR mRNA level. Conclusion: DsRNA-EGFR showed a blockbuster effect in downregulation of EGFR mRNA level and protein produc-tion, and inhibition of tumor growth in vivo.

  6. SOT1, a pentatricopeptide repeat protein with a small MutS-related domain, is required for correct processing of plastid 23S-4.5S rRNA precursors in Arabidopsis thaliana.

    Science.gov (United States)

    Wu, Wenjuan; Liu, Sheng; Ruwe, Hannes; Zhang, Delin; Melonek, Joanna; Zhu, Yajuan; Hu, Xupeng; Gusewski, Sandra; Yin, Ping; Small, Ian D; Howell, Katharine A; Huang, Jirong

    2016-03-01

    Ribosomal RNA processing is essential for plastid ribosome biogenesis, but is still poorly understood in higher plants. Here, we show that SUPPRESSOR OF THYLAKOID FORMATION1 (SOT1), a plastid-localized pentatricopeptide repeat (PPR) protein with a small MutS-related domain, is required for maturation of the 23S-4.5S rRNA dicistron. Loss of SOT1 function leads to slower chloroplast development, suppression of leaf variegation, and abnormal 23S and 4.5S processing. Predictions based on the PPR motif sequences identified the 5' end of the 23S-4.5S rRNA dicistronic precursor as a putative SOT1 binding site. This was confirmed by electrophoretic mobility shift assay, and by loss of the abundant small RNA 'footprint' associated with this site in sot1 mutants. We found that more than half of the 23S-4.5S rRNA dicistrons in sot1 mutants contain eroded and/or unprocessed 5' and 3' ends, and that the endonucleolytic cleavage product normally released from the 5' end of the precursor is absent in a sot1 null mutant. We postulate that SOT1 binding protects the 5' extremity of the 23S-4.5S rRNA dicistron from exonucleolytic attack, and favours formation of the RNA structure that allows endonucleolytic processing of its 5' and 3' ends. PMID:26800847

  7. A complex small RNA repertoire is generated by a plant/fungal-like machinery and effected by a metazoan-like Argonaute in the single-cell human parasite Toxoplasma gondii.

    Science.gov (United States)

    Braun, Laurence; Cannella, Dominique; Ortet, Philippe; Barakat, Mohamed; Sautel, Céline F; Kieffer, Sylvie; Garin, Jérôme; Bastien, Olivier; Voinnet, Olivier; Hakimi, Mohamed-Ali

    2010-05-27

    In RNA silencing, small RNAs produced by the RNase-III Dicer guide Argonaute-like proteins as part of RNA-induced silencing complexes (RISC) to regulate gene expression transcriptionally or post-transcriptionally. Here, we have characterized the RNA silencing machinery and exhaustive small RNAome of Toxoplasma gondii, member of the Apicomplexa, a phylum of animal- and human-infecting parasites that cause extensive health and economic damages to human populations worldwide. Remarkably, the small RNA-generating machinery of Toxoplasma is phylogenetically and functionally related to that of plants and fungi, and accounts for an exceptionally diverse array of small RNAs. This array includes conspicuous populations of repeat-associated small interfering RNA (siRNA), which, as in plants, likely generate and maintain heterochromatin at DNA repeats and satellites. Toxoplasma small RNAs also include many microRNAs with clear metazoan-like features whose accumulation is sometimes extremely high and dynamic, an unexpected finding given that Toxoplasma is a unicellular protist. Both plant-like heterochromatic small RNAs and metazoan-like microRNAs bind to a single Argonaute protein, Tg-AGO. Toxoplasma miRNAs co-sediment with polyribosomes, and thus, are likely to act as translational regulators, consistent with the lack of catalytic residues in Tg-AGO. Mass spectrometric analyses of the Tg-AGO protein complex revealed a common set of virtually all known RISC components so far characterized in human and Drosophila, as well as novel proteins involved in RNA metabolism. In agreement with its loading with heterochromatic small RNAs, Tg-AGO also associates substoichiometrically with components of known chromatin-repressing complexes. Thus, a puzzling patchwork of silencing processor and effector proteins from plant, fungal and metazoan origin accounts for the production and action of an unsuspected variety of small RNAs in the single-cell parasite Toxoplasma and possibly in other

  8. Elevated paternal glucocorticoid exposure alters the small noncoding RNA profile in sperm and modifies anxiety and depressive phenotypes in the offspring.

    Science.gov (United States)

    Short, A K; Fennell, K A; Perreau, V M; Fox, A; O'Bryan, M K; Kim, J H; Bredy, T W; Pang, T Y; Hannan, A J

    2016-01-01

    Recent studies have suggested that physiological and behavioral traits may be transgenerationally inherited through the paternal lineage, possibly via non-genomic signals derived from the sperm. To investigate how paternal stress might influence offspring behavioral phenotypes, a model of hypothalamic-pituitary-adrenal (HPA) axis dysregulation was used. Male breeders were administered water supplemented with corticosterone (CORT) for 4 weeks before mating with untreated female mice. Female, but not male, F1 offspring of CORT-treated fathers displayed altered fear extinction at 2 weeks of age. Only male F1 offspring exhibited altered patterns of ultrasonic vocalization at postnatal day 3 and, as adults, showed decreased time in open on the elevated-plus maze and time in light on the light-dark apparatus, suggesting a hyperanxiety-like behavioral phenotype due to paternal CORT treatment. Interestingly, expression of the paternally imprinted gene Igf2 was increased in the hippocampus of F1 male offspring but downregulated in female offspring. Male and female F2 offspring displayed increased time spent in the open arm of the elevated-plus maze, suggesting lower levels of anxiety compared with control animals. Only male F2 offspring showed increased immobility time on the forced-swim test and increased latency to feed on the novelty-supressed feeding test, suggesting a depression-like phenotype in these animals. Collectively, these data provide evidence that paternal CORT treatment alters anxiety and depression-related behaviors across multiple generations. Analysis of the small RNA profile in sperm from CORT-treated males revealed marked effects on the expression of small noncoding RNAs. Sperm from CORT-treated males contained elevated levels of three microRNAs, miR-98, miR-144 and miR-190b, which are predicted to interact with multiple growth factors, including Igf2 and Bdnf. Sustained elevation of glucocorticoids is therefore involved in the transmission of paternal

  9. Investigation of radiation-induced transcriptome profile of radioresistant non-small cell lung cancer A549 cells using RNA-seq.

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    Hee Jung Yang

    Full Text Available Radioresistance is a main impediment to effective radiotherapy for non-small cell lung cancer (NSCLC. Despite several experimental and clinical studies of resistance to radiation, the precise mechanism of radioresistance in NSCLC cells and tissues still remains unclear. This result could be explained by limitation of previous researches such as a partial understanding of the cellular radioresistance mechanism at a single molecule level. In this study, we aimed to investigate extensive radiation responses in radioresistant NSCLC cells and to identify radioresistance-associating factors. For the first time, using RNA-seq, a massive sequencing-based approach, we examined whole-transcriptome alteration in radioresistant NSCLC A549 cells under irradiation, and verified significant radiation-altered genes and their chromosome distribution patterns. Also, bioinformatic approaches (GO analysis and IPA were performed to characterize the radiation responses in radioresistant A549 cells. We found that epithelial-mesenchymal transition (EMT, migration and inflammatory processes could be meaningfully related to regulation of radiation responses in radioresistant A549 cells. Based on the results of bioinformatic analysis for the radiation-induced transcriptome alteration, we selected seven significant radiation-altered genes (SESN2, FN1, TRAF4, CDKN1A, COX-2, DDB2 and FDXR and then compared radiation effects in two types of NSCLC cells with different radiosensitivity (radioresistant A549 cells and radiosensitive NCI-H460 cells. Interestingly, under irradiation, COX-2 showed the most significant difference in mRNA and protein expression between A549 and NCI-H460 cells. IR-induced increase of COX-2 expression was appeared only in radioresistant A549 cells. Collectively, we suggest that COX-2 (also known as prostaglandin-endoperoxide synthase 2 (PTGS2 could have possibility as a putative biomarker for radioresistance in NSCLC cells.

  10. Effect of MicroRNA-335 on the Metastasis, Invasion and Proliferation of Cells in Patients with Non-small Cell Lung Cancer

    Institute of Scientific and Technical Information of China (English)

    Wang He; Liu Zhili; Wang Zhaoxia

    2013-01-01

    Objective: To investigate the effect of microRNA-335 on the metastasis, invasion and proliferation of cells in patients with non-small-cell lung cancer (NSCLC). Methods:Real-time PCR was performed to detect the expression differences of microRNA-335 between 12 pairs of NSCLC and normal cancerous peripheral tissues, and between SPCA-1 cells of NSCLC and 16HBE of normal pulmonary epithelial cells, while miR-335 expression in SPCA-1 cells were down-regulated and proved by Lipofectamine 2000 transient transfection and real-time PCR, respectively. Scratch test, Transwell invasion assay as well as MTT and clone formation assays were applied to respectively determine the effect of miR-335 on the metastasis, invasion and proliferation of SPCA-1 cells. Results:Compared with para-carcinoma tissues and 16HBE cells, miR-335 expression was evidently higher in NSCLC and SPCA-1 cells. However, it decreased remarkably after transient transfection of anti-miR-335 by SPCA-1 cells with Lipofectamine 2000 for 24 h. Metastasis and invasion of SPCA-1 cells could be inhibited by suppressing miR-335 expression with suppression rates being (42.8±2.7)%and (73.25±4.4)%, respectively. However, the inhibition of miR-335 expression had no effect on the proliferation of SPCA-1 cells. Conclusion:miR-335 expresses highly in NSCLC and its low expression can obviously inhibit the metastasis and invasion of SPCA-1 cells, but has no effect on the proliferation.

  11. Expanding the RpoS/σS-network by RNA sequencing and identification of σS-controlled small RNAs in Salmonella.

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    Corinne Lévi-Meyrueis

    Full Text Available The RpoS/σS sigma subunit of RNA polymerase (RNAP controls a global adaptive response that allows many Gram-negative bacteria to survive starvation and various stresses. σS also contributes to biofilm formation and virulence of the food-borne pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium. In this study, we used directional RNA-sequencing and complementary assays to explore the σS-dependent transcriptome of S. Typhimurium during late stationary phase in rich medium. This study confirms the large regulatory scope of σS and provides insights into the physiological functions of σS in Salmonella. Extensive regulation by σS of genes involved in metabolism and membrane composition, and down-regulation of the respiratory chain functions, were important features of the σS effects on gene transcription that might confer fitness advantages to bacterial cells and/or populations under starving conditions. As an example, we show that arginine catabolism confers a competitive fitness advantage in stationary phase. This study also provides a firm basis for future studies to address molecular mechanisms of indirect regulation of gene expression by σS. Importantly, the σS-controlled downstream network includes small RNAs that might endow σS with post-transcriptional regulatory functions. Of these, four (RyhB-1/RyhB-2, SdsR, SraL were known to be controlled by σS and deletion of the sdsR locus had a competitive fitness cost in stationary phase. The σS-dependent control of seven additional sRNAs was confirmed in Northern experiments. These findings will inspire future studies to investigate molecular mechanisms and the physiological impact of post-transcriptional regulation by σS.

  12. Characterization of 12S rRNA gene for meat identification of common wild and domestic small herbivores as an aid to wildlife forensic

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    E. Joseph

    2013-10-01

    Full Text Available Aim: Chital and sambar are the common wild small herbivores, which are vulnerable to poaching for their meat. Many times poachers claim the wild meat to be that of goat or sheep. Hence, authentic evidences are required to stop such wildlife crime. The present investigation was carried out to study the species specific PCR-RFLP patterns for meat identification of chital and sambar and then differentiation from the meat of goat and sheep. Materials and Methods: Extracted DNA from meat samples were subjected to PCR using the universal primers of 12S rRNA gene. The PCR products were subjected to RFLP and sequencing. Results: The size of amplified PCR products was similar (440 bp in each species and sequence alignment showed more than 89 % similarities among these species. However, phylogenetic analysis revealed that Chital and Sambar are in one cluster while Goat and sheep are in other cluster. To differentiate between species, restriction digestion of the PCR products was carried out to produce characteristic PCR-RFLP patterns for each species. Restriction digestion with RsaI and AluI enzymes produced distinct PCR-RFLP patterns that differentiated the meat of wild species (chital-sambar from that of domestic species (goat-sheep. BsrI restriction digestion revealed unique PCR-RFLP pattern in chital differentiating it from the meat of other three species. Restriction digestion with DdeI enzyme led to the production of distinct PCR-RFLP patterns for chital and sambar to identify their meat individually. Conclusion: This study showed the effectiveness of 12S rRNA gene polymorphism in meat identification. The data can be used as evidence against the poachers to convict the wildlife crime in the court of law [Vet World 2013; 6(5.000: 254-259

  13. Translation inhibition of the developmental cycle protein HctA by the small RNA IhtA is conserved across Chlamydia.

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    Jeremiah Tattersall

    Full Text Available The developmental cycle of the obligate intracellular pathogen Chlamydia trachomatis serovar L2 is controlled in part by the small non-coding RNA (sRNA, IhtA. All Chlamydia alternate in a regulated fashion between the infectious elementary body (EB and the replicative reticulate body (RB which asynchronously re-differentiates back to the terminal EB form at the end of the cycle. The histone like protein HctA is central to RB:EB differentiation late in the cycle as it binds to and occludes the genome, thereby repressing transcription and translation. The sRNA IhtA is a critical component of this regulatory loop as it represses translation of hctA until late in infection at which point IhtA transcription decreases, allowing HctA expression to occur and RB to EB differentiation to proceed. It has been reported that IhtA is expressed during infection by the human pathogens C. trachomatis serovars L2, D and L2b and C. pneumoniae. We show in this work that IhtA is also expressed by the animal pathogens C. caviae and C. muridarum. Expression of HctA in E. coli is lethal and co-expression of IhtA relieves this phenotype. To determine if regulation of HctA by IhtA is a conserved mechanism across pathogenic chlamydial species, we cloned hctA and ihtA from C. trachomatis serovar D, C. muridarum, C. caviae and C. pneumoniae and assayed for rescue of growth repression in E. coli co-expression studies. In each case, co-expression of ihtA with the cognate hctA resulted in relief of growth repression. In addition, expression of each chlamydial species IhtA rescued the lethal phenotype of C. trachomatis serovar L2 HctA expression. As biolayer interferometry studies indicate that IhtA interacts directly with hctA message for all species tested, we predict that conserved sequences of IhtA are necessary for function and/or binding.

  14. Translation inhibition of the developmental cycle protein HctA by the small RNA IhtA is conserved across Chlamydia.

    Science.gov (United States)

    Tattersall, Jeremiah; Rao, Geeta Vittal; Runac, Justin; Hackstadt, Ted; Grieshaber, Scott S; Grieshaber, Nicole A

    2012-01-01

    The developmental cycle of the obligate intracellular pathogen Chlamydia trachomatis serovar L2 is controlled in part by the small non-coding RNA (sRNA), IhtA. All Chlamydia alternate in a regulated fashion between the infectious elementary body (EB) and the replicative reticulate body (RB) which asynchronously re-differentiates back to the terminal EB form at the end of the cycle. The histone like protein HctA is central to RB:EB differentiation late in the cycle as it binds to and occludes the genome, thereby repressing transcription and translation. The sRNA IhtA is a critical component of this regulatory loop as it represses translation of hctA until late in infection at which point IhtA transcription decreases, allowing HctA expression to occur and RB to EB differentiation to proceed. It has been reported that IhtA is expressed during infection by the human pathogens C. trachomatis serovars L2, D and L2b and C. pneumoniae. We show in this work that IhtA is also expressed by the animal pathogens C. caviae and C. muridarum. Expression of HctA in E. coli is lethal and co-expression of IhtA relieves this phenotype. To determine if regulation of HctA by IhtA is a conserved mechanism across pathogenic chlamydial species, we cloned hctA and ihtA from C. trachomatis serovar D, C. muridarum, C. caviae and C. pneumoniae and assayed for rescue of growth repression in E. coli co-expression studies. In each case, co-expression of ihtA with the cognate hctA resulted in relief of growth repression. In addition, expression of each chlamydial species IhtA rescued the lethal phenotype of C. trachomatis serovar L2 HctA expression. As biolayer interferometry studies indicate that IhtA interacts directly with hctA message for all species tested, we predict that conserved sequences of IhtA are necessary for function and/or binding.

  15. 小干扰RNA对人淀粉样前体蛋白基因的沉默作用%Silencing effect of amyloid precursor protein triggered by small interfering RNA

    Institute of Scientific and Technical Information of China (English)

    邱昕; 潘纪安; 陈宇; 张苏明; 郭德银

    2005-01-01

    [Objective] Much needs to be learned about human amyloid precursor protein (APP) gene functions because APP may be etiologically involved in Alzheimer disease(AD). However, physiological function of the APP remains to be elucidated. As a novel technique, RNA interference (RNAi) is quite appropriate to analyze APP gene function. [Methods] In order to observe silencing effect of small interference RNA (siRNA) directed against APP gene, a designed oligonucleotide was transcribed endogenously into small hairpin RNA (shRNA) and two enhanced green fluorescence protein (EGFP) carried two APP isoforms recombinant plasmids were constructed. It will demonstrate the silencing effect by cotransfection of siRNA expression plasmid and GFP-APP recombinant plasmids into COS-7 cells because EGFP will act as a reporter gene. [Results] The siRNA directed against APP could silence APP gene effectively. [Conclusion] Therefore, siRNA directed against APP will play a crucial role in the functional exploration of APP gene.%目的观察小干扰RNA(siRNA)对人淀粉样前体蛋白(APP)基因的沉默作用.方法该研究将设计好的一对寡核甘酸在细胞内源性地转录成小发夹RNA(shRNA),同时构建了携带两个APP亚型的增强型绿色荧光蛋白(EGFP)的GFP-APP重组质粒.利用EGFP作为报告基因,在COS-7细胞内通过共转染siRNA表达质粒和GFP-APP的重组质粒来观察沉默效应.结果针对APP的siRNA可有效地下调APP基因.结论针对APP的siRNA将不仅在研究APP基因功能和深入研究阿尔茨海默病(AD)的病理分子机制上,而且在运用siRNA这种新型的反义核酸药物治疗AD方面扮演重要角色.

  16. MicroRNA-187-5p suppresses cancer cell progression in non-small cell lung cancer (NSCLC) through down-regulation of CYP1B1.

    Science.gov (United States)

    Mao, Ming; Wu, Zhouqing; Chen, Jiakuan

    2016-09-16

    Lung cancer remains a leading cause of cancer-associated mortality worldwide and non-small lung cancer (NSCLC) is responsible for over 80% of lung cancer-related deaths. Identifying novel molecular biomarker that can inhibit the progression of lung cancer will facilitate the development of new treatment strategies. Herein, we demonstrated that miR-187-5p is a tumor-suppressor miRNA in NSCLC progression. We found that expression of miR-187-5p was decreased obviously in NSCLC tissues. Down-regulation of miR-187-5p was associated with TNM stage and postoperative survival. Overexpression of miR-187-5p inhibited the growth and metastasis of NSCLC cells. The CYP1B1 was a direct target of miR-187-5p and promoted the growth and metastasis of NSCLC cells. Further study showed that CYP1B1 could reverse the inhibitory effect of miR-187-5p on growth and metastasis of NSCLC cells. Taken together, our data highlight the pivotal role of miR-187-5p in the progression of NSCLC. Thus, miR-187-5p may be a potential prognostic marker and of treatment relevance for NSCLC progression intervention. PMID:27495872

  17. Downregulation of Cyclophilin A by siRNA diminishes non-small cell lung cancer cell growth and metastasis via the regulation of matrix metallopeptidase 9

    Directory of Open Access Journals (Sweden)

    Qian Zhe

    2012-10-01

    Full Text Available Abstract Background Cyclophilin A (CypA is a cytosolic protein possessing peptidyl-prolyl isomerase activity that was recently reported to be overexpressed in several cancers. Here, we explored the biology and molecular mechanism of CypA in non-small cell lung cancer (NSCLC. Methods The expression of CypA in human NSCLC cell lines was detected by real-time reverse transcription PCR. The RNA interference-mediated knockdown of CypA was established in two NSCLC cell lines (95C and A549. 239836 CypA inhibitor was also used to suppress CypA activity. Tumorigenesis was assessed based on cellular proliferation, colony formation assays, and anchorage-independent growth assays; metastasis was assessed based on wound healing and transwell assays. Results Suppression of CypA expression inhibited the cell growth and colony formation of A549 and 95C cells. CypA knockdown resulted in the inhibition of cell motility and invasion. Significantly, we show for the first time that CypA increased NSCLC cell invasion by regulating the activity of secreted matrix metallopeptidase 9 (MMP9. Likewise, suppression of CypA with 239836 CypA inhibitor decreased cell proliferation and MMP9 activity. Conclusions The suppression of CypA expression was correlated with decreased NSCLC cell tumorigenesis and metastasis.

  18. MicroRNA-449a is downregulated in non-small cell lung cancer and inhibits migration and invasion by targeting c-Met.

    Directory of Open Access Journals (Sweden)

    Wenting Luo

    Full Text Available MicroRNA-449a is expressed at a low level in several tumors and cancer cell lines, and induces G1 arrest, apoptosis, and senescence. To identify the function of miR-449a in non-small cell lung cancer (NSCLC, we discussed the potential relevance of miR-449a to clinicopathological characteristics and prognosis in NSCLC. We also investigated the impact of miR-449a on migration and invasion in NSCLC cells. The expression of miR-449a in NSCLC tissues and cell lines was detected using RT-qPCR. In vitro, gain-of-function, loss-of-function experiments, and fluorescence assays were performed to identify the potential target of miR-449a and the function of miR-449a in NSCLC cells. MiR-449a was downregulated in both NSCLC tissues and cell lines. Moreover, a low expression level of miR-449a appeared to be correlated with lymph node metastasis and poor survival. In vitro, miR-449 regulated cell migration and invasion in NSCLC cells as a potential tumor suppressor, at least in part by targeting c-Met. Furthermore, reciprocal expression of miR-449a and c-Met was shown in NSCLC tissue samples. This study indicates that miR-449a might be associated with NSCLC progression, and suggests a crucial role for miR-449a in NSCLC.

  19. Small kinetochore associated protein (SKAP promotes UV-induced cell apoptosis through negatively regulating pre-mRNA processing factor 19 (Prp19.

    Directory of Open Access Journals (Sweden)

    Shan Lu

    Full Text Available Apoptosis is a regulated cellular suicide program that is critical for the development and maintenance of healthy tissues. Previous studies have shown that small kinetochore associated protein (SKAP cooperates with kinetochore and mitotic spindle proteins to regulate mitosis. However, the role of SKAP in apoptosis has not been investigated. We have identified a new interaction involving SKAP, and we propose a mechanism through which SKAP regulates cell apoptosis. Our experiments demonstrate that both overexpression and knockdown of SKAP sensitize cells to UV-induced apoptosis. Further study has revealed that SKAP interacts with Pre-mRNA processing Factor 19 (Prp19. We find that UV-induced apoptosis can be inhibited by ectopic expression of Prp19, whereas silencing Prp19 has the opposite effect. Additionally, SKAP negatively regulates the protein levels of Prp19, whereas Prp19 does not alter SKAP expression. Finally, rescue experiments demonstrate that the pro-apoptotic role of SKAP is executed through Prp19. Taken together, these findings suggest that SKAP promotes UV-induced cell apoptosis by negatively regulating the anti-apoptotic protein Prp19.

  20. Upregulation of long intergenic noncoding RNA 00673 promotes tumor proliferation via LSD1 interaction and repression of NCALD in non-small-cell lung cancer.

    Science.gov (United States)

    Shi, Xuefei; Ma, Chenhui; Zhu, Qingqing; Yuan, Dongmei; Sun, Ming; Gu, Xiaoling; Wu, Guannan; Lv, Tangfeng; Song, Yong

    2016-05-01

    Despite improvements in diagnostics and treatment of non-small cell lung cancer (NSCLC), it remains the leading causes of cancer-related mortality worldwide. In more recent years, mutiple lines of evidence have highlighted long noncoding RNAs (lncRNAs) serve as novel class of regulators of cancer biological processes, including proliferation, apoptosis and metastasis. LncRNAs serve as a novel class of regulators of cancer biological processes in cancer, but little is known of their expression and potential functions in NSCLC. We identified an oncogene, linc00673, whose expression level was upregulated by bioinformatics analyses and qRT-PCR analyses in NSCLC. The effects of linc00673 on tumor progression were investigated in vitro and in vivo. Linc00673 knockdown significantly inhibited cell proliferation and colony-forming ability, and suppressed S-phase entry in vitro and shRNA linc00673 mediated knockdown significantly inhibit tumor growth in vivo, meanwhile, linc00673 overexpression increased tumor cell growth. Analysis of RNAseq data revealed linc00673 could modulate the transcription of a large amount of genes including oncogene and tumor suppressor gene, so we investigated the role and regulatory mechanism of linc00673 in NSCLC proliferation. Further mechanistic analyses indicated that the oncogenic activity of linc00673 is partially attributable to its repression of NCALD through association with the epigenetic repressor LSD1. Taken together, these findings suggested that linc00673 could play crucial role in NSCLC progression and might be a potential therapeutic target for patients with NSCLC. PMID:27027352

  1. Time-Course Small RNA Profiling Reveals Rice miRNAs and Their Target Genes in Response to Rice Stripe Virus Infection

    Science.gov (United States)

    Choi, Hoseong; Kim, Kook-Hyung

    2016-01-01

    It has been known that many microRNAs (miRNAs) are involved in the regulation for the plant development and defense mechanism by regulating the expression of the target gene. Several previous studies has demonstrated functional roles of miRNAs in antiviral defense mechanisms. In this study, we employed high-throughput sequencing technology to identify rice miRNAs upon rice stripe virus (RSV) infection at three different time points. Six libraries from mock and RSV-infected samples were subjected for small RNA sequencing. Bioinformatic analyses revealed 374 known miRNAs and 19 novel miRNAs. Expression of most identified miRNAs was not dramatically changed at 3 days post infection (dpi) and 7 dpi by RSV infection. However, many numbers of miRNAs were up-regulated in mock and RSV-infected samples at 15 dpi by RSV infection. Moreover, expression profiles of identified miRNAs revealed that only few numbers of miRNAs were strongly regulated by RSV infection. In addition, 15 resistance genes were targets of six miRNAs suggesting that those identified miRNAs and 15 NBS-LRR resistance genes might be involved in RSV infection. Taken together, our results provide novel insight into the dynamic expression profiles of rice miRNAs upon RSV infection and clues for the understanding of the regulatory roles of miRNAs via time-course. PMID:27626631

  2. Small RNA and degradome deep sequencing reveals drought-and tissue-specific micrornas and their important roles in drought-sensitive and drought-tolerant tomato genotypes.

    Science.gov (United States)

    Candar-Cakir, Bilgin; Arican, Ercan; Zhang, Baohong

    2016-08-01

    Drought stress has adverse impacts on plant production and productivity. MicroRNAs (miRNAs) are one class of noncoding RNAs regulating gene expression post-transcriptionally. In this study, we employed small RNA and degradome sequencing to systematically investigate the tissue-specific miRNAs responsible to drought stress, which are understudied in tomato. For this purpose, root and upground tissues of two different drought-responsive tomato genotypes (Lycopersicon esculentum as sensitive and L. esculentum var. cerasiforme as tolerant) were subjected to stress with 5% polyethylene glycol for 7 days. A total of 699 conserved miRNAs belonging to 578 families were determined and 688 miRNAs were significantly differentially expressed between different treatments, tissues and genotypes. Using degradome sequencing, 44 target genes were identified associated with 36 miRNA families. Drought-related miRNAs and their targets were enriched functionally by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Totally, 53 miRNAs targeted 23 key drought stress- and tissue development-related genes, including DRP (dehydration-responsive protein), GTs (glycosyltransferases), ERF (ethylene responsive factor), PSII (photosystem II) protein, HD-ZIP (homeodomain-leucine zipper), MYB and NAC-domain transcription factors. miR160, miR165, miR166, miR171, miR398, miR408, miR827, miR9472, miR9476 and miR9552 were the key miRNAs functioning in regulation of these genes and involving in tomato response to drought stress. Additionally, plant hormone signal transduction pathway genes were differentially regulated by miR169, miR172, miR393, miR5641, miR5658 and miR7997 in both tissues of both sensitive and tolerant genotypes. These results provide new insight into the regulatory role of miRNAs in drought response with plant hormone signal transduction and drought-tolerant tomato breeding. PMID:26857916

  3. FHIT promoter methylation status, low protein and high mRNA levels in patients with non-small cell lung cancer.

    Science.gov (United States)

    Czarnecka, Karolina H; Migdalska-Sęk, Monika; Domańska, Daria; Pastuszak-Lewandoska, Dorota; Dutkowska, Agata; Kordiak, Jacek; Nawrot, Ewa; Kiszałkiewicz, Justyna; Antczak, Adam; Brzeziańska-Lasota, Ewa

    2016-09-01

    FHIT is a tumor suppressor gene that is frequently silenced in non-small cell lung cancer (NSCLC) and also in preneoplastic lesions. Promoter hypermethylation was previously observed in NSCLC, and its epigenetic silencing, observed on mRNA or protein level, was proposed to predict NSCLC outcome. In the present study we evaluated the relationship between FHIT expression on mRNA level and promoter methylation, or immunoexpression level. The aim of this study was to analyze the usefulness of FHIT as early differentiating biomarker in NSCLC patients. Lung tissue specimens were obtained from 59 patients with diagnosed NSCLC (SCC=34, AC=20, LCC=5). FHIT promoter methylation was assessed in methylation-specific PCR. Relative expression analysis of FHIT was performed in real-time PCR (qPCR) and protein immunoexpression by ELISA assay. Significant differences in FHIT expression between NSCLC histopathological groups (SCC, AC, LCC) were observed (p=0.000009), with the lowest level in SCC. FHIT expression was significantly higher (p=0.034) in men vs. women. Methylated FHIT alleles were present both in NSCLC and control specimens. Mean MI value was higher in control tissue vs. neoplasm, and in men vs. women and it increased with patient age. Significant increase in MI level was observed in N0 group vs. N1 and N2, according to the TNM staging (p=0.0073). Differences in FHIT expression levels between AC, LCC and SCC indicated the usefulness of this gene as a diagnostic marker for NSCLC subtype differentiation. FHIT promoter hypermethylation both in cancer and control tissue indicated the presence of epigenetic alterations in early stage of NSCLC development. Differences in gene promoter methylation between cancer patients with and without node infiltration might be considered as a prognostic marker. Significantly lower FHIT protein immunoexpression was revealed in the group with long and intense history of smoking assessed as PYs (PY<40 vs. PY≥40, p=0.01). These results

  4. mRNA turnover rate limits siRNA and microRNA efficacy

    OpenAIRE

    Larsson, Erik; Sander, Chris; Marks, Debora

    2010-01-01

    What determines how strongly an mRNA responds to a microRNA or an siRNA? We know that properties of the sequence match between the small RNA and the mRNA are crucial. However, large-scale validations of siRNA efficacies have shown that certain transcripts remain recalcitrant to perturbation even after repeated redesign of the siRNA (Krueger et al, 2007). Weak response to RNAi may thus be an inherent property of the mRNA, but the underlying factors have proven difficult to uncover. siRNAs indu...

  5. The microRNA miR-34a inhibits non-small cell lung cancer (NSCLC) growth and the CD44hi stem-like NSCLC cells.

    Science.gov (United States)

    Shi, Yang; Liu, Can; Liu, Xin; Tang, Dean G; Wang, Junchen

    2014-01-01

    Lung cancer is among the most lethal malignancies with a high metastasis and recurrence rate, which is probably due to the existence of lung cancer stem cells (CSCs). CSCs in many tumors including non-small cell lung cancer (NSCLC) have been identified using adhesion molecular CD44, either individually or in combination with other marker(s). MicroRNAs (miRNAs) regulate both normal stem cells and CSCs and dysregulation of miRNAs has been implicated in tumorigenesis. Recently, miR-34a was found to be downregulated in NSCLC cells but the biological functions of miR-34a in regulating NSCLC cell behavior have not been extensively studied. Here we show that transfection of synthetic miR-34a, but not the negative control (NC) miRNA oligonucleotides (oligos) in three NSCLC cell lines, i.e., A549, H460, and H1299, inhibited their holoclone formation, clonogenic expansion, and tumor regeneration in vivo. Furthermore, the lentiviral vector-mediated overexpression of miR-34a in purified CD44hi H460 cells also inhibited tumor outgrowth. In contrast, expression of miR-34a antagomirs (i.e., antisense oligos) in the CD44lo H460 cells promoted tumor development. Our study shows that miR-34a is a negative regulator of the tumorigenic properties of NSCLC cells and CD44hi lung CSCs, and establishes a strong rationale for developing miR-34a as a novel therapeutic agent against NSCLC. PMID:24595209

  6. The microRNA miR-34a inhibits non-small cell lung cancer (NSCLC growth and the CD44hi stem-like NSCLC cells.

    Directory of Open Access Journals (Sweden)

    Yang Shi

    Full Text Available Lung cancer is among the most lethal malignancies with a high metastasis and recurrence rate, which is probably due to the existence of lung cancer stem cells (CSCs. CSCs in many tumors including non-small cell lung cancer (NSCLC have been identified using adhesion molecular CD44, either individually or in combination with other marker(s. MicroRNAs (miRNAs regulate both normal stem cells and CSCs and dysregulation of miRNAs has been implicated in tumorigenesis. Recently, miR-34a was found to be downregulated in NSCLC cells but the biological functions of miR-34a in regulating NSCLC cell behavior have not been extensively studied. Here we show that transfection of synthetic miR-34a, but not the negative control (NC miRNA oligonucleotides (oligos in three NSCLC cell lines, i.e., A549, H460, and H1299, inhibited their holoclone formation, clonogenic expansion, and tumor regeneration in vivo. Furthermore, the lentiviral vector-mediated overexpression of miR-34a in purified CD44hi H460 cells also inhibited tumor outgrowth. In contrast, expression of miR-34a antagomirs (i.e., antisense oligos in the CD44lo H460 cells promoted tumor development. Our study shows that miR-34a is a negative regulator of the tumorigenic properties of NSCLC cells and CD44hi lung CSCs, and establishes a strong rationale for developing miR-34a as a novel therapeutic agent against NSCLC.

  7. Fluorescence Spectroscopy of tRNA[superscript Phe] Y Base in the Presence of Mg[superscript 2+] and Small Molecule Ligands

    Science.gov (United States)

    Kirk, Sarah R.; Silverstein, Todd P.; McFarlane Holman, Karen L.

    2008-01-01

    This laboratory project is one component of a semester-long advanced biochemistry laboratory course that uses several complementary techniques to study tRNA[superscript Phe] conformational changes induced by ligand binding. In this article we describe a set of experiments in which students use fluorescence spectroscopy to study tRNA[superscript…

  8. Structural organization of the genes encoding the small nuclear RNAs U1 to U6 of Tetrahymena thermophila is very similar to that of plant small nuclear RNA genes

    DEFF Research Database (Denmark)

    Orum, H; Nielsen, Henrik; Engberg, J

    1992-01-01

    of other organisms revealed several similarities to plant snRNA genes. These similarities include the overall promoter architecture as well as specific sequence elements. The structural organization of the 3' flanking region of some of the T. thermophila snRNA genes is not observed in other organisms...

  9. 半乳凝素-3和微小 RNA-21在非小细胞肺癌中的表达和临床意义∗%The expression level and clinical significance of Galectin-3 and miRNA-21 in non-small-cell lung carcinoma

    Institute of Scientific and Technical Information of China (English)

    易震南; 伍俊; 黄仁清; 黎东明; 张绪超

    2015-01-01

    Objective To study the expression level and clinical significance of Galectin-3 and miRNA-21 in non-small-cell lung carcinoma(NSCLC).Methods One hundred and fifty patients with NSCLC were chosen as cancer group,and 1 50 patients with benign pulmonary diseases were chosen as control group.The expression level of Galectin-3 and that of miRNA-21 between two groups were compared,and the relevance between expression level of Galectin-3 and that of miRNA-21 and clinical feature were analysed.Results In cancer group,the expression level of Galectin-3 was 6.75±2.38,and that of control group was 1.12 ±0.29;the expression level of miRNA-21 was 5.91 ± 1.59,and that of control group was 0.97 ± 0.1 7,and the difference between two groups had statistical significance(P 0.05 ).Conclusion The expression level of Galectin-3 and that of miRNA-21 can be applied in the diagnosis and prognosis of non-small-cell lung carcinoma.%目的:研究半乳凝素-3(Galectin-3)和微小 RNA-21(miRNA-21)在非小细胞肺癌(NSCLC)组织中的表达和临床意义。方法选取150例 NSCLC 患者作为肺癌组,同一时期来该院进行治疗的150例肺部良性病变患者作为对照组。比较两组患者的 Galectin-3和 miRNA-21的表达水平,分析它们与患者临床特征的相关性。结果肺癌组患者的 Galectin-3为6.75±2.38, miRNA-21为5.91±1.59;肺癌组患者的 Galectin-3为1.12±0.29,miRNA-21为0.97±0.17,差异具有统计学意义(P 0.05)。结论Ga-lectin-3和 miRNA-21的表达水平在 NSCLC 的诊断和预后判断中具有一定的应用价值。

  10. Changes in messenger RNA of pancreatic enzymes and intestinal cholecystokinin after a 7-day bile-pancreatic juice diversion from the proximal small intestine in rats.

    Science.gov (United States)

    Hara, H; Ochi, Y; Kasai, T

    1997-06-01

    We have previously demonstrated the bile-pancreatic juice (BPJ)-independent stimulation of pancreatic enzyme secretion in chronic BPJ-diverted rats. Pancreatic and intestinal adaptation to 7-day BPJ diversion was next examined. Pancreatic enzyme mRNA and cholecystokinin mRNA in the jejunal mucosa were measured in rats with BPJ diverted into the ileum (PBD rats) in comparison with the figures for rats with BPJ returned to the duodenum (normal rats) or laparotomized (Intact) rats under well-nourished conditions. Amylase mRNA in the pancreas was lower and trypsinogen plus chymotrypsinogen mRNA was higher in the PBD rats than in the intact rats. The change in pancreatic mRNA was similar to that in the specific activities of the enzymes after a chronic BPJ diversion. This finding suggests that these pancreatic enzymes were regulated by the mRNA level. The portal concentration of cholecystokinin in the postabsorptive period (exogenously non-stimulated status) was 4-fold higher in the PBD group than in the normal and intact groups. Cholecystokinin mRNA in the jejunal mucosa of PBD rats was somewhat higher than that of intact rats. These results suggest that intestinal cholecystokinin was predominantly increased at the translational or later stage by chronic BPJ diversion.

  11. Generation of siRNA Nanosheets for Efficient RNA Interference

    Science.gov (United States)

    Kim, Hyejin; Lee, Jae Sung; Lee, Jong Bum

    2016-04-01

    After the discovery of small interference RNA (siRNA), nanostructured siRNA delivery systems have been introduced to achieve an efficient regulation of the target gene expression. Here we report a new siRNA-generating two dimensional nanostructure in a formation of nanosized sheet. Inspired by tunable mechanical and functional properties of the previously reported RNA membrane, siRNA nanosized sheets (siRNA-NS) with multiple Dicer cleavage sites were prepared. The siRNA-NS has two dimensional structure, providing a large surface area for Dicer to cleave the siRNA-NS for the generation of functional siRNAs. Furthermore, downregulation of the cellular target gene expression was achieved by delivery of siRNA-NS without chemical modification of RNA strands or conjugation to other substances.

  12. Deciphering the RNA landscape by RNAome sequencing

    NARCIS (Netherlands)

    K.W.J. Derks (Kasper); B. Misovic (Branislav); M.C.G.N. van den hout (Mirjam); C. Kockx (Christel); C.P. Gomez (Cesar Payan); R.W.W. Brouwer; H. Vrieling (Harry); J.H.J. Hoeijmakers (Jan); W.F.J. van IJcken (Wilfred); J. Pothof (Joris)

    2015-01-01

    textabstractCurrent RNA expression profiling methods rely on enrichment steps for specific RNA classes, thereby not detecting all RNA species in an unperturbed manner. We report strand-specific RNAome sequencing that determines expression of small and large RNAs from rRNA-depleted total RNA in a sin

  13. The restorative effect of mouse intestinal RNA on the small intestine of mice of the same strain after γ ray irradiation

    International Nuclear Information System (INIS)

    Mouse intestinal RNA was injected into the mice of the same strain within 1-3 h after different doses of abdominal or whole body 60Co γ irradiation, so as to explore the initial effective time of mouse intestinal RNA and the affection of radiation condition on its restorative effect, by measuring the survival of mouse intestinal crypt. The results showed (1) A decrease in the survival of mouse intestinal crypt began 6h after the irradiation, and the lowest survival rate appeared on the fourth day. (2) The survival of mouse intestinal crypt of the abdominal irradiated mice increased 21.4% at 6h after intestinal RNA injection as compared with that of the irradiated control group. (3) The dose modifying factor (DMF) of normal mouse intestinal RNA in the promotion of the recovery of the duodenum, jejunum and ileum of mice after whole body irradiation 1.17, 1.12 and 1.10 respectively. The above results suggest that mouse intestinal RNA can raise not only the survival of jejunum crypt of the mice of the same strain after abdominal irradiation but also the survival of crypt of the duodenum, jejunum and ileum of the mouse after whole body irradiation, which may be observed 6h after the irradiation

  14. Identification and preliminary characterization of a SigB regulated small non-coding RNA in Listeria monocytogenes

    DEFF Research Database (Denmark)

    Nielsen, Jesper Sejrup; Olsen, Anders Steno; Bonde, Mette;

    by an alternative sigma factor. Using this approach we have successfully identified a novel sRNA of ~75 nucleotides in L. monocytogenes that is specifically regulated by SigB. This sRNA, which we have termed SigB1 is expressed from the 3'-UTR of a large operon. SigB1 is expressed in a SigB dependant manner only...... in response to known SigB associated stresses such as salt- and ethanol-stress or entrance into stationary phase. Using transcriptional promoter-lacZ reporter assays, we have confirmed that SigB1 is not the result of an RNA-processing event. Interestingly, SigB1 does not contain any obvious Hfq binding sites...

  15. Systemic Delivery of Small Interfering RNA Targeting the Interleukin-2/15 Receptor β Chain Prevents Disease Progression in Experimental Arthritis

    OpenAIRE

    Tiantian Zhang; Xuehua Bai; Xiaohua Mao

    2013-01-01

    The role of interleukin (IL)-15 in the pathogenesis of rheumatoid arthritis (RA) is well established; however, systemic knockdown of IL-15 receptor (IL-15R) for reduction in inflammation at local sites has not been demonstrated. In this study, the therapeutic effect of intravenously administered siRNA targeting the β chain of IL-15R which is shared by the receptor for IL-2 was examined in rats with adjuvant-induced arthritis (AA). Polyethylenimine (PEI)-complexed siRNA nanoparticles could eas...

  16. Insulin-like Growth Factor Receptor 1 mRNA Expression as a Prognostic Marker in Advanced Non-small Cell Lung Cancer

    DEFF Research Database (Denmark)

    Vilmar, Adam; Santoni-Rugiu, Eric; Cillas, Jesus Garcia-Fon;

    2014-01-01

    reaction (qRT-PCR) and immunohistochemistry (IHC). MATERIALS AND METHODS: Analyses of IGF1R were performed on patients with advanced NSCLC, included in a randomized chemotherapy trial, having large, representative tissue samples. IGF1R mRNA and protein expression were correlated to clinical end.......039 and 10.9 vs. 14.3 months, p=0.038, respectively). IGF1R protein expression showed a similar, although non-significant tendency. CONCLUSION: IGF1R mRNA expression may be a prognostic biomarker in advanced NSCLC and should be investigated in a larger population....

  17. Insulin-like growth factor receptor 1 mRNA expression as a prognostic marker in advanced non-small cell lung cancer

    DEFF Research Database (Denmark)

    Vilmar, Adam; Santoni-Rugiu, Eric; Cillas, Jesus Garcia-Fon;

    2014-01-01

    reaction (qRT-PCR) and immunohistochemistry (IHC). MATERIALS AND METHODS: Analyses of IGF1R were performed on patients with advanced NSCLC, included in a randomized chemotherapy trial, having large, representative tissue samples. IGF1R mRNA and protein expression were correlated to clinical end.......039 and 10.9 vs. 14.3 months, p=0.038, respectively). IGF1R protein expression showed a similar, although non-significant tendency. CONCLUSION: IGF1R mRNA expression may be a prognostic biomarker in advanced NSCLC and should be investigated in a larger population....

  18. Construction and identification of the recombinant adenovirus vector carrying a small interfering RNA targeting the peroxisome proliferator-activated receptor-γ

    Institute of Scientific and Technical Information of China (English)

    LIU Ming; WANG Yi-sheng; LI Yue-bai; ZHAO Guo-qiang

    2012-01-01

    Background Steroid-induced osteonecrosis of the femoral head (ONFH) is a common clinical disease,with a high disability rate.At present,efficient prevention and treatment of steroid-induced ONFH is still lacking.The peroxisome proliferator-activated receptor-γ (PPARγ) is recognized as an important pathogenic gene for the development of steroid-induced ONFH.RNA interference (RNAi) is a tool for functional gene analysis,which has been successfully used to down-regulate the levels of specific target proteins.Therefore,down-regulation of PPARγ expression by RNAi may prevent the incidence of steroid-induced ONFH.Methods According to the principles of siRNA design,three duplex siRNA sequences (971-989,1253-1271 and 1367-1385) derived from the PPARy gene (NM_001082148) were synthesized.These duplexes were annealed,purified and ligated into 1.0-cytomegalovirus (CMV) shuttle vector.The shuttle vector was transfected into HEK293 cells.The HEK293 generated recombinant adenovirus vector carrying PPARγ siRNA sequences was purified and the titer of recombinant adenovirus was determined.Results After the annealing of single-strand DNA oligo encoding short hairpin RNA (shRNA) sequences,products were identified by gel electrophoresis.These products were ligated into the 1.0-CMV shuttle vector and the recombinant shuttle vectors 1.0-CMV-971,1.0-CMV-1253 and 1.0-CMV-1367 were constructed.These sequences of these recombinant vectors were confirmed.We then successfully constructed the recombinant adenovirus vector carrying siRNA targeting PPARγ.After purification,the virus titer was higher than 1010 plaque forming unit (PFU)/ml.Conclusion In this study,three recombinant adenovirus shuttle vectors carrying siRNA targeting PPARγ,including shuttle vectors 1.0-CMV-971,1.0-CMV-1253 and 1.0-CMV-1367,were successfully constructed and high titers of recombinant adenovirus were obtained.

  19. Glucose uptake regulation in E. coli by the small RNA SgrS: comparative analysis of E. coli K-12 (JM109 and MG1655 and E. coli B (BL21

    Directory of Open Access Journals (Sweden)

    Ng Weng-Ian

    2010-09-01

    Full Text Available Abstract Background The effect of high glucose concentration on the transcription levels of the small RNA SgrS and the messenger RNA ptsG, (encoding the glucose transporter IICBGlc, was studied in both E. coli K-12 (MG1655 and JM109 and E. coli B (BL21. It is known that the transcription level of sgrS increases when E. coli K-12 (MG1655 and JM109 is exposed to the non-metabolized glucose alpha methyl glucoside (αMG or when the bacteria with a defective glycolysis pathway is grown in presence of glucose. The increased level of sRNA SgrS reduces the level of the ptsG mRNA and consequently lowers the level of the glucose transporter IICBGlc. The suggested trigger for this action is the accumulation of the corresponding phospho-sugars. Results In the course of the described work, it was found that E. coli B (BL21 and E. coli K-12 (JM109 and MG1655 responded similarly to αMG: both strains increased SgrS transcription and reduced ptsG transcription. However, the two strains reacted differently to high glucose concentration (40 g/L. E. coli B (BL21 reacted by increasing sgrS transcription and reducing ptsG transcription while E. coli K-12 (JM109 and MG1655 did not respond to the high glucose concentration, and, therefore, transcription of sgrS was not detected and ptsG mRNA level was not affected. Conclusions The results suggest that E. coli B (BL21 tolerates high glucose concentration not only by its more efficient central carbon metabolism, but also by controlling the glucose transport into the cells regulated by the sRNA SgrS, which may suggest a way to control glucose consumption and increase its efficient utilization.

  20. Association of interleukin 1 beta (IL-1β polymorphism with mRNA expression and risk of non small cell lung cancer

    Directory of Open Access Journals (Sweden)

    Imtiyaz A. Bhat

    2014-12-01

    Conclusion: We conclude that lung cancer risk genotype interleukin 1 beta-31TT results in increased expression of interleukin 1 beta mRNA in lung cancer patients. Our data suggest that this genotype (IL1β -31TT in the interleukin 1 beta regulatory region provide a microenvironment with elevated inflammatory stimuli and thus increasing the risk for lung cancer.

  1. Adeno-Associated Virus Type 5-Mediated Intraarticular Administration of Tumor Necrosis Factor Small Interfering RNA Improves Collagen-Induced Arthritis

    NARCIS (Netherlands)

    M. Khoury; G. Courties; S. Fabre; C. Bouffi; C.A. Seemayer; M.J. Vervoordeldonk; P.P. Tak; C. Jorgensen; F. Apparailly

    2010-01-01

    Objective. RNA interference (RNAi) is a powerful tool for sequence-specific gene silencing, and interest in its application in human diseases is growing. Given the success of recent strategies for administering gene therapy in rheumatoid arthritis using recombinant vectors such as adeno-associated v

  2. Inhibition of Nonsmall Cell Lung Cancer Cell Migration by Protein Arginine Methyltransferase 1-small Hairpin RNA Through Inhibiting Epithelial-mesenchymal Transition, Extracellular Matrix Degradation, and Src Phosphorylation In Vitro

    Directory of Open Access Journals (Sweden)

    Ting Zhang

    2015-01-01

    Full Text Available Background: Protein arginine methyltransferases 1 (PRMT1 is over-expressed in a variety of cancers, including lung cancer, and is correlated with a poor prognosis of tumor development. This study aimed to investigate the role of PRMT1 in nonsmall cell lung cancer (NSCLC migration in vitro. Methods: In this study, PRMT1 expression in the NSCLC cell line A549 was silenced using lentiviral vector-mediated short hairpin RNAs. Cell migration was measured using both scratch wound healing and transwell cell migration assays. The mRNA expression levels of matrix metalloproteinase 2 (MMP-2 and tissue inhibitor of metalloproteinase 1, 2 (TIMP1, 2 were measured using quantitative real-time reverse transcription-polymerase chain reaction. The expression levels of protein markers for epithelial-mesenchymal transition (EMT (E-cadherin, N-cadherin, focal adhesion kinase (FAK, Src, AKT, and their corresponding phosphorylated states were detected by Western blot. Results: Cell migration was significantly inhibited in the PRMT1 silenced group compared to the control group. The mRNA expression of MMP-2 decreased while TIMP1 and TIMP2 increased significantly. E-cadherin mRNA expression also increased while N-cadherin decreased. Only phosphorylated Src levels decreased in the silenced group while FAK or AKT remained unchanged. Conclusions: PRMT1-small hairpin RNA inhibits the migration abilities of NSCLC A549 cells by inhibiting EMT, extracellular matrix degradation, and Src phosphorylation in vitro.

  3. Combined Targeted DNA Sequencing in Non-Small Cell Lung Cancer (NSCLC Using UNCseq and NGScopy, and RNA Sequencing Using UNCqeR for the Detection of Genetic Aberrations in NSCLC.

    Directory of Open Access Journals (Sweden)

    Xiaobei Zhao

    Full Text Available The recent FDA approval of the MiSeqDx platform provides a unique opportunity to develop targeted next generation sequencing (NGS panels for human disease, including cancer. We have developed a scalable, targeted panel-based assay termed UNCseq, which involves a NGS panel of over 200 cancer-associated genes and a standardized downstream bioinformatics pipeline for detection of single nucleotide variations (SNV as well as small insertions and deletions (indel. In addition, we developed a novel algorithm, NGScopy, designed for samples with sparse sequencing coverage to detect large-scale copy number variations (CNV, similar to human SNP Array 6.0 as well as small-scale intragenic CNV. Overall, we applied this assay to 100 snap-frozen lung cancer specimens lacking same-patient germline DNA (07-0120 tissue cohort and validated our results against Sanger sequencing, SNP Array, and our recently published integrated DNA-seq/RNA-seq assay, UNCqeR, where RNA-seq of same-patient tumor specimens confirmed SNV detected by DNA-seq, if RNA-seq coverage depth was adequate. In addition, we applied the UNCseq assay on an independent lung cancer tumor tissue collection with available same-patient germline DNA (11-1115 tissue cohort and confirmed mutations using assays performed in a CLIA-certified laboratory. We conclude that UNCseq can identify SNV, indel, and CNV in tumor specimens lacking germline DNA in a cost-efficient fashion.

  4. Small interfering RNA targeted to IGF-IR delays tumor growth and induces proinflammatory cytokines in a mouse breast cancer model.

    Directory of Open Access Journals (Sweden)

    Tiphanie Durfort

    Full Text Available Insulin-like growth factor I (IGF-I and its type I receptor (IGF-IR play significant roles in tumorigenesis and in immune response. Here, we wanted to know whether an RNA interference approach targeted to IGF-IR could be used for specific antitumor immunostimulation in a breast cancer model. For that, we evaluated short interfering RNA (siRNAs for inhibition of in vivo tumor growth and immunological stimulation in immunocompetent mice. We designed 2'-O-methyl-modified siRNAs to inhibit expression of IGF-IR in two murine breast cancer cell lines (EMT6, C4HD. Cell transfection of IGF-IR siRNAs decreased proliferation, diminished phosphorylation of downstream signaling pathway proteins, AKT and ERK, and caused a G0/G1 cell cycle block. The IGF-IR silencing also induced secretion of two proinflammatory cytokines, TNF- α and IFN-γ. When we transfected C4HD cells with siRNAs targeting IGF-IR, mammary tumor growth was strongly delayed in syngenic mice. Histology of developing tumors in mice grafted with IGF-IR siRNA treated C4HD cells revealed a low mitotic index, and infiltration of lymphocytes and polymorphonuclear neutrophils, suggesting activation of an antitumor immune response. When we used C4HD cells treated with siRNA as an immunogen, we observed an increase in delayed-type hypersensitivity and the presence of cytotoxic splenocytes against wild-type C4HD cells, indicative of evolving immune response. Our findings show that silencing IGF-IR using synthetic siRNA bearing 2'-O-methyl nucleotides may offer a new clinical approach for treatment of mammary tumors expressing IGF-IR. Interestingly, our work also suggests that crosstalk between IGF-I axis and antitumor immune response can mobilize proinflammatory cytokines.

  5. RNA Crystallization

    Science.gov (United States)

    Golden, Barbara L.; Kundrot, Craig E.

    2003-01-01

    RNA molecules may be crystallized using variations of the methods developed for protein crystallography. As the technology has become available to syntheisize and purify RNA molecules in the quantities and with the quality that is required for crystallography, the field of RNA structure has exploded. The first consideration when crystallizing an RNA is the sequence, which may be varied in a rational way to enhance crystallizability or prevent formation of alternate structures. Once a sequence has been designed, the RNA may be synthesized chemically by solid-state synthesis, or it may be produced enzymatically using RNA polymerase and an appropriate DNA template. Purification of milligram quantities of RNA can be accomplished by HPLC or gel electrophoresis. As with proteins, crystallization of RNA is usually accomplished by vapor diffusion techniques. There are several considerations that are either unique to RNA crystallization or more important for RNA crystallization. Techniques for design, synthesis, purification, and crystallization of RNAs will be reviewed here.

  6. [small beta]-Turn mimetic-based stabilizers of protein-protein interactions for the study of the non-canonical roles of leucyl-tRNA synthetase

    DEFF Research Database (Denmark)

    Kim, Chanwoo; Jung, Jinjoo; Thanh Tung, Truong;

    2016-01-01

    For the systematic perturbation of protein-protein interactions, we designed and synthesized tetra-substituted hexahydro-4H-pyrazino[2,1-c][1,2,4]triazine-4,7(6H)-diones as [small beta]-turn mimetics. We then devised a new synthetic route to obtain [small beta]-turn mimetic scaffolds via tandem N...

  7. Knockdown of Ki-67 by dicer-substrate small interfering RNA sensitizes bladder cancer cells to curcumin-induced tumor inhibition.

    Directory of Open Access Journals (Sweden)

    Sivakamasundari Pichu

    Full Text Available Transitional cell carcinoma (TCC of the urinary bladder is the most common cancer of the urinary tract. Most of the TCC cases are of the superficial type and are treated with transurethral resection (TUR. However, the recurrence rate is high and the current treatments have the drawback of inducing strong systemic toxicity or cause painful cystitis. Therefore, it would be of therapeutic value to develop novel concepts and identify novel drugs for the treatment of bladder cancer. Ki-67 is a large nucleolar phosphoprotein whose expression is tightly linked to cell proliferation, and curcumin, a phytochemical derived from the rhizome Curcuma longa, has been shown to possess powerful anticancer properties. In this study, we evaluated the combined efficacy of curcumin and a siRNA against Ki-67 mRNA (Ki-67-7 in rat (AY-27 and human (T-24 bladder cancer cells. The anticancer effects were assessed by the determination of cell viability, apoptosis and cell cycle analysis. Ki-67-7 (10 nM and curcumin (10 µM, when treated independently, were moderately effective. However, in their combined presence, proliferation of bladder cancer cells was profoundly (>85% inhibited; the rate of apoptosis in the combined presence of curcumin and Ki-67-7 (36% was greater than that due to Ki-67-7 (14% or curcumin (13% alone. A similar synergy between curcumin and Ki-67-7 in inducing cell cycle arrest was also observed. Western blot analysis suggested that pretreatment with Ki-67-7 sensitized bladder cancer cells to curcumin-mediated apoptosis and cell cycle arrest by p53- and p21-independent mechanisms. These data suggest that a combination of anti-Ki-67 siRNA and curcumin could be a viable treatment against the proliferation of bladder cancer cells.

  8. A Pathway Involving Farnesoid X Receptor and Small Heterodimer Partner Positively Regulates Hepatic Sirtuin 1 Levels via MicroRNA-34a Inhibition*

    OpenAIRE

    Lee, Jiyoung; Padhye, Amruta; Sharma, Abhilasha; Song, Guisheng; Miao, Ji; Mo, Yin-Yuan; Wang, Li; Kemper, Jongsook Kim

    2010-01-01

    Sirtuin 1 (SIRT1) is a NAD-dependent deacetylase that is critically involved in diverse cellular processes including metabolic disease, cancer, and possibly aging. Despite extensive studies on SIRT1 function, how SIRT1 levels are regulated remains relatively unknown. Here, we report that the nuclear bile acid receptor farnesoid X receptor (FXR) inhibits microRNA-34a (miR-34a) in the liver, which results in a positive regulation of SIRT1 levels. Activation of FXR by the synthetic agonist GW406...

  9. Small Interfering RNA-mediated Caveolin-1 Knockout on Plasminogen Activator Inhibitor-1 Expression in Insulin-stimulated Human Vascular Endothelial Cells

    Institute of Scientific and Technical Information of China (English)

    Huiling YANG; Gebo WEN; Weixin HU; Shuya HE; Zhihua QUAN; Weixia PENG; Bin YAN; Jianghua LIU; Fang WEN; Renxian CAO; Yangyan XU

    2007-01-01

    Using human vascular endothelial cells (ECV304) as the target,we studied the effect of caveolin(CAV)-1 in the course of insulin-stimulated expression of plasminogen activator inhibitor(PAI)-1.The appropriate single-stranded oligonucleotides representing the RNAi CAV-1 gene were analyzed by Ambion software.After annealing to generate double-stranded oligonucleotides (ds oligo),it was cloned into the pENTR/U6 entry vector containing RNA polymerase Ⅲ expression element by T4 DNA ligase.The short hairpin (shRNA) sequences transferred from the pENTR/U6 entry were cloned into the pLenti6/BLOCK-iTDEST vector with an LR recombination reaction.After identification by sequencing,we successfully constructed the CAV-1 RNAi lentiviral expression system using Gateway technology.Silencing efficiency was assayed by real-time reverse transcription-polymerase chain reaction,immunofluorescence staining and Western blotting.ECV304 cells were cultured in the medium containing different concentrations of insulin(1×10-9 to 1×10-7M)with the CAV-1 gene silenced or not.The expression level and subcellular localization of PAI-1 and CAV-1 were compared using reverse transcription-polymerase chain reaction,immunofluorescence staining and Western blot assay.The results showed that the potent inhibition of CAV-1 expression could reach 85%,and it was specific to the CAV-1-derived shRNA,not the S100A13-derived shRNA.There was no dramatic difference in PAI-1 expression between the RNAi+ and RNAi-ECV304 cells incubated with physiological insulin,but PAI-1 protein did accumulate under the cell membrane.As the concentration of insulin increased,the expression of PAI-1 was up-regulated,whereas the expression of CAV-1 attenuated.Furthermore,PAl-1 clearly augmented after CAV-1 knockdown.These results indicated that hyperinsulinism could promote PAI-1 expression by inhibiting CAV-1,and stabilizing or up-regulating CAV-1 expression in endothelial cells might reduce complications of the great vessels

  10. siRNA-induced in vivo downregulation of L-type calcium channels in rat small mesenteric arteries

    DEFF Research Database (Denmark)

    Matchkov, Vladimir; Larsen, Per; Kold-Petersen, Henrik Houmann;

    2008-01-01

    of the mesenteric artery of anestisized Wistar rats were transfected with siRNAs targeting different exons of the α1C gene or with control non-related siRNAs. The effect of transfection was analyzed after 3 and 10 days using quantitative PCR and immunohistochemistry. The functional effects of transfection were...... to noradrenaline, vasopressin and caffeine were also suppressed in Cav1.2 downregulated arteries. The reduced force development was accompanied with reduced nifedipine sensitive [Ca2+]i increase. The responses to K+-depolarization and agonist-stimulation in arteries transfected with siRNA directed against α1C were...

  11. Silencing expression of the catalytic subunit of DNA-dependent protein kinase by small interfering RNA sensitizes human cells for radiation-induced chromosome damage, cell killing, and mutation

    Science.gov (United States)

    Peng, Yuanlin; Zhang, Qinming; Nagasawa, Hatsumi; Okayasu, Ryuichi; Liber, Howard L.; Bedford, Joel S.

    2002-01-01

    Targeted gene silencing in mammalian cells by RNA interference (RNAi) using small interfering RNAs (siRNAs) was recently described by Elbashir et al. (S. M. Elbashir et al., Nature (Lond.), 411: 494-498, 2001). We have used this methodology in several human cell strains to reduce expression of the Prkdc (DNA-PKcs) gene coding for the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) that is involved in the nonhomologous end joining of DNA double-strand breaks. We have also demonstrated a radiosensitization for several phenotypic endpoints of radiation damage. In low-passage normal human fibroblasts, siRNA knock-down of DNA-PKcs resulted in a reduced capacity for restitution of radiation-induced interphase chromosome breaks as measured by premature chromosome condensation, an increased yield of acentric chromosome fragments at the first postirradiation mitosis, and an increased radiosensitivity for cell killing. For three strains of related human lymphoblasts, DNA-PKcs-targeted siRNA transfection resulted in little or no increase in radiosensitivity with respect to cell killing, a 1.5-fold decrease in induced mutant yield in TK6- and p53-null NH32 cells, but about a 2-fold increase in induced mutant yield in p53-mutant WTK1 cells at both the hypoxanthine quanine phosphoribosyl transferase (hprt) and the thymidine kinase loci.

  12. Evaluating the Development of Small Interfering RNA Expression Vector from Its Functional Elements%从功能部件看小干扰RNA表达载体的发展

    Institute of Scientific and Technical Information of China (English)

    侯莹; 孙西魁; 唐明青

    2015-01-01

    RNA interference as a potent gene silencing approach plays important parts in gene regulation, functional study and gene therapy. The small interfering RNA expression vector achieves a sustainable, stable and controllable application of the RNAi technology, and becomes a promising way for gene silencing. Although the interfering RNA expression vector has progressed to the second generation, and many commercial products were availabe, the discrepancies among efficiency, safety and controllability of these vectors still exist. The development of the interfering RNA expression vector seems get into a lag phase. Therefore, this article analysed the history and development of these small interference RNA expression vectors on the basis of their functional blocks, providing a theoretical fundation for the vector’s optimization and selection.%RNA干扰作为转录后基因沉默的有效途径,在基因调控、功能分析及疾病防治等领域发挥重要作用。小干扰RNA表达载体的诞生实现了RNA干扰技术持续、稳定和可控性应用,是实现基因沉默的理想选择。目前干扰性小RNA表达载体虽已发展到第二代,也开发出多种商品化的产品,但依然未能很好地解决其高效、安全、可控性方面的矛盾,发展陷入了瓶颈期。因此,从载体自身角度出发,通过系统分析其功能部件的特点,纵观小RNA载体的历史渊源、发展现状、存在问题和发展方向等问题,为干扰性小RNA表达载体的优化与选择提供相关理论指导。

  13. Assessment of fecal pollution sources in a small northern-plains watershed using PCR and phylogenetic analyses of Bacteroidetes 16S rRNA gene

    Science.gov (United States)

    Lamendella, R.; Domingo, J.W.S.; Oerther, D.B.; Vogel, J.R.; Stoeckel, D.M.

    2007-01-01

    We evaluated the efficacy, sensitivity, host-specificity, and spatial/temporal dynamics of human- and ruminant-specific 16S rRNA gene Bacteroidetes markers used to assess the sources of fecal pollution in a fecally impacted watershed. Phylogenetic analyses of 1271 fecal and environmental 16S rRNA gene clones were also performed to study the diversity of Bacteroidetes in this watershed. The host-specific assays indicated that ruminant feces were present in 28-54% of the water samples and in all sampling seasons, with increasing frequency in downstream sites. The human-targeted assays indicated that only 3-5% of the water samples were positive for human fecal signals, although a higher percentage of human-associated signals (19-24%) were detected in sediment samples. Phylogenetic analysis indicated that 57% of all water clones clustered with yet-to-be-cultured Bacteroidetes species associated with sequences obtained from ruminant feces, further supporting the prevalence of ruminant contamination in this watershed. However, since several clusters contained sequences from multiple sources, future studies need to consider the potential cosmopolitan nature of these bacterial populations when assessing fecal pollution sources using Bacteroidetes markers. Moreover, additional data is needed in order to understand the distribution of Bacteroidetes host-specific markers and their relationship to water quality regulatory standards. ?? 2006 Federation of European Microbiological Societies.

  14. The mRNA and Protein Levels of Tubulin and β-Actin Are Greatly Reduced in the Proximal Duodenum of Mice Relative to the Rest of the Small Intestines.

    Science.gov (United States)

    Yu, Sungsook; Hwang, Hyekyung E; Yun, Nakhyeon; Goldenring, James R; Nam, Ki Taek

    2015-09-01

    To accurately quantify mRNA and protein levels, it is critical to choose appropriate internal standards. As the expression of housekeeping genes is assumed to remain constant, they are often employed to normalize signals to correct for sample-to-sample variations. However, recent studies have documented that β-actin and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression levels change in response to various stimuli during proliferation, activation, and differentiation. We investigated levels of α-, β-, γ-tubulin, β-actin, and GAPDH vary across the gastrointestinal tract of mice. We found that different regions of the small intestines had dramatically different expression profiles, as measured by western blot, quantitative Reverse transcription polymerase chain reaction (RT-PCR), and immunohistochemical staining. These results revealed that the expression levels of tubulins and β-actin were dramatically lower in the proximal duodenum, relative to the rest of the small intestines. These varying levels of housekeeping genes may reflect differences in the activities of specialized tissues and suggest unique requirements for tubulins in these tissue types. We conclude that the use of a single housekeeping gene to normalize gene expression in the gastrointestinal tracts of mice may introduce errors, as measured differences in gene expression may reflect regulation of the internal control rather than the mRNA or protein under investigation.

  15. Inhibition of Nonsmall Cell Lung Cancer Cell Migration by Protein Arginine Methyltransferase 1-small Hairpin RNA Through Inhibiting Epithelial-mesenchymal Transition,Extracellular Matrix Degradation, and Src Phosphorylation In Vitro

    Institute of Scientific and Technical Information of China (English)

    Ting Zhang; Ge Cui; Yun-Liang Yao; Yue Guo; Qi-Chun Wang; Xi-Ning Li; Wen-Ming Feng

    2015-01-01

    Background:Protein arginine methyltransferases 1 (PRMT1) is over-expressed in a variety of cancers,including lung cancer,and is correlated with a poor prognosis of tumor development.This study aimed to investigate the role of PRMT1 in nonsmall cell lung cancer (NSCLC) migration in vitro.Methods:In this study,PRMT1 expression in the NSCLC cell line A549 was silenced using lentiviral vector-mediated short hairpin RNAs.Cell migration was measured using both scratch wound healing and transwell cell migration assays.The mRNA expression levels of matrix metalloproteinase 2 (MMP-2) and tissue inhibitor ofmetalloproteinase 1,2 (TIMP l,2) were measured using quantitative real-time reverse transcription-polymerase chain reaction.The expression levels of protein markers for epithelial-mesenchymal transition (EMT) (E-cadherin,N-cadherin),focal adhesion kinase (FAK),Src,AKT,and their corresponding phosphorylated states were detected by Western blot.Results:Cell migration was significantly inhibited in the PRMT1 silenced group compared to the control group.The mRNA expression of MMP-2 decreased while TIMP 1 and TIMP2 increased significantly.E-cadherin mRNA expression also increased while N-cadherin decreased.Only phosphorylated Src levels decreased in the silenced group while FAK or AKT remained unchanged.Conclusions:PRMT1-small hairpin RNA inhibits the migration abilities of NSCLC A549 cells by inhibiting EMT,extracellular matrix degradation,and Src phosphorylation in vitro.

  16. A miRNA-tRNA mix-up: tRNA origin of proposed miRNA.

    Science.gov (United States)

    Schopman, Nick C T; Heynen, Stephan; Haasnoot, Joost; Berkhout, Ben

    2010-01-01

    The rapid release of new data from DNA genome sequencing projects has led to a variety of misannotations in public databases. Our results suggest that next generation sequencing approaches are particularly prone to such misannotations. Two related miRNA candidates did recently enter the miRBase database, miR-1274b and miR-1274a, but they share identical 18-nucleotide stretches with tRNA (Lys3) and tRNA (Lys5) , respectively. The possibility that the small RNA fragments that led to the description of these two miRNAs originated from the two tRNAs was examined. The ratio of the miR-1274b:miR-1274a fragments does closely resemble the known tRNA lys3:lys5 ratio in the cell. Furthermore, the proposed miRNA hairpins have a very low prediction score and the proposed miRNA genes are in fact endogenous retroviral elements. We searched for other miRNA-mimics in the human genome and found more examples of tRNA-miRNA mimicry. We propose that the corresponding miRNAs should be validated in more detail, as the small RNA fragments that led to their description are likely derived from tRNA processing. PMID:20818168

  17. 微量RNA的cDNA PCR文库的构建%The Construction of cDNA PCR Library from a Small Amount of RNA

    Institute of Scientific and Technical Information of China (English)

    李晶泉; 袁晓东; 汤敏谦

    2001-01-01

    By the method of PCR (Polymerase Chain Reaction),we have constructed the cDNA PCR library from mRNA.The cDNA PCR library can amplify the original cDNA up to hundreds of times.With the total RNA of human K562 cultured cell,the cDNA of β-Actin has been obtained by the methods of cDNA PCR library and reverse transcription respectively.As contrast,the amount of β-Actin′s cDNA from the cDNA PCR library is much higher than from reverse transcription.75pg total RNA of human K562 Cultured cell is employed to construct 50μl cDNA PCR library,and the cDNA of β-Actin can even be detected by using 1μl of the library as template to perform the PCR.Therefore cDNA PCR library can greatly enlarge the amount of information.%使用PCR(polymerase chain reaction)技术,调制了mRNA的cDNA PCR文库,实验证明,cDNA PCR文库能使原cDNA的量放大数百倍。同时,使用人体K562培养细胞的总RNA,对cDNA PCR文库法和反转录中的β-Actin的cDNA量进行了比较,cDNA PCR文库法中的β-Actin的cDNA量大大高于反转录中的β-Actin的cDNA量。使用75pg的人体K562培养细胞的总RNA,调制成50μl的cDNA PCR文库,使用1μl的cDNA PCR文库进行PCR反应时,可对文库中的β-Actin的cDNA进行PCR检测。因此,cDNA PCR文库显示了良好的信息放大性能。

  18. Every base matters: assessing small subunit rRNA primers for marine microbiomes with mock communities, time series and global field samples.

    Science.gov (United States)

    Parada, Alma E; Needham, David M; Fuhrman, Jed A

    2016-05-01

    Microbial community analysis via high-throughput sequencing of amplified 16S rRNA genes is an essential microbiology tool. We found the popular primer pair 515F (515F-C) and 806R greatly underestimated (e.g. SAR11) or overestimated (e.g. Gammaproteobacteria) common marine taxa. We evaluated marine samples and mock communities (containing 11 or 27 marine 16S clones), showing alternative primers 515F-Y (5'-GTGYCAGCMGCCGCGGTAA) and 926R (5'-CCGYCAATTYMTTTRAGTTT) yield more accurate estimates of mock community abundances, produce longer amplicons that can differentiate taxa unresolvable with 515F-C/806R, and amplify eukaryotic 18S rRNA. Mock communities amplified with 515F-Y/926R yielded closer observed community composition versus expected (r(2)  = 0.95) compared with 515F-Y/806R (r(2)  ∼ 0.5). Unexpectedly, biases with 515F-Y/806R against SAR11 in field samples (∼4-10-fold) were stronger than in mock communities (∼2-fold). Correcting a mismatch to Thaumarchaea in the 515F-C increased their apparent abundance in field samples, but not as much as using 926R rather than 806R. With plankton samples rich in eukaryotic DNA (> 1 μm size fraction), 18S sequences averaged ∼17% of all sequences. A single mismatch can strongly bias amplification, but even perfectly matched primers can exhibit preferential amplification. We show that beyond in silico predictions, testing with mock communities and field samples is important in primer selection.

  19. Suppression of Melanin Formation of A375 Cells by Small Interfering RNA%siRNA抑制A375细胞中黑色素形成

    Institute of Scientific and Technical Information of China (English)

    窦克军; 孙春宝

    2007-01-01

    以小干扰RNA(siRNA)技术抑制恶性黑素瘤细胞株A375的黑素皮质素受体1(melanocortin-1-receptor,MC1R)、小眼球相关转录因子(microphthalmia-associated transcription factor,MITF)基因表达,从而抑制细胞黑色素的生成.通过RT-PCR检测MC1R、MITF、酪氨酸酶(tyrosinase,TYR)、酪氨酸酶相关蛋白l(tyrosinase related protein-1,TRP-1)基因mRNA水平的变化,通过测定黑色素含量的变化检测蛋白水平的改变.并用台盼蓝活细胞计数法检测其对细胞增殖的影响.经RT-PCR检测,特异性siRNA作用的靶基因显著下调,黑色素含量明显降低.细胞增殖的变化说明,脂质体包裹的siRNA对A375细胞的毒性远小于熊果苷等化学合成药物.结果显示,利用siRNA对A375细胞中靶基因的调控,有效的抑制了黑色素的生成,siRNA技术与传统的调控黑色素的方法(化学合成物--褪色剂)比较,具有用量小、抑制效率高、细胞毒性低等优点.同时,为进一步研究黑色素形成通路中各基因之间的的关系提供了新思路.

  20. 靶向siRNA抑制幽门螺杆菌vacA表达%Small interfering RNA targeted inhibition of vacA expression in Helicobacter pylori

    Institute of Scientific and Technical Information of China (English)

    赵刚; 詹文华; 严燕国; 马晋平; 彭俊生; 董文广; 蔡世荣; 何裕隆

    2006-01-01

    目的:观察靶向siRNA能否抑制幽门螺杆菌(Hpylori)vacA基因表达.方法:合成靶向Hylori vacA基因5对特异siRNA(实验组:vacA-s1,vacA-s2,vacA-s3,vacA-s4,vacA-s5)和1对非特异siRNA(对照组).通过电穿孔法使siRNA转化至H pylori内,观察转化效率和转化1,6,12,24,48 h mRNA和蛋白表达的抑制率,并将PCR产物克隆和测序.结果:电穿孔转化效率平均为89%.vacA-s2、vacA-s4组在转化1 h vacA mRNA表达抑制率达最大值,1,6,12 h抑制率分别为65%和77%,43%和50%,17%和9%,24和48 h时vacA mRNA表达无抑制效应,与vacA-s1、vacA-s3、vacA-s5、vacA-s6组相比有显著差异(P<0.05),因为vacA-s1、vacA-3、vacA-s5、vacA-s6组转化后各时间点mRNA表达无变化.vacA-s2和vacA-s4组在转化1,6,12,24 h时vacA蛋白表达抑制率分别为26%和17%,47%和40%,70%和75%,33%和30%,与vacA-s1、vacA-s3、vacA-s5、vacA-s6组相比有显著差异(P<0.05);转化48 h则无抑制效应.PCR产物克隆和测序与相应报道序列比较,同源性为99%.结论:靶向siRNA可以通过电穿孔法转化并特异地抑制Hpylori vacA基因表达.

  1. MicroRNA-related single-nucleotide polymorphism of XPO5 is strongly correlated with the prognosis and chemotherapy response in advanced non-small-cell lung cancer patients.

    Science.gov (United States)

    Geng, Ji-Qun; Wang, Xiao-Chen; Li, Long-Fei; Zhao, Jun; Wu, Song; Yu, Gui-Ping; Zhu, Kou-Jun

    2016-02-01

    This study was performed to investigate if the microRNA-related single-nucleotide polymorphisms (miR-SNPs) of XPO5 gene predicted the prognosis and pathological features of advanced non-small-cell lung cancer patients receiving chemotherapy. A total of 131 advanced non-small-cell lung cancer (NSCLC) patients were recruited. MicroRNA (miRNA) binding site prediction software was adopted for the prediction and screening of SNPs in XPO5 and miRNA binding regions. Polymerase chain reaction (PCR) amplification was further performed. Time-dependent survival-free curves were constructed using the Kaplan-Meier technique. Univariate and the multivariate survival analyses were conducted for confirmation of prognostic factor for advanced NSCLC patients receiving chemotherapy. There were no significant differences of SNP distribution frequencies between groups, without statistical significance (P > 0.05). Included clinical pathological features and chemotherapy regimens showed no apparent statistical significance in influencing the curative effect of chemotherapy in advanced NSCLC patients (all P > 0.05). While the objective response rate (ORR) in patients who carried AA and AC genotype was 35.48 and 51.22 %, respectively, with statistically significant difference (P < 0.05). Univariate survival analysis indicated that patients who carried AA genotype showed a significantly lower 5-year survival rate to those who carried AC genotype (P < 0.05). And, considering pathological features, statistical significance was found in patients with different pathological types, lymph node metastasis, differentiation degree, T staging, and pathological staging (all P < 0.05). Multivariate analysis results indicated that the SNP sites of rs11077 might be an independent prognostic factor of advanced NSCLC patients receiving chemotherapy (risk ratio [RR] = 0.346; 95 % confidence interval [95 % CI] = 0.174-0.685, P = 0.002). Other clinical features were all

  2. MicroRNA-100 is a potential molecular marker of non-small cell lung cancer and functions as a tumor suppressor by targeting polo-like kinase 1

    International Nuclear Information System (INIS)

    Polo-like kinase 1 (PLK1) is highly expressed in many human cancers and regulates critical steps in mitotic progression. Previously, we have reported that PLK1 was overexpressed in non-small cell lung cancer (NSCLC), but the underlying molecular mechanisms are not well understood. By using microRNA (miR) target prediction algorithms, we identified miR-100 that might potentially bind the 3’-untranslated region of PLK1 transcripts. The purpose of this study was to investigate the roles of miR-100 and its association with PLK1 in NSCLC development. Taqman real-time quantitative RT-PCR assay was performed to detect miR-100 expression 10 NSCLC tissues and corresponding nontumor tissues. Additionally, the expression of miR-100 in 110 NSCLC tissues and its correlation with clinicopathological factors or prognosis of patients was analyzed. Finally, the effects of miR-100 expression on growth, apoptosis and cell cycle of NSCLC cells by posttranscriptionally regulating PLK1 expression were determined. MiR-100 was significantly downregulated in NSCLC tissues, and low miR-100 expression was found to be closely correlated with higher clinical stage, advanced tumor classification and lymph node metastasis of patients. The overall survival of NSCLC patients with low miR-100 was significantly lower than that of those patients with high miR-100, and univariate and multivariate analyses indicated that low miR-100 expression might be a poor prognostic factor. Also, miR-100 mimics could lead to growth inhibition, G2/M cell cycle arrest and apoptosis enhancement in NSCLC cells. Meanwhile, miR-100 mimics could significantly inhibit PLK1 mRNA and protein expression and reduce the luciferase activity of a PLK1 3’ untranslated region-based reporter construct in A549 cells. Furthermore, small interfering RNA (siRNA)-mediated PLK1 downregulation could mimic the effects of miR-100 mimics while PLK1 overexpression could partially rescue the phenotypical changes of NSCLC cells induced by mi

  3. Small interfering RNA targeting LASS2 gene enhances invasion capacity of hepatocellular carcinoma cells%靶向LASS2基因的小干扰RNA对人肝癌细胞侵袭能力的影响

    Institute of Scientific and Technical Information of China (English)

    唐宁; 游海燕; 金洁; 于彬; 覃文新

    2009-01-01

    目的:研究靶向人源性长寿保障基因2(homosapiens longevity assurance homologue 2,LASS 2)的小干扰RNA(small interfering RNA,siRNA)对人肝癌细胞MHCC97-L体外侵袭能力的影响.方法:通过脂质体转染将靶向LASS2的siRNA转染MHCC97-L细胞,运用实时荧光定量PCR(real-time fluorogentic quantitative PCR,RFQ-PCR)检测转染后LASS2 mRNA的表达,筛选有效的siRNA片段;运用 BCECF氢离子敏感荧光探针检测MHCC97-L细胞跨膜运输氢离子的功能;采用Western印迹法分析MHCC97-L细胞及上清液中基质金属蛋白酶2(matrix metalloproteinase 2,MMP-2)的表达情况;应用明胶酶谱法检测MHCC97-L细胞上清液中MMP-2的活性; Transwell法检测细胞体外侵袭能力.结果:筛选得到靶向LASS2的有效siRNA-1片段.MHCC97-L细胞转染LASS2 siRNA-1后,其跨膜运输氢离子的能力增强,与空白对照组比差异有统计学意义(P<0.05);MMP-2蛋白的表达及分泌无明显变化,但分泌的MMP-2活性形式增加,与空白对照组和无关序列组比明显提高,差异有统计学意义(P<0.05);且细胞的体外侵袭能力明显高于无关序列组和空白对照组(P<0.05).结论:靶向LASS2的siRNA能有效下调其表达,并能提高MHCC97-L细胞在转染LASS2 siRNA后的体外侵袭能力.

  4. A direct link between the global regulator PhoP and the Csr regulon in Y. pseudotuberculosis through the small regulatory RNA CsrC.

    Science.gov (United States)

    Nuss, Aaron M; Schuster, Franziska; Kathrin Heroven, Ann; Heine, Wiebke; Pisano, Fabio; Dersch, Petra

    2014-01-01

    In this study we investigated the influence of the global response regulator PhoP on the complex regulatory cascade controlling expression of early stage virulence genes of Yersinia pseudotuberculosis via the virulence regulator RovA. Our analysis revealed the following novel features: (1) PhoP activates expression of the CsrC RNA in Y. pseudotuberculosis, leading to activation of RovA synthesis through the CsrABC-RovM cascade, (2) activation of csrC transcription is direct and PhoP is shown to bind to two separate PhoP box-like sites, (3) PhoP-mediated activation results in transcription from two different promoters closely downstream of the PhoP binding sites, leading to two distinct CsrC RNAs, and (4) the stability of the CsrC RNAs differs significantly between the Y. pseudotuberculosis strains YPIII and IP32953 due to a 20 nucleotides insertion in CsrC(IP32953), which renders the transcript more susceptible to degradation. In summary, our study showed that PhoP-mediated influence on the regulatory cascade controlling the Csr system and RovA in Y. pseudotuberculosis varies within the species, suggesting that the Csr system is a focal point to readjust and adapt the genus to different hosts and reservoirs. PMID:24786463

  5. Morphology, morphogenesis and small subunit rRNA gene sequence of a soil hypotrichous ciliate, Perisincirra paucicirrata (Ciliophora, Kahliellidae), from the shoreline of the Yellow River, North China.

    Science.gov (United States)

    Li, Fengchao; Xing, Yi; Li, Jiamei; Al-Rasheid, Khaled A S; He, Songke; Shao, Chen

    2013-01-01

    The morphology, morphogenesis, and 18S rRNA gene sequence of a soil hypotrichous ciliate Perisincirra paucicirrata, isolated from north China, were investigated. Perisincirra paucicirrata differs from its congeners in: (1) having a body length to width ratio in vivo of 4:1, (2) its adoral zone occupying between 15% and 25% of the total body length, and (3) the presence of two parabuccal cirri, three left (with 10-16 cirri each) and two right marginal rows (with 14-24 cirri each), and three dorsal kineties. Our study offers a first attempt to begin to map the morphogenetic processes of the genus, which are mainly characterised by the following: the formation of four frontal ventral transverse anlagens for each daughter cell, with the proter's anlage I originating from the reorganised anterior part of the parental paroral; the paroral and endoral anlage developed from the reorganised old endoral and do not contribute the first frontal cirrus; the frontoventral transverse anlage I contributing the left frontal cirrus; anlage II generating the middle frontal and the buccal cirri; anlage III developing the right frontal cirrus and the anterior parabuccal cirrus; and anlage IV contributing the posterior parabuccal cirrus. As an additional contribution, we judge that the inner one or the two right rows of P. kahli and P. longicirrata are marginal rows. Phylogenetic analysis based on SSU rDNA sequences suggests that Perisincirra is related to sporadotrichids, but provides no credible evidence for its taxonomic position.

  6. The microRNA miR-34a Inhibits Non-Small Cell Lung Cancer (NSCLC) Growth and the CD44hi Stem-Like NSCLC Cells

    OpenAIRE

    Shi, Yang; Liu, Can; Liu, Xin; Tang, Dean G.; Wang, Junchen

    2014-01-01

    Lung cancer is among the most lethal malignancies with a high metastasis and recurrence rate, which is probably due to the existence of lung cancer stem cells (CSCs). CSCs in many tumors including non-small cell lung cancer (NSCLC) have been identified using adhesion molecular CD44, either individually or in combination with other marker(s). MicroRNAs (miRNAs) regulate both normal stem cells and CSCs and dysregulation of miRNAs has been implicated in tumorigenesis. Recently, miR-34a was found...

  7. Pharmacological and small interference RNA-mediated inhibition of breast cancer-associated fatty acid synthase (oncogenic antigen-519) synergistically enhances Taxol (paclitaxel)-induced cytotoxicity.

    Science.gov (United States)

    Menendez, Javier A; Vellon, Luciano; Colomer, Ramon; Lupu, Ruth

    2005-05-20

    combined treatment of C75 and Taxol inactivated the anti-apoptotic AKT (protein kinase B) kinase more than either agent alone, as evidenced by a synergistic down-regulation of AKT phosphorylation at its activating site Ser(473) without affecting AKT protein levels. To rule out a role for non-FAS C75-mediated effects, we finally used the potent and highly sequence-specific mechanism of RNA interference (RNAi) to block FAS-dependent signaling. Importantly, SK-Br3 and multi-drug resistant MCF-7/AdrR cells transiently transfected with sequence-specific double-stranded RNA oligonucleotides targeting FAS gene demonstrated hypersensitivity to Taxol-induced apoptotic cell death. Our findings establish for the first time that FAS blockade augments the cytotoxicity of anti-mitotic drug Taxol against breast cancer cells and that this chemosensitizing effect is schedule-dependent. We suggest that the alternate activation of both the pro-apoptotic p38 MAPK-p53 signaling and the cytoprotective MEK1/2 --> ERK1/2 cascade, as well as the inactivation of the anti-apoptotic AKT activity may explain, at least in part, the sequence-dependent enhancement of Taxol-induced cytotoxicity and apoptosis that follows inhibition of FAS activity in breast cancer cells. If chemically stable FAS inhibitors demonstrate systemic anticancer effects of FAS inhibition in vivo, these findings may render FAS as a valuable molecular target to enhance the efficacy of taxanes-based chemotherapy in human breast cancer.

  8. The Value of MicroRNA-155 as a Prognostic Factor for Survival in Non-Small Cell Lung Cancer: A Meta-Analysis.

    Directory of Open Access Journals (Sweden)

    Fei Wang

    Full Text Available Recent studies have shown that miR-155 play a positive role in the development of carcinoma. This meta-analysis aimed to identify the role of miR-155 in the survival of non-small cell lung cancer patients.Eligible studies were identified through database searches. Relevant data were extracted from each eligible study to assess the correlation between miR-155 expression and survival in lung carcinoma patients. The hazard ratios (HRs and 95% confidence intervals (CIs of the patients' outcomes in relation to miR-155 were calculated. A total of 6 studies were included for this meta-analysis. For overall survival (OS, recurrence-free survival (RFS, disease-free survival (DFS, and cancer-specific survival (CSS, the combined HRs and 95% CIs were not statistically significant. Additionally, in Asian and America subgroups, greater expression levels of miR-155 were related to poor prognoses for lung cancer (HR 1.71 95% CI: 1.22-2.40, P = 0.002, HR 2.35 95% CI: 1.42-3.89 P = 0.001, while no significant relationship was present in a Europe subgroup (HR 0.75 95%CI: 0.27-2.10, P = 0.587.These results suggest that miR-155 expression is not significantly related to non-small cell lung cancer patients except in patients from Asian and America.

  9. A Feed-Forward Loop Consisting of the Response Regulator RpaB and the Small RNA PsrR1 Controls Light Acclimation of Photosystem I Gene Expression in the Cyanobacterium Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Kadowaki, Taro; Nagayama, Ryuta; Georg, Jens; Nishiyama, Yoshitaka; Wilde, Annegret; Hess, Wolfgang R; Hihara, Yukako

    2016-04-01

    Since cyanobacteria need to decrease PSI content to avoid absorption of excess light energy, down-regulation of PSI gene expression is one of the key characteristics of the high-light (HL) acclimation response. The transcriptional regulator RpaB and the small RNA PsrR1 (photosynthesis regulatory RNA1) have been suggested to be the two most critical factors for this response in Synechocystis sp. PCC 6803. In this study, we found that the HLR1 DNA-binding motif, the recognition sequence for RpaB, is highly conserved in the core promoter region of the psrR1 gene among cyanobacterial species. Gel mobility shift assay revealed that RpaB binds to the HLR1 sequence of psrR1 in vitro. RNA gel blot analysis together with chromatin affinity purification (ChAP) analysis suggested that PSI genes are activated and the psrR1 gene is repressed by the binding of RpaB under low-light (LL) conditions. A decrease in DNA binding affinity of RpaB occurs within 5 min after the shift from LL to HL conditions, leading to the prompt decrease in PSI promoter activity together with derepression of psrR1 gene expression. Accumulating PsrR1 molecules then prevent translation from pre-existing PSI transcripts. By this dual repression at transcriptional and post-transcriptional levels, rapid and strict down-regulation of PSI expression under HL is secured. Our findings suggest that RpaB and PsrR1 constitute a feed-forward loop for the regulation of PSI gene expression to achieve a rapid acclimation response to the damaging HL conditions.

  10. Upregulated long non-coding RNA AGAP2-AS1 represses LATS2 and KLF2 expression through interacting with EZH2 and LSD1 in non-small-cell lung cancer cells.

    Science.gov (United States)

    Li, W; Sun, M; Zang, C; Ma, P; He, J; Zhang, M; Huang, Z; Ding, Y; Shu, Y

    2016-01-01

    Recently, long non-coding RNAs (lncRNAs) are identified as new crucial regulators of diverse cellular processes, including cell proliferation, differentiation and cancer cells metastasis. Accumulating evidence has revealed that aberrant lncRNA expression plays important roles in carcinogenesis and tumor progression. However, the expression pattern and biological function of lncRNAs in non-small-cell lung cancer (NSCLC) remain largely unknown. In this study, we performed comprehensive analysis of lncRNA expression in human NSCLC samples by using microarray data from Gene Expression Omnibus. After validation in a cohort of 80 pairs of NSCLC tissues, we identified a differentially expressed novel oncogenic lncRNA termed as AGAP2-AS1. The AGAP2-AS1 expression level was significantly upregulated in NSCLC tissues and negatively correlated with poor prognostic outcomes in patients. In vitro loss- and gain-of-function assays revealed that AGAP2-AS1 knockdown inhibited cell proliferation, migration and invasion, and induced cell apoptosis. In vivo assays also confirmed the ability of AGAP2-AS1 to promote tumor growth. Furthermore, mechanistic investigation showed that AGAP2-AS1 could bind with enhancer of zeste homolog 2 and lysine (K)-specific demethylase 1A, and recruit them to KLF2 and LATS2 promoter regions to repress their transcription. Taken together, our findings indicate that AGAP2-AS1 may act as an oncogene by repressing tumor-suppressor LATS2 and KLF2 transcription. By clarifying the AGAP2-AS1 mechanisms underlying NSCLC development and progression, these findings might promote the development of novel therapeutic strategies for this disease. PMID:27195672

  11. Expression and clinical significance of plasma small RNA in patients with pancreatic cancer%微小 RNA 在胰腺癌患者血浆中的表达情况及临床意义

    Institute of Scientific and Technical Information of China (English)

    潘文征; 唐万燕; 袁伟; 于琦; 左雯; 徐昌青; 马洁

    2014-01-01

    Objective The aim of this study was to identify six miRNAs expressed in plasma of patients with pancreatic cancer ( PCa ) and analyze their value as a diagnostic index of pancreatic cancer . Methods Plasma total RNAs were extracted from 30 PCa patients and 26 normal controls, and the abundance of six microRNAs was measured using real-time PCR.The possibility to combine them with CA19-9 as diagnostic biomarkers was analyzed .Results The expression level of miR-21, miR-210, miR-155, miR-20a, miR-25 and miR-196a in plasma of patients with pancreatic cancer were 1.65 ×106 , 5.98 × 104, 2.83 ×103, 3.47 ×106, 2.76 ×106, and 1.03 ×103 (copies/μl), while the normal controls were 4.08 ×105, 2.54 ×104, 8.55 ×102, 1.79 ×106, 9.32 ×105, and 4.67 ×102(copies/μl), respectively, with a significant difference between the two groups (P<0.05).The areas under the ROC curve of miR-21, miR-210, miR-155, miR-20a, miR-25 and miR-196a were 0.893, 0.810, 0.820, 0.766, 0.816 and 0.729, respectively.MiR-21 had the highest diagnostic value when it was used as diagnostic marker alone . The combination of miR-155 and miR-25 was more effective to distinguish PCa from normal than to be used alone, and the area under the ROC curve was 0.913(95%CI 0.838-0.988).When CA199 associated with miR-210 and miR-25, respectively, the areas under the ROC curves were 0.96 (95%CI was 0-1.0) and 0.942(95% CI was 0.876-1.0), which were higher than CA199 alone (0.862,95%CI was 0.748-0.975).There was a high improvement in diagnostic sensitivity and accuracy when miR-210 and miR-25 were combind with CA19-9, respectively.Conclusions Plasma miR-21, miR-155, miR-25, miR-210 have diagnostic value for pancreatic cancer , and deserve further study .%目的:探讨微小RNA ( miRNA)在胰腺癌患者血浆中的表达情况及临床意义。方法采用实时定量PCR方法检测30例胰腺癌和26例健康志愿者血浆中miR-21、miR-210、miR-155、miR-20a、miR-25和miR-196a的

  12. Construction of lentiviral mediated CyPA siRNA and its functions in non-small cell lung cancer%亲环素A基因在非小细胞肺癌中的功能

    Institute of Scientific and Technical Information of China (English)

    冯艳铭; 燕贞; 吴逸明; 徐正顺; 吴卫东

    2010-01-01

    目的 研究亲环素A(CyPA)基因在非小细胞肺癌中的功能.方法 设计并合成4对针对CyPA siRNA靶序列的寡核苷酸单链,退火形成双链DNA,与经Hpal和Xhol酶切后的pGCL-GFP载体连接产生Lv-shCyPA慢病毒载体,该载体含U6启动子和绿色荧光蛋白(GFP),经聚合酶链反应(PCR)筛选阳性克隆并测序鉴定.将Lv-shCyPA、pHelper1.0和pHelper2.0质粒共感染包装细胞293T细胞,包装成假病毒颗粒并感染非小细胞肺癌A549细胞,经免疫印迹法(Western blot)检测筛选CyPAsiRNA有效靶点和慢病毒颗粒,采用流式细胞技术分析细胞周期和凋亡率,进行裸鼠移植瘤实验.结果 PCR和测序结果证实,Lv-shCyPA慢病毒载体构建正确,经Western blot法筛选最有效的CyPA siRNA病毒颗粒滴度为2×10 9TU/ml,感染A549细胞凋亡率(5.01%±0.57%)增加,细胞周期G2-M期比例(11.40%±0.68%)减少,裸鼠移植瘤瘤体生长明显减缓,与未感染病毒的A549细胞细胞凋亡率(0.35%±0.17%)及G2-M期比例(14.52%±1.19%)相比,差异均有统计学意义(P<0.05).结论 CyPA基因沉默在非小细胞肺癌的发生中有一定的抑瘤作用,对非小细胞肺癌的预防和分子流行学研究提供了新的理论依据.%Objective To construct a lentiviral-vector-mediated CyPA small interference RNA (siRNA) and study its function in non-small cell lung cancer.Methods First,four target sequences were selected according to CyPA mRNA sequence.The complementary DNA containing both sense and antisense oligonucleotides were designed,synthesized and cloned into the pGCL-GFP vector,which contained U6 promoter and green fluorescent protein (GFP).The resulting lentiviral vector containing CyPA shRNA was named Lv-shCyPA,and it was confirmed by PCR and sequencing.Next,it was cotransfected by Lipofectamine2000 along with pHelper1.0 and pHelper 2.0 into 293T cells to package lentivirus particles.At the same time,the packed virus infected non-small cell lung cancer cell (A549

  13. RNA oxidation

    DEFF Research Database (Denmark)

    Kjaer, L. K.; Cejvanovic, V.; Henriken, T.;

    2015-01-01