Ewing's Sarcoma: An Analysis of miRNA Expression Profiles and Target Genes in Paraffin-Embedded Primary Tumor Tissue.

Science.gov (United States)

Parafioriti, Antonina; Bason, Caterina; Armiraglio, Elisabetta; Calciano, Lucia; Daolio, Primo Andrea; Berardocco, Martina; Di Bernardo, Andrea; Colosimo, Alessia; Luksch, Roberto; Berardi, Anna C

2016-04-30

The molecular mechanism responsible for Ewing's Sarcoma (ES) remains largely unknown. MicroRNAs (miRNAs), a class of small non-coding RNAs able to regulate gene expression, are deregulated in tumors and may serve as a tool for diagnosis and prediction. However, the status of miRNAs in ES has not yet been thoroughly investigated. This study compared global miRNAs expression in paraffin-embedded tumor tissue samples from 20 ES patients, affected by primary untreated tumors, with miRNAs expressed in normal human mesenchymal stromal cells (MSCs) by microarray analysis. A miRTarBase database was used to identify the predicted target genes for differentially expressed miRNAs. The miRNAs microarray analysis revealed distinct patterns of miRNAs expression between ES samples and normal MSCs. 58 of the 954 analyzed miRNAs were significantly differentially expressed in ES samples compared to MSCs. Moreover, the qRT-PCR analysis carried out on three selected miRNAs showed that miR-181b, miR-1915 and miR-1275 were significantly aberrantly regulated, confirming the microarray results. Bio-database analysis identified BCL-2 as a bona fide target gene of the miR-21, miR-181a, miR-181b, miR-29a, miR-29b, miR-497, miR-195, miR-let-7a, miR-34a and miR-1915. Using paraffin-embedded tissues from ES patients, this study has identified several potential target miRNAs and one gene that might be considered a novel critical biomarker for ES pathogenesis.

Expression analysis of miRNA and target mRNAs in esophageal cancer

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Meng, X.R. [Oncology Department, The First Affiliated Hospital of Zhengzhou University, Zhengzhou (China); Lu, P. [Gastrointestinal Surgery Department, People' s Hospital of Zhengzhou, Zhengzhou (China); Mei, J.Z.; Liu, G.J. [Medical Oncology Department, People' s Hospital of Zhengzhou, Zhengzhou (China); Fan, Q.X. [Oncology Department, The First Affiliated Hospital of Zhengzhou University, Zhengzhou (China)

2014-08-01

We aimed to investigate miRNAs and related mRNAs through a network-based approach in order to learn the crucial role that they play in the biological processes of esophageal cancer. Esophageal squamous-cell carcinoma (ESCC) and adenocarcinoma (EAC)-related miRNA and gene expression data were downloaded from the Gene Expression Omnibus database, and differentially expressed miRNAs and genes were selected. Target genes of differentially expressed miRNAs were predicted and their regulatory networks were constructed. Differentially expressed miRNA analysis selected four miRNAs associated with EAC and ESCC, among which hsa-miR-21 and hsa-miR-202 were shared by both diseases. hsa-miR-202 was reported for the first time to be associated with esophageal cancer in the present study. Differentially expressed miRNA target genes were mainly involved in cancer-related and signal-transduction pathways. Functional categories of these target genes were related to transcriptional regulation. The results may indicate potential target miRNAs and genes for future investigations of esophageal cancer.

Identification of novel miRNAs and miRNA dependent developmental shifts of gene expression in Arabidopsis thaliana.

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Shuhua Zhan

Full Text Available microRNAs (miRNAs are small, endogenous RNAs of 20 approximately 25 nucleotides, processed from stem-loop regions of longer RNA precursors. Plant miRNAs act as negative regulators of target mRNAs predominately by slicing target transcripts, and a number of miRNAs play important roles in development. We analyzed a number of published datasets from Arabidopsis thaliana to characterize novel miRNAs, novel miRNA targets, and miRNA-regulated developmental changes in gene expression. These data include microarray profiling data and small RNA (sRNA deep sequencing data derived from miRNA biogenesis/transport mutants, microarray profiling data of mRNAs in a developmental series, and computational predictions of conserved genomic stem-loop structures. Our conservative analyses identified five novel mature miRNAs and seven miRNA targets, including one novel target gene. Two complementary miRNAs that target distinct mRNAs were encoded by one gene. We found that genes targeted by known miRNAs, and genes up-regulated or down-regulated in miRNA mutant inflorescences, are highly expressed in the wild type inflorescence. In addition, transcripts upregulated within the mutant inflorescences were abundant in wild type leaves and shoot meristems and low in pollen and seed. Downregulated transcripts were abundant in wild type pollen and seed and low in shoot meristems, roots and leaves. Thus, disrupting miRNA function causes the inflorescence transcriptome to resemble the leaf and meristem and to differ from pollen and seed. Applications of our computational approach to other species and the use of more liberal criteria than reported here will further expand the number of identified miRNAs and miRNA targets. Our findings suggest that miRNAs have a global role in promoting vegetative to reproductive transitions in A. thaliana.

Targeted gene deletion of miRNAs in mice by TALEN system.

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Takada, Shuji; Sato, Tempei; Ito, Yoshiaki; Yamashita, Satoshi; Kato, Tomoko; Kawasumi, Miyuri; Kanai-Azuma, Masami; Igarashi, Arisa; Kato, Tomomi; Tamano, Moe; Asahara, Hiroshi

2013-01-01

Mice are among the most valuable model animal species with an enormous amount of heritage in genetic modification studies. However, targeting genes in mice is sometimes difficult, especially for small genes, such as microRNAs (miRNAs) and targeting genes in repeat sequences. Here we optimized the application of TALEN system for mice and successfully obtained gene targeting technique in mice for intergenic region and series of microRNAs. Microinjection of synthesized RNA of TALEN targeting each gene in one cell stage of embryo was carried out and injected oocytes were transferred into pseudopregnant ICR female mice, producing a high success rate of the targeted deletion of miRNA genes. In our condition, TALEN RNA without poly(A) tail worked better than that of with poly(A) tail. This mutated allele in miRNA was transmitted to the next generation, suggesting the successful germ line transmission of this targeting method. Consistent with our notion of miRNAs maturation mechanism, in homozygous mutant mice of miR-10a, the non- mutated strand of miRNAs expression was completely diminished. This method will lead us to expand and accelerate our genetic research using mice in a high throughput way.

Identification of miRNAs and their targets through high-throughput sequencing and degradome analysis in male and female Asparagus officinalis.

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Chen, Jingli; Zheng, Yi; Qin, Li; Wang, Yan; Chen, Lifei; He, Yanjun; Fei, Zhangjun; Lu, Gang

2016-04-12

MicroRNAs (miRNAs), a class of non-coding small RNAs (sRNAs), regulate various biological processes. Although miRNAs have been identified and characterized in several plant species, miRNAs in Asparagus officinalis have not been reported. As a dioecious plant with homomorphic sex chromosomes, asparagus is regarded as an important model system for studying mechanisms of plant sex determination. Two independent sRNA libraries from male and female asparagus plants were sequenced with Illumina sequencing, thereby generating 4.13 and 5.88 million final clean reads, respectively. Both libraries predominantly contained 24-nt sRNAs, followed by 21-nt sRNAs. Further analysis identified 154 conserved miRNAs, which belong to 26 families, and 39 novel miRNA candidates seemed to be specific to asparagus. Comparative profiling revealed that 63 miRNAs exhibited significant differential expression between male and female plants, which was confirmed by real-time quantitative PCR analysis. Among them, 37 miRNAs were significantly up-regulated in the female library, whereas the others were preferentially expressed in the male library. Furthermore, 40 target mRNAs representing 44 conserved and seven novel miRNAs were identified in asparagus through high-throughput degradome sequencing. Functional annotation showed that these target mRNAs were involved in a wide range of developmental and metabolic processes. We identified a large set of conserved and specific miRNAs and compared their expression levels between male and female asparagus plants. Several asparagus miRNAs, which belong to the miR159, miR167, and miR172 families involved in reproductive organ development, were differentially expressed between male and female plants, as well as during flower development. Consistently, several predicted targets of asparagus miRNAs were associated with floral organ development. These findings suggest the potential roles of miRNAs in sex determination and reproductive developmental processes in

Ewing’s Sarcoma: An Analysis of miRNA Expression Profiles and Target Genes in Paraffin-Embedded Primary Tumor Tissue

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Antonina Parafioriti

2016-04-01

Full Text Available The molecular mechanism responsible for Ewing’s Sarcoma (ES remains largely unknown. MicroRNAs (miRNAs, a class of small non-coding RNAs able to regulate gene expression, are deregulated in tumors and may serve as a tool for diagnosis and prediction. However, the status of miRNAs in ES has not yet been thoroughly investigated. This study compared global miRNAs expression in paraffin-embedded tumor tissue samples from 20 ES patients, affected by primary untreated tumors, with miRNAs expressed in normal human mesenchymal stromal cells (MSCs by microarray analysis. A miRTarBase database was used to identify the predicted target genes for differentially expressed miRNAs. The miRNAs microarray analysis revealed distinct patterns of miRNAs expression between ES samples and normal MSCs. 58 of the 954 analyzed miRNAs were significantly differentially expressed in ES samples compared to MSCs. Moreover, the qRT-PCR analysis carried out on three selected miRNAs showed that miR-181b, miR-1915 and miR-1275 were significantly aberrantly regulated, confirming the microarray results. Bio-database analysis identified BCL-2 as a bona fide target gene of the miR-21, miR-181a, miR-181b, miR-29a, miR-29b, miR-497, miR-195, miR-let-7a, miR-34a and miR-1915. Using paraffin-embedded tissues from ES patients, this study has identified several potential target miRNAs and one gene that might be considered a novel critical biomarker for ES pathogenesis.

Targeted gene deletion of miRNAs in mice by TALEN system.

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Shuji Takada

Full Text Available Mice are among the most valuable model animal species with an enormous amount of heritage in genetic modification studies. However, targeting genes in mice is sometimes difficult, especially for small genes, such as microRNAs (miRNAs and targeting genes in repeat sequences. Here we optimized the application of TALEN system for mice and successfully obtained gene targeting technique in mice for intergenic region and series of microRNAs. Microinjection of synthesized RNA of TALEN targeting each gene in one cell stage of embryo was carried out and injected oocytes were transferred into pseudopregnant ICR female mice, producing a high success rate of the targeted deletion of miRNA genes. In our condition, TALEN RNA without poly(A tail worked better than that of with poly(A tail. This mutated allele in miRNA was transmitted to the next generation, suggesting the successful germ line transmission of this targeting method. Consistent with our notion of miRNAs maturation mechanism, in homozygous mutant mice of miR-10a, the non- mutated strand of miRNAs expression was completely diminished. This method will lead us to expand and accelerate our genetic research using mice in a high throughput way.

Identifying MicroRNAs and Transcript Targets in Jatropha Seeds

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Galli, Vanessa; Guzman, Frank; de Oliveira, Luiz F. V.; Loss-Morais, Guilherme; Körbes, Ana P.; Silva, Sérgio D. A.; Margis-Pinheiro, Márcia M. A. N.; Margis, Rogério

2014-01-01

MicroRNAs, or miRNAs, are endogenously encoded small RNAs that play a key role in diverse plant biological processes. Jatropha curcas L. has received significant attention as a potential oilseed crop for the production of renewable oil. Here, a sRNA library of mature seeds and three mRNA libraries from three different seed development stages were generated by deep sequencing to identify and characterize the miRNAs and pre-miRNAs of J. curcas. Computational analysis was used for the identification of 180 conserved miRNAs and 41 precursors (pre-miRNAs) as well as 16 novel pre-miRNAs. The predicted miRNA target genes are involved in a broad range of physiological functions, including cellular structure, nuclear function, translation, transport, hormone synthesis, defense, and lipid metabolism. Some pre-miRNA and miRNA targets vary in abundance between the three stages of seed development. A search for sequences that produce siRNA was performed, and the results indicated that J. curcas siRNAs play a role in nuclear functions, transport, catalytic processes and disease resistance. This study presents the first large scale identification of J. curcas miRNAs and their targets in mature seeds based on deep sequencing, and it contributes to a functional understanding of these miRNAs. PMID:24551031

Turmeric (Curcuma longa): miRNAs and their regulating targets are involved in development and secondary metabolite pathways.

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Singh, Noopur; Sharma, Ashok

Turmeric has been used as a therapeutic herb over centuries in traditional medicinal systems due to the presence of several secondary metabolite compounds. microRNAs are known to regulate gene expression at the post-transcriptional level by transcriptional cleavage or translation repression. miRNAs have been demonstrated to play an active role in secondary metabolism regulation. The present work was focused on the identification of the miRNAs involved in the regulation of secondary metabolite and development process of turmeric. Eighteen miRNA families were identified for turmeric. Sixteen miRNA families were observed to regulate 238 target transcripts. LncRNAs targets of the putative miRNA candidates were also predicted. Our results indicated their role in binding, reproduction, stress, and other developmental processes. Gene annotation and pathway analysis illustrated the biological function of the targets regulated by the putative miRNAs. The miRNA-mediated gene regulatory network also revealed co-regulated targets that were regulated by two or more miRNA families. miR156 and miR5015 were observed to be involved in rhizome development. miR5021 showed regulation for terpenoid backbone biosynthesis and isoquinoline alkaloid biosynthesis pathways. The flavonoid biosynthesis pathway was observed to be regulated by miR2919. The analysis revealed the probable involvement of three miRNAs (miR1168.2, miR156b and miR1858) in curcumin biosynthesis. Other miRNAs were found to be involved in the growth and developmental process of turmeric. Phylogenetic analysis of selective miRNAs was also performed. Copyright © 2017 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.

VIRmiRNA: a comprehensive resource for experimentally validated viral miRNAs and their targets.

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Qureshi, Abid; Thakur, Nishant; Monga, Isha; Thakur, Anamika; Kumar, Manoj

2014-01-01

Viral microRNAs (miRNAs) regulate gene expression of viral and/or host genes to benefit the virus. Hence, miRNAs play a key role in host-virus interactions and pathogenesis of viral diseases. Lately, miRNAs have also shown potential as important targets for the development of novel antiviral therapeutics. Although several miRNA and their target repositories are available for human and other organisms in literature, but a dedicated resource on viral miRNAs and their targets are lacking. Therefore, we have developed a comprehensive viral miRNA resource harboring information of 9133 entries in three subdatabases. This includes 1308 experimentally validated miRNA sequences with their isomiRs encoded by 44 viruses in viral miRNA ' VIRMIRNA: ' and 7283 of their target genes in ' VIRMIRTAR': . Additionally, there is information of 542 antiviral miRNAs encoded by the host against 24 viruses in antiviral miRNA ' AVIRMIR': . The web interface was developed using Linux-Apache-MySQL-PHP (LAMP) software bundle. User-friendly browse, search, advanced search and useful analysis tools are also provided on the web interface. VIRmiRNA is the first specialized resource of experimentally proven virus-encoded miRNAs and their associated targets. This database would enhance the understanding of viral/host gene regulation and may also prove beneficial in the development of antiviral therapeutics. Database URL: http://crdd.osdd.net/servers/virmirna. © The Author(s) 2014. Published by Oxford University Press.

Investigation of miRNA Biology by Bioinformatic Tools and Impact of miRNAs in Colorectal Cancer: Regulatory Relationship of c-Myc and p53 with miRNAs

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Yaguang Xi

2007-01-01

Full Text Available MicroRNAs (miRNAs are a class of small non-coding RNAs that mediate gene expression at the posttranscriptional and translational levels and have been demonstrated to be involved in diverse biological functions. Mounting evidence in recent years has shown that miRNAs play key roles in tumorigenesis due to abnormal expression of and mutations in miRNAs. High throughput miRNA expression profiling of several major tumor types has identified miRNAs associated with clinical diagnosis and prognosis of cancer treatment. Previously our group has discovered a novel regulatory relationship between tumor suppressor gene p53 with miRNAs expression and a number of miRNA promoters contain putative p53 binding sites. In addition, others have reported that c-myc can mediate a large number of miRNAs expression. In this review, we will emphasize algorithms to identify mRNA targets of miRNAs and the roles of miRNAs in colorectal cancer. In particular, we will discuss a novel regulatory relationship of miRNAs with tumor suppressor p53 and c-myc. miRNAs are becoming promising novel targets and biomarkers for future cancer therapeutic development and clinical molecular diagnosis.

MiRNA-155 and miRNA-132 as potential diagnostic biomarkers for pulmonary tuberculosis: A preliminary study.

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Zheng, Meng-Li; Zhou, Nai-Kang; Luo, Cheng-Hua

2016-11-01

In our study, we aimed to profile a panel microRNAs (miRNAs) as potential biomarkers for the early diagnosis of pulmonary tuberculosis (PTB) and to illuminate the molecular mechanisms in the development of PTB. Firstly, gene expression profile of E-GEOD-49951 was downloaded from ArrayExpress database, and quantile-adjusted conditional maximum likelihood method was utilized to identify statistical difference between miRNAs of Mycobacterium tuberculosis (MTB)-infected individuals and healthy subjects. Furthermore, in order to assess the performance of our methodology, random forest (RF) classification model was utilized to identify the top 10 miRNAs with better Area Under The Curve (AUC) using 10-fold cross-validation method. Additionally, Monte Carlo Cross-Validation was repeated 50 times to explore the best miRNAs. In order to learn more about the differentially-expressed miRNAs, the target genes of differentially-expressed miRNAs were retrieved from TargetScan database and Ingenuity Pathways Analysis (IPA) was used to screen out biological pathways where target genes were involved. After normalization, a total of 478 miRNAs with higher than 0.25-fold quantile average across all samples were required. Based on the differential expression analysis, 38 differentially expressed miRNAs were identified when the significance was set as false discovery rate (FDR) < 0.01. Among the top 10 differentially expressed miRNAs, miRNA-155 obtained a highest AUC value 0.976, showing a good performance between PTB and control groups. Similarly, miRNA-449a, miRNA-212 and miRNA-132 revealed also a good performance with AUC values 0.947, 0.931 and 0.930, respectively. Moreover, miRNA-155, miRNA-449a, miRNA-29b-1* and miRNA-132 appeared in 50, 49, 49 and 48 bootstraps. Thus, miRNA-155 and miRNA-132 might be important in the progression of PTB and thereby, might present potential signatures for diagnosis of PTB. Copyright © 2016 Elsevier Ltd. All rights reserved.

Genome-wide identification and characterization of microRNA genes and their targets in flax (Linum usitatissimum): Characterization of flax miRNA genes.

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Barvkar, Vitthal T; Pardeshi, Varsha C; Kale, Sandip M; Qiu, Shuqing; Rollins, Meaghen; Datla, Raju; Gupta, Vidya S; Kadoo, Narendra Y

2013-04-01

MicroRNAs (miRNAs) are small (20-24 nucleotide long) endogenous regulatory RNAs that play important roles in plant growth and development. They regulate gene expression at the post-transcriptional level by translational repression or target degradation and gene silencing. In this study, we identified 116 conserved miRNAs belonging to 23 families from the flax (Linum usitatissimum L.) genome using a computational approach. The precursor miRNAs varied in length; while most of the mature miRNAs were 21 nucleotide long, intergenic and showed conserved signatures of RNA polymerase II transcripts in their upstream regions. Promoter region analysis of the flax miRNA genes indicated prevalence of MYB transcription factor binding sites. Four miRNA gene clusters containing members of three phylogenetic groups were identified. Further, 142 target genes were predicted for these miRNAs and most of these represent transcriptional regulators. The miRNA encoding genes were expressed in diverse tissues as determined by digital expression analysis as well as real-time PCR. The expression of fourteen miRNAs and nine target genes was independently validated using the quantitative reverse transcription PCR (qRT-PCR). This study suggests that a large number of conserved plant miRNAs are also found in flax and these may play important roles in growth and development of flax.

Uncovering leaf rust responsive miRNAs in wheat (Triticum aestivum L.) using high-throughput sequencing and prediction of their targets through degradome analysis.

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Kumar, Dhananjay; Dutta, Summi; Singh, Dharmendra; Prabhu, Kumble Vinod; Kumar, Manish; Mukhopadhyay, Kunal

2017-01-01

Deep sequencing identified 497 conserved and 559 novel miRNAs in wheat, while degradome analysis revealed 701 targets genes. QRT-PCR demonstrated differential expression of miRNAs during stages of leaf rust progression. Bread wheat (Triticum aestivum L.) is an important cereal food crop feeding 30 % of the world population. Major threat to wheat production is the rust epidemics. This study was targeted towards identification and functional characterizations of micro(mi)RNAs and their target genes in wheat in response to leaf rust ingression. High-throughput sequencing was used for transcriptome-wide identification of miRNAs and their expression profiling in retort to leaf rust using mock and pathogen-inoculated resistant and susceptible near-isogenic wheat plants. A total of 1056 mature miRNAs were identified, of which 497 miRNAs were conserved and 559 miRNAs were novel. The pathogen-inoculated resistant plants manifested more miRNAs compared with the pathogen infected susceptible plants. The miRNA counts increased in susceptible isoline due to leaf rust, conversely, the counts decreased in the resistant isoline in response to pathogenesis illustrating precise spatial tuning of miRNAs during compatible and incompatible interaction. Stem-loop quantitative real-time PCR was used to profile 10 highly differentially expressed miRNAs obtained from high-throughput sequencing data. The spatio-temporal profiling validated the differential expression of miRNAs between the isolines as well as in retort to pathogen infection. Degradome analysis provided 701 predicted target genes associated with defense response, signal transduction, development, metabolism, and transcriptional regulation. The obtained results indicate that wheat isolines employ diverse arrays of miRNAs that modulate their target genes during compatible and incompatible interaction. Our findings contribute to increase knowledge on roles of microRNA in wheat-leaf rust interactions and could help in rust

Identification and target prediction of miRNAs specifically expressed in rat neural tissue

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Tu Kang

2009-05-01

Full Text Available Abstract Background MicroRNAs (miRNAs are a large group of RNAs that play important roles in regulating gene expression and protein translation. Several studies have indicated that some miRNAs are specifically expressed in human, mouse and zebrafish tissues. For example, miR-1 and miR-133 are specifically expressed in muscles. Tissue-specific miRNAs may have particular functions. Although previous studies have reported the presence of human, mouse and zebrafish tissue-specific miRNAs, there have been no detailed reports of rat tissue-specific miRNAs. In this study, Home-made rat miRNA microarrays which established in our previous study were used to investigate rat neural tissue-specific miRNAs, and mapped their target genes in rat tissues. This study will provide information for the functional analysis of these miRNAs. Results In order to obtain as complete a picture of specific miRNA expression in rat neural tissues as possible, customized miRNA microarrays with 152 selected miRNAs from miRBase were used to detect miRNA expression in 14 rat tissues. After a general clustering analysis, 14 rat tissues could be clearly classified into neural and non-neural tissues based on the obtained expression profiles with p values Conclusion Our work provides a global view of rat neural tissue-specific miRNA profiles and a target map of miRNAs, which is expected to contribute to future investigations of miRNA regulatory mechanisms in neural systems.

DNA methyltransferase 1-targeting miRNA-148aof dairymilk: apotential bioactive modifier of thehumanepigenome

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Bodo C. Melnik

2017-09-01

Full Text Available Background: The perception of milk has changed from a “simple food” to a more sophisticated bioactive functional signaling system that promotes mTORC1-driven postnatal anabolism, growth, and development of the newborn infant. Accumulating evidence supports the view that milk´s miRNAs significantly contribute to these processes. The most abundant miRNA of milk found in milk fat and milk exosomes is miRNA-148a, which targets DNA methyltransferase 1 (DNMT1, a pivotal epigenetic regulator that suppresses transcription. Furthermore, milk-derived miRNA-125b, miRNA-30d, and miRNA-25 target TP53, the guardian of the genome that interacts with DNMT1 and regulates metabolism, cell kinetics, and apoptosis. Thus, the question arose whether cow´s milk-derived miRNAs may modify epigenetic regulation of the human milk consumer. Methods: To understand the potential impact of dairy milk consumption on human epigenetics, we have analyzed all relevant research-based bioinformatics data related to milk, milk miRNAs, epigenetic regulation, and lactation performance with special attention to bovine miRNAs that modify gene expression of DNA methyltransferase 1 (DNMT1 and p53 (TP53, the two guardians of the mammalian genome. By means of translational research and comparative functional genomics, we investigated the potential impact of cow´s milk miRNAs on epigenetic regulation of human DNMT1, TP53, FOXP3, and FTO, which are critically involved in immunologic and metabolic programming respectively. miRNA sequences have been obtained from mirbase.org. miRNA-target site prediction has been performed using TargetScan release 7.0. Results: The most abundant miRNA of cow´s milk is miRNA-148a, which represents more than 10% of all miRNAs of cow´s milk, survives pasteurization and refrigerated storage. The seed sequence of human and bovine miRNA-148a-3p is identical. Furthermore, human and bovine DNMT1 mRNA share 88% identity. The miRNA-148a 7mer seed is conserved in

Isolation and Identification of miRNAs in Jatropha curcas

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Wang, Chun Ming; Liu, Peng; Sun, Fei; Li, Lei; Liu, Peng; Ye, Jian; Yue, Gen Hua

2012-01-01

MicroRNAs (miRNAs) are small noncoding RNAs that play crucial regulatory roles by targeting mRNAs for silencing. To identify miRNAs in Jatropha curcas L, a bioenergy crop, cDNA clones from two small RNA libraries of leaves and seeds were sequenced and analyzed using bioinformatic tools. Fifty-two putative miRNAs were found from the two libraries, among them six were identical to known miRNAs and 46 were novel. Differential expression patterns of 15 miRNAs in root, stem, leave, fruit and seed were detected using quantitative real-time PCR. Ten miRNAs were highly expressed in fruit or seed, implying that they may be involved in seed development or fatty acids synthesis in seed. Moreover, 28 targets of the isolated miRNAs were predicted from a jatropha cDNA library database. The miRNA target genes were predicted to encode a broad range of proteins. Sixteen targets had clear BLASTX hits to the Uniprot database and were associated with genes belonging to the three major gene ontology categories of biological process, cellular component, and molecular function. Four targets were identified for JcumiR004. By silencing JcumiR004 primary miRNA, expressions of the four target genes were up-regulated and oil composition were modulated significantly, indicating diverse functions of JcumiR004. PMID:22419887

Comparative profiling of miRNAs and target gene identification in distant-grafting between tomato and Lycium (goji berry

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A B M Khaldun

2016-10-01

Full Text Available Local translocation of small RNAs between cells is proved. Long distance translocation between rootstock and scion is also well documented in the homo-grafting system, but the process in distant-grafting is widely unexplored where rootstock and scion belonging to different genera. Micro RNAs are a class of small, endogenous, noncoding, gene silencing RNAs that regulate target genes of a wide range of important biological pathways in plants. In this study, tomato was grafted onto goji (Lycium chinense Mill. to reveal the insight of miRNAs regulation and expression patterns within a distant-grafting system. Goji is an important traditional Chinese medicinal plant with enriched phytochemicals. Illumina sequencing technology has identified 68 evolutionary known miRNAs of 37 miRNA families. Moreover, 168 putative novel miRNAs were also identified. Compared with control tomato, 43 (11 known and 32 novels and 163 (33 known and 130 novels miRNAs were expressed significantly different in shoot and fruit of grafted tomato, respectively. The fruiting stage was identified as the most responsive in the distant-grafting approach and 123 miRNAs were found as up-regulating in the grafted fruit which is remarkably higher compare to the grafted shoot tip (28. Potential targets of differentially expressed miRNAs were found to be involved in diverse metabolic and regulatory pathways. ADP binding activities, molybdopterin synthase complex and RNA helicase activity were found as enriched terms in GO (Gene Ontology analysis. Additionally, ‘metabolic pathways’ was revealed as the most significant pathway in KEGG (Kyoto Encyclopedia of Genes and Genomes analysis. The information of the small RNA transcriptomes that are obtained from this study might be the first miRNAs elucidation for a distant-grafting system, particularly between goji and tomato. The results from this study will provide the insights into the molecular aspects of miRNA-mediated regulation in the

DeepMirTar: a deep-learning approach for predicting human miRNA targets.

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Wen, Ming; Cong, Peisheng; Zhang, Zhimin; Lu, Hongmei; Li, Tonghua

2018-06-01

MicroRNAs (miRNAs) are small noncoding RNAs that function in RNA silencing and post-transcriptional regulation of gene expression by targeting messenger RNAs (mRNAs). Because the underlying mechanisms associated with miRNA binding to mRNA are not fully understood, a major challenge of miRNA studies involves the identification of miRNA-target sites on mRNA. In silico prediction of miRNA-target sites can expedite costly and time-consuming experimental work by providing the most promising miRNA-target-site candidates. In this study, we reported the design and implementation of DeepMirTar, a deep-learning-based approach for accurately predicting human miRNA targets at the site level. The predicted miRNA-target sites are those having canonical or non-canonical seed, and features, including high-level expert-designed, low-level expert-designed, and raw-data-level, were used to represent the miRNA-target site. Comparison with other state-of-the-art machine-learning methods and existing miRNA-target-prediction tools indicated that DeepMirTar improved overall predictive performance. DeepMirTar is freely available at https://github.com/Bjoux2/DeepMirTar_SdA. lith@tongji.edu.cn, hongmeilu@csu.edu.cn. Supplementary data are available at Bioinformatics online.

In silico profiling of miRNAs and their target polymorphisms in ...

African Journals Online (AJOL)

To assess, whether miRNA target SNPs are implicated in leukemia associated genes, we conducted an in silico approach along with the availability of publicly available web based tools for miRNA prediction and comprehensive genomic databases of SNPs. In this in-depth report, we attempted to use two computational ...

Predicting human miRNA target genes using a novel evolutionary methodology

KAUST Repository

Aigli, Korfiati; Kleftogiannis, Dimitrios A.; Konstantinos, Theofilatos; Spiros, Likothanassis; Athanasios, Tsakalidis; Seferina, Mavroudi

2012-01-01

The discovery of miRNAs had great impacts on traditional biology. Typically, miRNAs have the potential to bind to the 3'untraslated region (UTR) of their mRNA target genes for cleavage or translational repression. The experimental identification of their targets has many drawbacks including cost, time and low specificity and these are the reasons why many computational approaches have been developed so far. However, existing computational approaches do not include any advanced feature selection technique and they are facing problems concerning their classification performance and their interpretability. In the present paper, we propose a novel hybrid methodology which combines genetic algorithms and support vector machines in order to locate the optimal feature subset while achieving high classification performance. The proposed methodology was compared with two of the most promising existing methodologies in the problem of predicting human miRNA targets. Our approach outperforms existing methodologies in terms of classification performances while selecting a much smaller feature subset. © 2012 Springer-Verlag.

Predicting human miRNA target genes using a novel evolutionary methodology

KAUST Repository

Aigli, Korfiati

2012-01-01

The discovery of miRNAs had great impacts on traditional biology. Typically, miRNAs have the potential to bind to the 3\\'untraslated region (UTR) of their mRNA target genes for cleavage or translational repression. The experimental identification of their targets has many drawbacks including cost, time and low specificity and these are the reasons why many computational approaches have been developed so far. However, existing computational approaches do not include any advanced feature selection technique and they are facing problems concerning their classification performance and their interpretability. In the present paper, we propose a novel hybrid methodology which combines genetic algorithms and support vector machines in order to locate the optimal feature subset while achieving high classification performance. The proposed methodology was compared with two of the most promising existing methodologies in the problem of predicting human miRNA targets. Our approach outperforms existing methodologies in terms of classification performances while selecting a much smaller feature subset. © 2012 Springer-Verlag.

Human disease MiRNA inference by combining target information based on heterogeneous manifolds.

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Ding, Pingjian; Luo, Jiawei; Liang, Cheng; Xiao, Qiu; Cao, Buwen

2018-04-01

The emergence of network medicine has provided great insight into the identification of disease-related molecules, which could help with the development of personalized medicine. However, the state-of-the-art methods could neither simultaneously consider target information and the known miRNA-disease associations nor effectively explore novel gene-disease associations as a by-product during the process of inferring disease-related miRNAs. Computational methods incorporating multiple sources of information offer more opportunities to infer disease-related molecules, including miRNAs and genes in heterogeneous networks at a system level. In this study, we developed a novel algorithm, named inference of Disease-related MiRNAs based on Heterogeneous Manifold (DMHM), to accurately and efficiently identify miRNA-disease associations by integrating multi-omics data. Graph-based regularization was utilized to obtain a smooth function on the data manifold, which constitutes the main principle of DMHM. The novelty of this framework lies in the relatedness between diseases and miRNAs, which are measured via heterogeneous manifolds on heterogeneous networks integrating target information. To demonstrate the effectiveness of DMHM, we conducted comprehensive experiments based on HMDD datasets and compared DMHM with six state-of-the-art methods. Experimental results indicated that DMHM significantly outperformed the other six methods under fivefold cross validation and de novo prediction tests. Case studies have further confirmed the practical usefulness of DMHM. Copyright © 2018 Elsevier Inc. All rights reserved.

MatureBayes: a probabilistic algorithm for identifying the mature miRNA within novel precursors.

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Katerina Gkirtzou

Full Text Available BACKGROUND: MicroRNAs (miRNAs are small, single stranded RNAs with a key role in post-transcriptional regulation of thousands of genes across numerous species. While several computational methods are currently available for identifying miRNA genes, accurate prediction of the mature miRNA remains a challenge. Existing approaches fall short in predicting the location of mature miRNAs but also in finding the functional strand(s of miRNA precursors. METHODOLOGY/PRINCIPAL FINDINGS: Here, we present a computational tool that incorporates a Naive Bayes classifier to identify mature miRNA candidates based on sequence and secondary structure information of their miRNA precursors. We take into account both positive (true mature miRNAs and negative (same-size non-mature miRNA sequences examples to optimize sensitivity as well as specificity. Our method can accurately predict the start position of experimentally verified mature miRNAs for both human and mouse, achieving a significantly larger (often double performance accuracy compared with two existing methods. Moreover, the method exhibits a very high generalization performance on miRNAs from two other organisms. More importantly, our method provides direct evidence about the features of miRNA precursors which may determine the location of the mature miRNA. We find that the triplet of positions 7, 8 and 9 from the mature miRNA end towards the closest hairpin have the largest discriminatory power, are relatively conserved in terms of sequence composition (mostly contain a Uracil and are located within or in very close proximity to the hairpin loop, suggesting the existence of a possible recognition site for Dicer and associated proteins. CONCLUSIONS: This work describes a novel algorithm for identifying the start position of mature miRNA(s produced by miRNA precursors. Our tool has significantly better (often double performance than two existing approaches and provides new insights about the potential use

Integrative analyses of miRNA and proteomics identify potential biological pathways associated with onset of pulmonary fibrosis in the bleomycin rat model

International Nuclear Information System (INIS)

Fukunaga, Satoki; Kakehashi, Anna; Sumida, Kayo; Kushida, Masahiko; Asano, Hiroyuki; Gi, Min; Wanibuchi, Hideki

2015-01-01

To determine miRNAs and their predicted target proteins regulatory networks which are potentially involved in onset of pulmonary fibrosis in the bleomycin rat model, we conducted integrative miRNA microarray and iTRAQ-coupled LC-MS/MS proteomic analyses, and evaluated the significance of altered biological functions and pathways. We observed that alterations of miRNAs and proteins are associated with the early phase of bleomycin-induced pulmonary fibrosis, and identified potential target pairs by using ingenuity pathway analysis. Using the data set of these alterations, it was demonstrated that those miRNAs, in association with their predicted target proteins, are potentially involved in canonical pathways reflective of initial epithelial injury and fibrogenic processes, and biofunctions related to induction of cellular development, movement, growth, and proliferation. Prediction of activated functions suggested that lung cells acquire proliferative, migratory, and invasive capabilities, and resistance to cell death especially in the very early phase of bleomycin-induced pulmonary fibrosis. The present study will provide new insights for understanding the molecular pathogenesis of idiopathic pulmonary fibrosis. - Highlights: • We analyzed bleomycin-induced pulmonary fibrosis in the rat. • Integrative analyses of miRNA microarray and proteomics were conducted. • We determined the alterations of miRNAs and their potential target proteins. • The alterations may control biological functions and pathways in pulmonary fibrosis. • Our result may provide new insights of pulmonary fibrosis

Integrative analyses of miRNA and proteomics identify potential biological pathways associated with onset of pulmonary fibrosis in the bleomycin rat model

Energy Technology Data Exchange (ETDEWEB)

Fukunaga, Satoki [Department of Molecular Pathology, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585 (Japan); Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd., 3-1-98 Kasugade-Naka, Konohana-ku, Osaka 554-8558 (Japan); Kakehashi, Anna [Department of Molecular Pathology, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585 (Japan); Sumida, Kayo; Kushida, Masahiko; Asano, Hiroyuki [Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd., 3-1-98 Kasugade-Naka, Konohana-ku, Osaka 554-8558 (Japan); Gi, Min [Department of Molecular Pathology, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585 (Japan); Wanibuchi, Hideki, E-mail: wani@med.osaka-cu.ac.jp [Department of Molecular Pathology, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585 (Japan)

2015-08-01

To determine miRNAs and their predicted target proteins regulatory networks which are potentially involved in onset of pulmonary fibrosis in the bleomycin rat model, we conducted integrative miRNA microarray and iTRAQ-coupled LC-MS/MS proteomic analyses, and evaluated the significance of altered biological functions and pathways. We observed that alterations of miRNAs and proteins are associated with the early phase of bleomycin-induced pulmonary fibrosis, and identified potential target pairs by using ingenuity pathway analysis. Using the data set of these alterations, it was demonstrated that those miRNAs, in association with their predicted target proteins, are potentially involved in canonical pathways reflective of initial epithelial injury and fibrogenic processes, and biofunctions related to induction of cellular development, movement, growth, and proliferation. Prediction of activated functions suggested that lung cells acquire proliferative, migratory, and invasive capabilities, and resistance to cell death especially in the very early phase of bleomycin-induced pulmonary fibrosis. The present study will provide new insights for understanding the molecular pathogenesis of idiopathic pulmonary fibrosis. - Highlights: • We analyzed bleomycin-induced pulmonary fibrosis in the rat. • Integrative analyses of miRNA microarray and proteomics were conducted. • We determined the alterations of miRNAs and their potential target proteins. • The alterations may control biological functions and pathways in pulmonary fibrosis. • Our result may provide new insights of pulmonary fibrosis.

Identifying relevant group of miRNAs in cancer using fuzzy mutual information.

Science.gov (United States)

Pal, Jayanta Kumar; Ray, Shubhra Sankar; Pal, Sankar K

2016-04-01

MicroRNAs (miRNAs) act as a major biomarker of cancer. All miRNAs in human body are not equally important for cancer identification. We propose a methodology, called FMIMS, which automatically selects the most relevant miRNAs for a particular type of cancer. In FMIMS, miRNAs are initially grouped by using a SVM-based algorithm; then the group with highest relevance is determined and the miRNAs in that group are finally ranked for selection according to their redundancy. Fuzzy mutual information is used in computing the relevance of a group and the redundancy of miRNAs within it. Superiority of the most relevant group to all others, in deciding normal or cancer, is demonstrated on breast, renal, colorectal, lung, melanoma and prostate data. The merit of FMIMS as compared to several existing methods is established. While 12 out of 15 selected miRNAs by FMIMS corroborate with those of biological investigations, three of them viz., "hsa-miR-519," "hsa-miR-431" and "hsa-miR-320c" are possible novel predictions for renal cancer, lung cancer and melanoma, respectively. The selected miRNAs are found to be involved in disease-specific pathways by targeting various genes. The method is also able to detect the responsible miRNAs even at the primary stage of cancer. The related code is available at http://www.jayanta.droppages.com/FMIMS.html .

Integration of the Pokeweed miRNA and mRNA Transcriptomes Reveals Targeting of Jasmonic Acid-Responsive Genes

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Kira C. M. Neller

2018-05-01

Full Text Available The American pokeweed plant, Phytolacca americana, displays broad-spectrum resistance to plant viruses and is a heavy metal hyperaccumulator. However, little is known about the regulation of biotic and abiotic stress responses in this non-model plant. To investigate the control of miRNAs in gene expression, we sequenced the small RNA transcriptome of pokeweed treated with jasmonic acid (JA, a hormone that mediates pathogen defense and stress tolerance. We predicted 145 miRNAs responsive to JA, most of which were unique to pokeweed. These miRNAs were low in abundance and condition-specific, with discrete expression change. Integration of paired mRNA-Seq expression data enabled us to identify correlated, novel JA-responsive targets that mediate hormone biosynthesis, signal transduction, and pathogen defense. The expression of approximately half the pairs was positively correlated, an uncommon finding that we functionally validated by mRNA cleavage. Importantly, we report that a pokeweed-specific miRNA targets the transcript of OPR3, novel evidence that a miRNA regulates a JA biosynthesis enzyme. This first large-scale small RNA study of a Phytolaccaceae family member shows that miRNA-mediated control is a significant component of the JA response, associated with widespread changes in expression of genes required for stress adaptation.

miRvestigator: web application to identify miRNAs responsible for co-regulated gene expression patterns discovered through transcriptome profiling.

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Plaisier, Christopher L; Bare, J Christopher; Baliga, Nitin S

2011-07-01

Transcriptome profiling studies have produced staggering numbers of gene co-expression signatures for a variety of biological systems. A significant fraction of these signatures will be partially or fully explained by miRNA-mediated targeted transcript degradation. miRvestigator takes as input lists of co-expressed genes from Caenorhabditis elegans, Drosophila melanogaster, G. gallus, Homo sapiens, Mus musculus or Rattus norvegicus and identifies the specific miRNAs that are likely to bind to 3' un-translated region (UTR) sequences to mediate the observed co-regulation. The novelty of our approach is the miRvestigator hidden Markov model (HMM) algorithm which systematically computes a similarity P-value for each unique miRNA seed sequence from the miRNA database miRBase to an overrepresented sequence motif identified within the 3'-UTR of the query genes. We have made this miRNA discovery tool accessible to the community by integrating our HMM algorithm with a proven algorithm for de novo discovery of miRNA seed sequences and wrapping these algorithms into a user-friendly interface. Additionally, the miRvestigator web server also produces a list of putative miRNA binding sites within 3'-UTRs of the query transcripts to facilitate the design of validation experiments. The miRvestigator is freely available at http://mirvestigator.systemsbiology.net.

Targeting oncomiRNAs and mimicking tumor suppressor miRNAs: New trends in the development of miRNA therapeutic strategies in oncology (Review)

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GAMBARI, ROBERTO; BROGNARA, ELEONORA; SPANDIDOS, DEMETRIOS A.; FABBRI, ENRICA

2016-01-01

MicroRNA (miRNA or miR) therapeutics in cancer are based on targeting or mimicking miRNAs involved in cancer onset, progression, angiogenesis, epithelial-mesenchymal transition and metastasis. Several studies conclusively have demonstrated that miRNAs are deeply involved in tumor onset and progression, either behaving as tumor-promoting miRNAs (oncomiRNAs and metastamiRNAs) or as tumor suppressor miRNAs. This review focuses on the most promising examples potentially leading to the development of anticancer, miRNA-based therapeutic protocols. The inhibition of miRNA activity can be readily achieved by the use of miRNA inhibitors and oligomers, including RNA, DNA and DNA analogues (miRNA antisense therapy), small molecule inhibitors, miRNA sponges or through miRNA masking. On the contrary, the enhancement of miRNA function (miRNA replacement therapy) can be achieved by the use of modified miRNA mimetics, such as plasmid or lentiviral vectors carrying miRNA sequences. Combination strategies have been recently developed based on the observation that i) the combined administration of different antagomiR molecules induces greater antitumor effects and ii) some anti-miR molecules can sensitize drug-resistant tumor cell lines to therapeutic drugs. In this review, we discuss two additional issues: i) the combination of miRNA replacement therapy with drug administration and ii) the combination of antagomiR and miRNA replacement therapy. One of the solid results emerging from different independent studies is that miRNA replacement therapy can enhance the antitumor effects of the antitumor drugs. The second important conclusion of the reviewed studies is that the combination of anti-miRNA and miRNA replacement strategies may lead to excellent results, in terms of antitumor effects. PMID:27175518

Silencing of Stress-Regulated miRNAs in Plants by Short Tandem Target Mimic (STTM) Approach.

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Teotia, Sachin; Tang, Guiliang

2017-01-01

In plants, microRNAs (miRNAs) regulate more than hundred target genes comprising largely transcription factors that control growth and development as well as stress responses. However, the exact functions of miRNA families could not be deciphered because each miRNA family has multiple loci in the genome, thus are functionally redundant. Therefore, an ideal approach to study the function of a miRNA family is to silence the expression of all members simultaneously, which is a daunting task. However, this can be partly overcome by Target Mimic (TM) approach that can knockdown an entire miRNA family. STTM is a modification of TM approach and complements it. STTMs have been successfully used in monocots and dicots to block miRNA functions. miR159 has been shown to be differentially regulated by various abiotic stresses including ABA in various plant species. Here, we describe in detail the protocol for designing STTM construct to block miR159 functions in Arabidopsis, with the potential to apply this technique on a number of other stress-regulated miRNAs in plants.

Evaluation of circulating miRNAs during late pregnancy in the mare.

Directory of Open Access Journals (Sweden)

Shavahn C Loux

Full Text Available MicroRNAs (miRNAs are small, non-coding RNAs which are produced throughout the body. Individual tissues tend to have a specific expression profile and excrete many of these miRNAs into circulation. These circulating miRNAs may be diagnostically valuable biomarkers for assessing the presence of disease while minimizing invasive testing. In women, numerous circulating miRNAs have been identified which change significantly during pregnancy-related complications (e.g. chorioamnionitis, eclampsia, recurrent pregnancy loss; however, no prior work has been done in this area in the horse. To identify pregnancy-specific miRNAs, we collected serial whole blood samples in pregnant mares at 8, 9, 10 m of gestation and post-partum, as well as from non-pregnant (diestrous mares. In total, we evaluated a panel of 178 miRNAs using qPCR, eventually identifying five miRNAs of interest. One miRNA (miR-374b was differentially regulated through late gestation and four miRNAs (miR-454, miR-133b, miR-486-5p and miR-204b were differentially regulated between the pregnant and non-pregnant samples. We were able to identify putative targets for the differentially regulated miRNAs using two separate target prediction programs, miRDB and Ingenuity Pathway Analysis. The targets for the miRNAs differentially regulated during pregnancy were predicted to be involved in signaling pathways such as the STAT3 pathway and PI3/AKT signaling pathway, as well as more endocrine-based pathways, including the GnRH, prolactin and insulin signaling pathways. In summary, this study provides novel information about the changes occurring in circulating miRNAs during normal pregnancy, as well as attempting to predict the biological effects induced by these miRNAs.

The evolution of Homo sapiens denisova and Homo sapiens neanderthalensis miRNA targeting genes in the prenatal and postnatal brain.

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Gunbin, Konstantin V; Afonnikov, Dmitry A; Kolchanov, Nikolay A; Derevianko, Anatoly P; Rogaev, Eugeny I

2015-01-01

As the evolution of miRNA genes has been found to be one of the important factors in formation of the modern type of man, we performed a comparative analysis of the evolution of miRNA genes in two archaic hominines, Homo sapiens neanderthalensis and Homo sapiens denisova, and elucidated the expression of their target mRNAs in bain. A comparative analysis of the genomes of primates, including species in the genus Homo, identified a group of miRNA genes having fixed substitutions with important implications for the evolution of Homo sapiens neanderthalensis and Homo sapiens denisova. The mRNAs targeted by miRNAs with mutations specific for Homo sapiens denisova exhibited enhanced expression during postnatal brain development in modern humans. By contrast, the expression of mRNAs targeted by miRNAs bearing variations specific for Homo sapiens neanderthalensis was shown to be enhanced in prenatal brain development. Our results highlight the importance of changes in miRNA gene sequences in the course of Homo sapiens denisova and Homo sapiens neanderthalensis evolution. The genetic alterations of miRNAs regulating the spatiotemporal expression of multiple genes in the prenatal and postnatal brain may contribute to the progressive evolution of brain function, which is consistent with the observations of fine technical and typological properties of tools and decorative items reported from archaeological Denisovan sites. The data also suggest that differential spatial-temporal regulation of gene products promoted by the subspecies-specific mutations in the miRNA genes might have occurred in the brains of Homo sapiens denisova and Homo sapiens neanderthalensis, potentially contributing to the cultural differences between these two archaic hominines.

Bioinformatic identification and experimental validation of miRNAs from foxtail millet (Setaria italica).

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Han, Jun; Xie, Hao; Sun, Qingpeng; Wang, Jun; Lu, Min; Wang, Weixiang; Guo, Erhu; Pan, Jinbao

2014-08-10

MiRNAs are a novel group of non-coding small RNAs that negatively regulate gene expression. Many miRNAs have been identified and investigated extensively in plant species with sequenced genomes. However, few miRNAs have been identified in foxtail millet (Setaria italica), which is an ancient cereal crop of great importance for dry land agriculture. In this study, 271 foxtail millet miRNAs belonging to 44 families were identified using a bioinformatics approach. Twenty-three pairs of sense/antisense miRNAs belonging to 13 families, and 18 miRNA clusters containing members of 8 families were discovered in foxtail millet. We identified 432 potential targets for 38 miRNA families, most of which were predicted to be involved in plant development, signal transduction, metabolic pathways, disease resistance, and environmental stress responses. Gene ontology (GO) analysis revealed that 101, 56, and 23 target genes were involved in molecular functions, biological processes, and cellular components, respectively. We investigated the expression patterns of 43 selected miRNAs using qRT-PCR analysis. All of the miRNAs were expressed ubiquitously with many exhibiting different expression levels in different tissues. We validated five predicted targets of four miRNAs using the RNA ligase mediated rapid amplification of cDNA end (5'-RLM-RACE) method. Copyright © 2014 Elsevier B.V. All rights reserved.

Identification and Expression Analyses of miRNAs from Two Contrasting Flower Color Cultivars of Canna by Deep Sequencing.

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Roy, Sribash; Tripathi, Abhinandan Mani; Yadav, Amrita; Mishra, Parneeta; Nautiyal, Chandra Shekhar

2016-01-01

miRNAs are endogenous small RNA (sRNA) that play critical roles in plant development processes. Canna is an ornamental plant belonging to family Cannaceae. Here, we report for the first time the identification and differential expression of miRNAs in two contrasting flower color cultivars of Canna, Tropical sunrise and Red president. A total of 313 known miRNAs belonging to 78 miRNA families were identified from both the cultivars. Thirty one miRNAs (17 miRNA families) were specific to Tropical sunrise and 43 miRNAs (10 miRNA families) were specific to Red president. Thirty two and 18 putative new miRNAs were identified from Tropical sunrise and Red president, respectively. One hundred and nine miRNAs were differentially expressed in the two cultivars targeting 1343 genes. Among these, 16 miRNAs families targeting 60 genes were involved in flower development related traits and five miRNA families targeting five genes were involved in phenyl propanoid and pigment metabolic processes. We further validated the expression analysis of a few miRNA and their target genes by qRT-PCR. Transcription factors were the major miRNA targets identified. Target validation of a few randomly selected miRNAs by RLM-RACE was performed but was successful with only miR162. These findings will help in understanding flower development processes, particularly the color development in Canna.

miRNAs as therapeutic targets in ischemic heart disease.

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Frost, Robert J A; van Rooij, Eva

2010-06-01

Ischemic heart disease is a form of congestive heart failure that is caused by insufficient blood supply to the heart, resulting in a loss of viable tissue. In response to the injury, the non-ischemic myocardium displays signs of secondary remodeling, like interstitial fibrosis and hypertrophy of cardiac myocytes. This remodeling process further deteriorates pump function and increases susceptibility to arrhythmias. MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression in a sequence-dependent manner. Recently, several groups identified miRNAs as crucial gene regulators in response to myocardial infarction (MI) and during post-MI remodeling. In this review, we discuss how modulation of these miRNAs represents a promising new therapeutic strategy to improve the clinical outcome in ischemic heart disease.

Analysis of miRNAs targeting transcription factors in Persicaria minor induced by Fusarium oxysporum

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Samad, Abdul Fatah A.; Ali, Nazaruddin Muhammad; Ismail, Ismanizan; Murad, Abdul Munir Abdul

2016-11-01

A recent discovery showed small non-coding RNA known as microRNA has a crucial role in plant development and plant survival in extreme condition. In the past few years, researchers have managed to identify the various families of transcription factors that play a crucial role in regulating plant development and plant responses to stresses. This study focuses on the expression pattern of miRNA targeted transcription factor under biotic stress in a plant rich with secondary metabolite, Persicaria minor. A pathogenic fungus, Fusarium oxysporum was used in the biotic stress treatment since the previous study revealed this fungus could trigger plant defense system. Two small RNA libraries were constructed which consist of control and treated samples. In order to identify the potential target, psRobot target prediction software was used for each miRNA that shows significant change due to the infection. The result showed miR156b/c, miR172a, miR319, miR858, and miR894 were found to be targeting a wide range of transcription factors that involve in plant development and plant response towards stresses. The expression of miR156b/c and miR172 were up-regulated while the expression of miR319, miR858, and miR894 was found to be down-regulated. These results may provide a certain level of networking between those two regulatory molecules in plant genetic system under biotic stress.

A comprehensive survey of 3' animal miRNA modification events and a possible role for 3' adenylation in modulating miRNA targeting effectiveness.

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Burroughs, A Maxwell; Ando, Yoshinari; de Hoon, Michiel J L; Tomaru, Yasuhiro; Nishibu, Takahiro; Ukekawa, Ryo; Funakoshi, Taku; Kurokawa, Tsutomu; Suzuki, Harukazu; Hayashizaki, Yoshihide; Daub, Carsten O

2010-10-01

Animal microRNA sequences are subject to 3' nucleotide addition. Through detailed analysis of deep-sequenced short RNA data sets, we show adenylation and uridylation of miRNA is globally present and conserved across Drosophila and vertebrates. To better understand 3' adenylation function, we deep-sequenced RNA after knockdown of nucleotidyltransferase enzymes. The PAPD4 nucleotidyltransferase adenylates a wide range of miRNA loci, but adenylation does not appear to affect miRNA stability on a genome-wide scale. Adenine addition appears to reduce effectiveness of miRNA targeting of mRNA transcripts while deep-sequencing of RNA bound to immunoprecipitated Argonaute (AGO) subfamily proteins EIF2C1-EIF2C3 revealed substantial reduction of adenine addition in miRNA associated with EIF2C2 and EIF2C3. Our findings show 3' addition events are widespread and conserved across animals, PAPD4 is a primary miRNA adenylating enzyme, and suggest a role for 3' adenine addition in modulating miRNA effectiveness, possibly through interfering with incorporation into the RNA-induced silencing complex (RISC), a regulatory role that would complement the role of miRNA uridylation in blocking DICER1 uptake.

Computational tools for genome-wide miRNA prediction and study

KAUST Repository

Malas, T.B.

2012-11-02

MicroRNAs (miRNAs) are single-stranded non-coding RNA susually of 22 nucleotidesin length that play an important post-transcriptional regulation role in many organisms. MicroRNAs bind a seed sequence to the 3-untranslated region (UTR) region of the target messenger RNA (mRNA), inducing degradation or inhibition of translation and resulting in a reduction in the protein level. This regulatory mechanism is central to many biological processes and perturbation could lead to diseases such as cancer. Given the biological importance, of miRNAs, there is a great need to identify and study their targets and functions. However, miRNAs are very difficult to clone in the lab and this has hindered the identification of novel miRNAs. Next-generation sequencing coupled with new computational tools has recently evolved to help researchers efficiently identify large numbers of novel miRNAs. In this review, we describe recent miRNA prediction tools and discuss their priorities, advantages and disadvantages. Malas and Ravasi.

Computational tools for genome-wide miRNA prediction and study

KAUST Repository

Malas, T.B.; Ravasi, Timothy

2012-01-01

MicroRNAs (miRNAs) are single-stranded non-coding RNA susually of 22 nucleotidesin length that play an important post-transcriptional regulation role in many organisms. MicroRNAs bind a seed sequence to the 3-untranslated region (UTR) region of the target messenger RNA (mRNA), inducing degradation or inhibition of translation and resulting in a reduction in the protein level. This regulatory mechanism is central to many biological processes and perturbation could lead to diseases such as cancer. Given the biological importance, of miRNAs, there is a great need to identify and study their targets and functions. However, miRNAs are very difficult to clone in the lab and this has hindered the identification of novel miRNAs. Next-generation sequencing coupled with new computational tools has recently evolved to help researchers efficiently identify large numbers of novel miRNAs. In this review, we describe recent miRNA prediction tools and discuss their priorities, advantages and disadvantages. Malas and Ravasi.

miRNA Repertoires of Demosponges Stylissa carteri and Xestospongia testudinaria

KAUST Repository

Liew, Yi Jin

2016-02-12

MicroRNAs (miRNAs) are small regulatory RNAs that are involved in many biological process in eukaryotes. They play a crucial role in modulating genetic expression of their targets, which makes them integral components of transcriptional regulatory networks. As sponges (phylum Porifera) are commonly considered the most basal metazoan, the in-depth capture of miRNAs from these organisms provides additional clues to the evolution of miRNA families in metazoans. Here, we identified the core proteins involved in the biogenesis of miRNAs, and obtained evidence for bona fide miRNA sequences for two marine sponges Stylissa carteri and Xestospongia testudinaria (11 and 19 respectively). Our analysis identified several miRNAs that are conserved amongst demosponges, and revealed that all of the novel miRNAs identified in these two species are specific to the class Demospongiae.

miRNA Repertoires of Demosponges Stylissa carteri and Xestospongia testudinaria

KAUST Repository

Liew, Yi Jin; Ryu, Tae Woo; Aranda, Manuel; Ravasi, Timothy

2016-01-01

MicroRNAs (miRNAs) are small regulatory RNAs that are involved in many biological process in eukaryotes. They play a crucial role in modulating genetic expression of their targets, which makes them integral components of transcriptional regulatory networks. As sponges (phylum Porifera) are commonly considered the most basal metazoan, the in-depth capture of miRNAs from these organisms provides additional clues to the evolution of miRNA families in metazoans. Here, we identified the core proteins involved in the biogenesis of miRNAs, and obtained evidence for bona fide miRNA sequences for two marine sponges Stylissa carteri and Xestospongia testudinaria (11 and 19 respectively). Our analysis identified several miRNAs that are conserved amongst demosponges, and revealed that all of the novel miRNAs identified in these two species are specific to the class Demospongiae.

Identification of Viscum album L. miRNAs and prediction of their medicinal values.

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Wenyan Xie

Full Text Available MicroRNAs (miRNAs are a class of approximately 22 nucleotides single-stranded non-coding RNA molecules that play crucial roles in gene expression. It has been reported that the plant miRNAs might enter mammalian bloodstream and have a functional role in human metabolism, indicating that miRNAs might be one of the hidden bioactive ingredients in medicinal plants. Viscum album L. (Loranthaceae, European mistletoe has been widely used for the treatment of cancer and cardiovascular diseases, but its functional compounds have not been well characterized. We considered that miRNAs might be involved in the pharmacological activities of V. album. High-throughput Illumina sequencing was performed to identify the novel and conserved miRNAs of V. album. The putative human targets were predicted. In total, 699 conserved miRNAs and 1373 novel miRNAs have been identified from V. album. Based on the combined use of TargetScan, miRanda, PITA, and RNAhybrid methods, the intersection of 30697 potential human genes have been predicted as putative targets of 29 novel miRNAs, while 14559 putative targets were highly enriched in 33 KEGG pathways. Interestingly, these highly enriched KEGG pathways were associated with some human diseases, especially cancer, cardiovascular diseases and neurological disorders, which might explain the clinical use as well as folk medicine use of mistletoe. However, further experimental validation is necessary to confirm these human targets of mistletoe miRNAs. Additionally, target genes involved in bioactive components synthesis in V. album were predicted as well. A total of 68 miRNAs were predicted to be involved in terpenoid biosynthesis, while two miRNAs including val-miR152 and miR9738 were predicted to target viscotoxins and lectins, respectively, which increased the knowledge regarding miRNA-based regulation of terpenoid biosynthesis, lectin and viscotoxin expressions in V. album.

Identification and validation of Asteraceae miRNAs by the expressed sequence tag analysis.

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Monavar Feshani, Aboozar; Mohammadi, Saeed; Frazier, Taylor P; Abbasi, Abbas; Abedini, Raha; Karimi Farsad, Laleh; Ehya, Farveh; Salekdeh, Ghasem Hosseini; Mardi, Mohsen

2012-02-10

MicroRNAs (miRNAs) are small non-coding RNA molecules that play a vital role in the regulation of gene expression. Despite their identification in hundreds of plant species, few miRNAs have been identified in the Asteraceae, a large family that comprises approximately one tenth of all flowering plants. In this study, we used the expressed sequence tag (EST) analysis to identify potential conserved miRNAs and their putative target genes in the Asteraceae. We applied quantitative Real-Time PCR (qRT-PCR) to confirm the expression of eight potential miRNAs in Carthamus tinctorius and Helianthus annuus. We also performed qRT-PCR analysis to investigate the differential expression pattern of five newly identified miRNAs during five different cotyledon growth stages in safflower. Using these methods, we successfully identified and characterized 151 potentially conserved miRNAs, belonging to 26 miRNA families, in 11 genus of Asteraceae. EST analysis predicted that the newly identified conserved Asteraceae miRNAs target 130 total protein-coding ESTs in sunflower and safflower, as well as 433 additional target genes in other plant species. We experimentally confirmed the existence of seven predicted miRNAs, (miR156, miR159, miR160, miR162, miR166, miR396, and miR398) in safflower and sunflower seedlings. We also observed that five out of eight miRNAs are differentially expressed during cotyledon development. Our results indicate that miRNAs may be involved in the regulation of gene expression during seed germination and the formation of the cotyledons in the Asteraceae. The findings of this study might ultimately help in the understanding of miRNA-mediated gene regulation in important crop species. Copyright © 2011 Elsevier B.V. All rights reserved.

Identification of distinct miRNA target regulation between breast cancer molecular subtypes using AGO2-PAR-CLIP and patient datasets

NARCIS (Netherlands)

Farazi, Thalia A.; ten Hoeve, Jelle J.; Brown, Miguel; Mihailovic, Aleksandra; Horlings, Hugo M.; van de Vijver, Marc J.; Tuschl, Thomas; Wessels, Lodewyk F. A.

2014-01-01

Various microRNAs (miRNAs) are up- or downregulated in tumors. However, the repression of cognate miRNA targets responsible for the phenotypic effects of this dysregulation in patients remains largely unexplored. To define miRNA targets and associated pathways, together with their relationship to

TRAIL, Wnt, Sonic Hedgehog, TGFβ, and miRNA Signalings Are Potential Targets for Oral Cancer Therapy.

Science.gov (United States)

Farooqi, Ammad Ahmad; Shu, Chih-Wen; Huang, Hurng-Wern; Wang, Hui-Ru; Chang, Yung-Ting; Fayyaz, Sundas; Yuan, Shyng-Shiou F; Tang, Jen-Yang; Chang, Hsueh-Wei

2017-07-14

Clinical studies and cancer cell models emphasize the importance of targeting therapies for oral cancer. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is highly expressed in cancer, and is a selective killing ligand for oral cancer. Signaling proteins in the wingless-type mouse mammary tumor virus (MMTV) integration site family (Wnt), Sonic hedgehog (SHH), and transforming growth factor β (TGFβ) pathways may regulate cell proliferation, migration, and apoptosis. Accordingly, the genes encoding these signaling proteins are potential targets for oral cancer therapy. In this review, we focus on recent advances in targeting therapies for oral cancer and discuss the gene targets within TRAIL, Wnt, SHH, and TGFβ signaling for oral cancer therapies. Oncogenic microRNAs (miRNAs) and tumor suppressor miRNAs targeting the genes encoding these signaling proteins are summarized, and the interactions between Wnt, SHH, TGFβ, and miRNAs are interpreted. With suitable combination treatments, synergistic effects are expected to improve targeting therapies for oral cancer.

TRAIL, Wnt, Sonic Hedgehog, TGFβ, and miRNA Signalings Are Potential Targets for Oral Cancer Therapy

Science.gov (United States)

Farooqi, Ammad Ahmad; Shu, Chih-Wen; Huang, Hurng-Wern; Wang, Hui-Ru; Chang, Yung-Ting; Fayyaz, Sundas; Yuan, Shyng-Shiou F.; Tang, Jen-Yang

2017-01-01

Clinical studies and cancer cell models emphasize the importance of targeting therapies for oral cancer. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is highly expressed in cancer, and is a selective killing ligand for oral cancer. Signaling proteins in the wingless-type mouse mammary tumor virus (MMTV) integration site family (Wnt), Sonic hedgehog (SHH), and transforming growth factor β (TGFβ) pathways may regulate cell proliferation, migration, and apoptosis. Accordingly, the genes encoding these signaling proteins are potential targets for oral cancer therapy. In this review, we focus on recent advances in targeting therapies for oral cancer and discuss the gene targets within TRAIL, Wnt, SHH, and TGFβ signaling for oral cancer therapies. Oncogenic microRNAs (miRNAs) and tumor suppressor miRNAs targeting the genes encoding these signaling proteins are summarized, and the interactions between Wnt, SHH, TGFβ, and miRNAs are interpreted. With suitable combination treatments, synergistic effects are expected to improve targeting therapies for oral cancer. PMID:28708091

Deciphering the role of a miRNA in rice domestication

Directory of Open Access Journals (Sweden)

Swetha Chenna

2017-10-01

Full Text Available MicroRNAs (miRNAs are a class of 21 nt non-coding small RNAs (sRNAs produced from endogenously expressed MIR genes. miRNAs are mostly involved in development and disease resistance. We are interested in identifying key miRNAs that are differentially expressed among wild and cultivated rice species. Analysis of sRNA datasets from two wild species (O. nivara and O. rufipogon and one cultivated species of rice (O. sativa var. indica Pusa Basmati-1, revealed a surprisingly higher abundance of small RNAs originating from Chromosome 2 in wild rice species. This locus codes for a novel 22 nt miRNA. This novel miRNA was found to be highly abundant in flag leaf of wild species, a tissue that usually provides 70% of energy required for grain filling. This miRNA targets a group of proteins (Os03g0273200, Os01g0827300, Os01g0850700, Os11g0708100 and Os01g0842500 which are involved in secondary metabolite production, although a functional significance of this interaction has not been understood. The expression of these targets also differs across the species. Typical of 22 nt miRNAs, the identified miRNA also triggers a secondary cascade silencing by producing small interfering RNAs (siRNAs from target mRNAs in O. nivara. These secondary siRNAs are observed only among wild rice species but not in cultivated rice. Currently we are using a range of genetic, biochemical and molecular techniques to understand role of this novel miRNA in domestication of rice.

Combining miRNA and mRNA Expression Profiles in Wilms Tumor Subtypes

Directory of Open Access Journals (Sweden)

Nicole Ludwig

2016-03-01

Full Text Available Wilms tumor (WT is the most common childhood renal cancer. Recent findings of mutations in microRNA (miRNA processing proteins suggest a pivotal role of miRNAs in WT genesis. We performed miRNA expression profiling of 36 WTs of different subtypes and four normal kidney tissues using microarrays. Additionally, we determined the gene expression profile of 28 of these tumors to identify potentially correlated target genes and affected pathways. We identified 85 miRNAs and 2107 messenger RNAs (mRNA differentially expressed in blastemal WT, and 266 miRNAs and 1267 mRNAs differentially expressed in regressive subtype. The hierarchical clustering of the samples, using either the miRNA or mRNA profile, showed the clear separation of WT from normal kidney samples, but the miRNA pattern yielded better separation of WT subtypes. A correlation analysis of the deregulated miRNA and mRNAs identified 13,026 miRNA/mRNA pairs with inversely correlated expression, of which 2844 are potential interactions of miRNA and their predicted mRNA targets. We found significant upregulation of miRNAs-183, -301a/b and -335 for the blastemal subtype, and miRNAs-181b, -223 and -630 for the regressive subtype. We found marked deregulation of miRNAs regulating epithelial to mesenchymal transition, especially in the blastemal subtype, and miRNAs influencing chemosensitivity, especially in regressive subtypes. Further research is needed to assess the influence of preoperative chemotherapy and tumor infiltrating lymphocytes on the miRNA and mRNA patterns in WT.

In silico identification of miRNAs and their target genes and analysis of gene co-expression network in saffron (Crocus sativus L.) stigma

Science.gov (United States)

Zinati, Zahra; Shamloo-Dashtpagerdi, Roohollah; Behpouri, Ali

2016-01-01

As an aromatic and colorful plant of substantive taste, saffron (Crocus sativus L.) owes such properties of matter to growing class of the secondary metabolites derived from the carotenoids, apocarotenoids. Regarding the critical role of microRNAs in secondary metabolic synthesis and the limited number of identified miRNAs in C. sativus, on the other hand, one may see the point how the characterization of miRNAs along with the corresponding target genes in C. sativus might expand our perspectives on the roles of miRNAs in carotenoid/apocarotenoid biosynthetic pathway. A computational analysis was used to identify miRNAs and their targets using EST (Expressed Sequence Tag) library from mature saffron stigmas. Then, a gene co- expression network was constructed to identify genes which are potentially involved in carotenoid/apocarotenoid biosynthetic pathways. EST analysis led to the identification of two putative miRNAs (miR414 and miR837-5p) along with the corresponding stem- looped precursors. To our knowledge, this is the first report on miR414 and miR837-5p in C. sativus. Co-expression network analysis indicated that miR414 and miR837-5p may play roles in C. sativus metabolic pathways and led to identification of candidate genes including six transcription factors and one protein kinase probably involved in carotenoid/apocarotenoid biosynthetic pathway. Presence of transcription factors, miRNAs and protein kinase in the network indicated multiple layers of regulation in saffron stigma. The candidate genes from this study may help unraveling regulatory networks underlying the carotenoid/apocarotenoid biosynthesis in saffron and designing metabolic engineering for enhanced secondary metabolites. PMID:28261627

Dehydration-responsive miRNAs in foxtail millet: genome-wide identification, characterization and expression profiling.

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Yadav, Amita; Khan, Yusuf; Prasad, Manoj

2016-03-01

A set of novel and known dehydration-responsive miRNAs have been identified in foxtail millet. These findings provide new insights into understanding the functional role of miRNAs and their respective targets in regulating plant response to dehydration stress. MicroRNAs perform significant regulatory roles in growth, development and stress response of plants. Though the miRNA-mediated gene regulatory networks under dehydration stress remain largely unexplored in plant including foxtail millet (Setaria italica), which is a natural abiotic stress tolerant crop. To find out the dehydration-responsive miRNAs at the global level, four small RNA libraries were constructed from control and dehydration stress treated seedlings of two foxtail millet cultivars showing contrasting tolerance behavior towards dehydration stress. Using Illumina sequencing technology, 55 known and 136 novel miRNAs were identified, representing 22 and 48 miRNA families, respectively. Eighteen known and 33 novel miRNAs were differentially expressed during dehydration stress. After the stress treatment, 32 dehydration-responsive miRNAs were up-regulated in tolerant cultivar and 22 miRNAs were down-regulated in sensitive cultivar, suggesting that miRNA-mediated molecular regulation might play important roles in providing contrasting characteristics to these cultivars. Predicted targets of identified miRNAs were found to encode various transcription factors and functional enzymes, indicating their involvement in broad spectrum regulatory functions and biological processes. Further, differential expression patterns of seven known miRNAs were validated by northern blot and expression of ten novel dehydration-responsive miRNAs were confirmed by SL-qRT PCR. Differential expression behavior of five miRNA-target genes was verified under dehydration stress treatment and two of them also validated by RLM RACE. Overall, the present study highlights the importance of dehydration stress-associated post

Integrating miRNA and mRNA Expression Profiling Uncovers miRNAs Underlying Fat Deposition in Sheep

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Guangxian Zhou

2017-01-01

Full Text Available MicroRNAs (miRNAs are endogenous, noncoding RNAs that regulate various biological processes including adipogenesis and fat metabolism. Here, we adopted a deep sequencing approach to determine the identity and abundance of miRNAs involved in fat deposition in adipose tissues from fat-tailed (Kazakhstan sheep, KS and thin-tailed (Tibetan sheep, TS sheep breeds. By comparing HiSeq data of these two breeds, 539 miRNAs were shared in both breeds, whereas 179 and 97 miRNAs were uniquely expressed in KS and TS, respectively. We also identified 35 miRNAs that are considered to be putative novel miRNAs. The integration of miRNA-mRNA analysis revealed that miRNA-associated targets were mainly involved in the gene ontology (GO biological processes concerning cellular process and metabolic process, and miRNAs play critical roles in fat deposition through their ability to regulate fundamental pathways. These pathways included the MAPK signaling pathway, FoxO and Wnt signaling pathway, and focal adhesion. Taken together, our results define miRNA expression signatures that may contribute to fat deposition and lipid metabolism in sheep.

Genome-wide miRNA screening reveals miR-310 family members negatively regulate the immune response in Drosophila melanogaster via co-targeting Drosomycin.

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Li, Yao; Li, Shengjie; Li, Ruimin; Xu, Jiao; Jin, Ping; Chen, Liming; Ma, Fei

2017-03-01

Although innate immunity mediated by Toll signaling has been extensively studied in Drosophila melanogaster, the role of miRNAs in regulating the Toll-mediated immune response remains largely unknown. In this study, following Gram-positive bacterial challenge, we identified 93 differentially expressed miRNAs via genome-wide miRNA screening. These miRNAs were regarded as immune response related (IRR). Eight miRNAs were confirmed to be involved in the Toll-mediated immune response upon Gram-positive bacterial infection through genetic screening of 41 UAS-miRNA lines covering 60 miRNAs of the 93 IRR miRNAs. Interestingly, four out of these eight miRNAs, miR-310, miR-311, miR-312 and miR-313, are clustered miRNAs and belong to the miR-310 family. These miR-310 family members were shown to target and regulate the expression of Drosomycin, an antimicrobial peptide produced by Toll signaling. Taken together, our study implies important regulatory roles of miRNAs in the Toll-mediated innate immune response of Drosophila upon Gram-positive bacterial infection. Copyright © 2016 Elsevier Ltd. All rights reserved.

The miRNAs and their regulatory networks responsible for pollen abortion in Ogura-CMS Chinese cabbage revealed by high-throughput sequencing of miRNAs, degradomes, and transcriptomes.

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Wei, Xiaochun; Zhang, Xiaohui; Yao, Qiuju; Yuan, Yuxiang; Li, Xixiang; Wei, Fang; Zhao, Yanyan; Zhang, Qiang; Wang, Zhiyong; Jiang, Wusheng; Zhang, Xiaowei

2015-01-01

Chinese cabbage (Brassica rapa ssp. pekinensis) is one of the most important vegetables in Asia and is cultivated across the world. Ogura-type cytoplasmic male sterility (Ogura-CMS) has been widely used in the hybrid breeding industry for Chinese cabbage and many other cruciferous vegetables. Although, the cause of Ogura-CMS has been localized to the orf138 locus in the mitochondrial genome, however, the mechanism by which nuclear genes respond to the mutation of the mitochondrial orf138 locus is unclear. In this study, a series of whole genome small RNA, degradome and transcriptome analyses were performed on both Ogura-CMS and its maintainer Chinese cabbage buds using deep sequencing technology. A total of 289 known miRNAs derived from 69 families (including 23 new families first reported in B. rapa) and 426 novel miRNAs were identified. Among these novel miRNAs, both 3-p and 5-p miRNAs were detected on the hairpin arms of 138 precursors. Ten known and 49 novel miRNAs were down-regulated, while one known and 27 novel miRNAs were up-regulated in Ogura-CMS buds compared to the fertile plants. Using degradome analysis, a total of 376 mRNAs were identified as targets of 30 known miRNA families and 100 novel miRNAs. A large fraction of the targets were annotated as reproductive development related. Our transcriptome profiling revealed that the expression of the targets was finely tuned by the miRNAs. Two novel miRNAs were identified that were specifically highly expressed in Ogura-CMS buds and sufficiently suppressed two pollen development essential genes: sucrose transporter SUC1 and H (+) -ATPase 6. These findings provide clues for the contribution of a potential miRNA regulatory network to bud development and pollen engenderation. This study contributes new insights to the communication between the mitochondria and chromosome and takes one step toward filling the gap in the regulatory network from the orf138 locus to pollen abortion in Ogura-CMS plants from a miRNA

The miRNAs and their regulatory networks responsible for pollen abortion in Ogura-CMS Chinese cabbage revealed by high-throughput sequencing of miRNAs, degradomes and transcriptomes

Directory of Open Access Journals (Sweden)

Xiaochun eWei

2015-10-01

Full Text Available Chinese cabbage (Brassica rapa ssp. pekinensis is one of the most important vegetables in Asia and is cultivated across the world. Ogura-type cytoplasmic male sterility (Ogura-CMS has been widely used in the hybrid breeding industry for Chinese cabbage and many other cruciferous vegetables. Although, the cause of Ogura-CMS has been localized to the orf138 locus in the mitochondrial genome, however, the mechanism by which nuclear genes respond to the mutation of the mitochondrial orf138 locus is unclear. In this study, a series of whole genome small RNA, degradome and transcriptome analyses were performed on both Ogura-CMS and its maintainer Chinese cabbage buds using deep sequencing technology. A total of 289 known miRNAs derived from 69 families (including 23 new families first reported in B. rapa and 426 novel miRNAs were identified. Among these novel miRNAs, both 3-p and 5-p miRNAs were detected on the hairpin arms of 138 precursors. Ten known and 49 novel miRNAs were down-regulated, while one known and 27 novel miRNAs were up-regulated in Ogura-CMS buds compared to the fertile plants. Using degradome analysis, a total of 376 mRNAs were identified as targets of 30 known miRNA families and 100 novel miRNAs. A large fraction of the targets were annotated as reproductive development related. Our transcriptome profiling revealed that the expression of the targets was finely tuned by the miRNAs. Two novel miRNAs were identified that were specifically highly expressed in Ogura-CMS buds and sufficiently suppressed two pollen development essential genes: sucrose transporter SUC1 and H+-ATPase 6. These findings provide clues for the contribution of a potential miRNA regulatory network to bud development and pollen engenderation. This study contributes new insights to the communication between the mitochondria and chromosome and takes one step toward filling the gap in the regulatory network from the orf138 locus to pollen abortion in Ogura-CMS plants

miRNA-target chimeras reveal miRNA 3'-end pairing as a major determinant of Argonaute target specificity

DEFF Research Database (Denmark)

Moore, Michael J; Scheel, Troels K H; Luna, Joseph M

2015-01-01

microRNAs (miRNAs) act as sequence-specific guides for Argonaute (AGO) proteins, which mediate posttranscriptional silencing of target messenger RNAs. Despite their importance in many biological processes, rules governing AGO-miRNA targeting are only partially understood. Here we report a modifie...

Altered expression of four miRNA (miR-1238-3p, miR-202-3p, miR-630 and miR-766-3p) and their potential targets in peripheral blood from vitiligo patients.

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Shang, Zhiwei; Li, Hongwen

2017-10-01

Vitiligo is an acquired skin disease with pigmentary disorder. Autoimmune destruction of melanocytes is thought to be major factor in the etiology of vitiligo. miRNA-based regulators of gene expression have been reported to play crucial roles in autoimmune disease. Therefore, we attempt to profile the miRNA expressions and predict their potential targets, assessing the biological functions of differentially expressed miRNA. Total RNA was extracted from peripheral blood of vitiligo (experimental group, n = 5) and non-vitiligo (control group, n = 5) age-matched patients. Samples were hybridized to a miRNA array. Box, scatter and principal component analysis plots were performed, followed by unsupervised hierarchical clustering analysis to classify the samples. Quantitative reverse transcription polymerase chain reaction (RT-PCR) was conducted for validation of microarray data. Three different databases, TargetScan, PITA and microRNA.org, were used to predict the potential target genes. Gene ontology (GO) annotation and pathway analysis were performed to assess the potential functions of predicted genes of identified miRNA. A total of 100 (29 upregulated and 71 downregulated) miRNA were filtered by volcano plot analysis. Four miRNA were validated by quantitative RT-PCR as significantly downregulated in the vitiligo group. The functions of predicted target genes associated with differentially expressed miRNA were assessed by GO analysis, showing that the GO term with most significantly enriched target genes was axon guidance, and that the axon guidance pathway was most significantly correlated with these miRNA. In conclusion, we identified four downregulated miRNA in vitiligo and assessed the potential functions of target genes related to these differentially expressed miRNA. © 2017 Japanese Dermatological Association.

A comprehensive survey of 3′ animal miRNA modification events and a possible role for 3′ adenylation in modulating miRNA targeting effectiveness

Science.gov (United States)

Burroughs, A. Maxwell; Ando, Yoshinari; de Hoon, Michiel J.L.; Tomaru, Yasuhiro; Nishibu, Takahiro; Ukekawa, Ryo; Funakoshi, Taku; Kurokawa, Tsutomu; Suzuki, Harukazu; Hayashizaki, Yoshihide; Daub, Carsten O.

2010-01-01

Animal microRNA sequences are subject to 3′ nucleotide addition. Through detailed analysis of deep-sequenced short RNA data sets, we show adenylation and uridylation of miRNA is globally present and conserved across Drosophila and vertebrates. To better understand 3′ adenylation function, we deep-sequenced RNA after knockdown of nucleotidyltransferase enzymes. The PAPD4 nucleotidyltransferase adenylates a wide range of miRNA loci, but adenylation does not appear to affect miRNA stability on a genome-wide scale. Adenine addition appears to reduce effectiveness of miRNA targeting of mRNA transcripts while deep-sequencing of RNA bound to immunoprecipitated Argonaute (AGO) subfamily proteins EIF2C1–EIF2C3 revealed substantial reduction of adenine addition in miRNA associated with EIF2C2 and EIF2C3. Our findings show 3′ addition events are widespread and conserved across animals, PAPD4 is a primary miRNA adenylating enzyme, and suggest a role for 3′ adenine addition in modulating miRNA effectiveness, possibly through interfering with incorporation into the RNA-induced silencing complex (RISC), a regulatory role that would complement the role of miRNA uridylation in blocking DICER1 uptake. PMID:20719920

Role of miRNAs in Epicardial Adipose Tissue in CAD Patients with T2DM

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Yang Liu

2016-01-01

Full Text Available Background. Epicardial adipose tissue (EAT is identified as an atypical fat depot surrounding the heart with a putative role in the involvement of metabolic disorders, including obesity, type-2 diabetes mellitus, and atherosclerosis. We profiled miRNAs in EAT of metabolic patients with coronary artery disease (CAD and type-2 diabetes mellitus (T2DM versus metabolically healthy patients by microarray. Compared to metabolically healthy patients, we identified forty-two miRNAs that are differentially expressed in patients with CAD and T2DM from Xinjiang, China. Eleven miRNAs were selected as potential novel miRNAs according to P value and fold change. Then the potential novel miRNAs targeted genes were predicted via TargetScan, PicTar, and miRTarbase, and the function of the target genes was predicted via Gene Ontology (GO analysis while the enriched KEGG pathway analyses of the miRNAs targeted genes were performed by bioinformatics software DAVID. Then protein-protein interaction networks of the targeted gene were conducted by online software STRING. Finally, using microarray, bioinformatics approaches revealed the possible molecular mechanisms pathogenesis of CAD and T2DM. A total of 11 differentially expressed miRNAs were identified and among them, hsa-miR-4687-3p drew specific attention. Bioinformatics analysis revealed that insulin signaling pathway is the central way involved in the progression of metabolic disorders. Conclusions. The current findings support the fact that miRNAs are involved in the pathogenesis of metabolic disorders in EAT of CAD patients with T2DM, and validation of the results of these miRNAs by independent and prospective study is certainly warranted.

Shrimp miRNAs regulate innate immune response against white spot syndrome virus infection.

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Kaewkascholkul, Napol; Somboonviwat, Kulwadee; Asakawa, Shuichi; Hirono, Ikuo; Tassanakajon, Anchalee; Somboonwiwat, Kunlaya

2016-07-01

MicroRNAs are short noncoding RNAs of RNA interference pathways that regulate gene expression through partial complementary base-pairing to target mRNAs. In this study, miRNAs that are expressed in white spot syndrome virus (WSSV)-infected Penaeus monodon, were identified using next generation sequencing. Forty-six miRNA homologs were identified from WSSV-infected shrimp hemocyte. Stem-loop real-time RT-PCR analysis showed that 11 out of 16 selected miRNAs were differentially expressed upon WSSV infection. Of those, pmo-miR-315 and pmo-miR-750 were highly responsive miRNAs. miRNA target prediction revealed that the miRNAs were targeted at 5'UTR, ORF, and 3'UTR of several immune-related genes such as genes encoding antimicrobial peptides, signaling transduction proteins, heat shock proteins, oxidative stress proteins, proteinases or proteinase inhibitors, proteins in blood clotting system, apoptosis-related proteins, proteins in prophenoloxidase system, pattern recognition proteins and other immune molecules. The highly conserved miRNA homolog, pmo-bantam, was characterized for its function in shrimp. The pmo-bantam was predicted to target the 3'UTR of Kunitz-type serine protease inhibitor (KuSPI). Binding of pmo-bantam to the target sequence of KuSPI gene was analyzed by luciferase reporter assay. Correlation of pmo-bantam and KuSPI expression was observed in lymphoid organ of WSSV-infected shrimp. These results implied that miRNAs might play roles as immune gene regulators in shrimp antiviral response. Copyright © 2016. Published by Elsevier Ltd.

In silico profiling of miRNAs and their target polymorphisms in ...

African Journals Online (AJOL)

C. George Priya Doss

2013-02-20

Feb 20, 2013 ... breast, and colorectal, and altered patterns of miRNA expres- sion may ... World Wide Web to predict miRNA-target mRNA sites; how- ever, it is ..... RB, SJST, and SKR were involved in design, acquisition of data, analysis and ...

Identification, characterization and expression analysis of pigeonpea miRNAs in response to Fusarium wilt.

Science.gov (United States)

Hussain, Khalid; Mungikar, Kanak; Kulkarni, Abhijeet; Kamble, Avinash

2018-05-05

Upon confrontation with unfavourable conditions, plants invoke a very complex set of biochemical and physiological reactions and alter gene expression patterns to combat the situations. MicroRNAs (miRNAs), a class of small non-coding RNA, contribute extensively in regulation of gene expression through translation inhibition or degradation of their target mRNAs during such conditions. Therefore, identification of miRNAs and their targets holds importance in understanding the regulatory networks triggered during stress. Structure and sequence similarity based in silico prediction of miRNAs in Cajanus cajan L. (Pigeonpea) draft genome sequence has been carried out earlier. These annotations also appear in related GenBank genome sequence entries. However, there are no reports available on context dependent miRNA expression and their targets in pigeonpea. Therefore, in the present study we addressed these questions computationally, using pigeonpea EST sequence information. We identified five novel pigeonpea miRNA precursors, their mature forms and targets. Interestingly, only one of these miRNAs (miR169i-3p) was identified earlier in draft genome sequence. We then validated expression of these miRNAs, experimentally. It was also observed that these miRNAs show differential expression patterns in response to Fusarium inoculation indicating their biotic stress responsive nature. Overall these results will help towards better understanding the regulatory network of defense during pigeonpea -pathogen interactions and role of miRNAs in the process. Copyright © 2018 Elsevier B.V. All rights reserved.

Small RNA Sequencing Reveals Differential miRNA Expression in the Early Development of Broccoli (Brassica oleracea var. italica) Pollen.

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Li, Hui; Wang, Yu; Wu, Mei; Li, Lihong; Jin, Chuan; Zhang, Qingli; Chen, Chengbin; Song, Wenqin; Wang, Chunguo

2017-01-01

Pollen development is an important and complex biological process in the sexual reproduction of flowering plants. Although the cytological characteristics of pollen development are well defined, the regulation of its early stages remains largely unknown. In the present study, miRNAs were explored in the early development of broccoli ( Brassica oleracea var. italica ) pollen. A total of 333 known miRNAs that originated from 235 miRNA families were detected. Fifty-five novel miRNA candidates were identified. Sixty of the 333 known miRNAs and 49 of the 55 predicted novel miRNAs exhibited significantly differential expression profiling in the three distinct developmental stages of broccoli pollen. Among these differentially expressed miRNAs, miRNAs that would be involved in the developmental phase transition from uninucleate microspores to binucleate pollen grains or from binucleate to trinucleate pollen grains were identified. miRNAs that showed significantly enriched expression in a specific early stage of broccoli pollen development were also observed. In addition, 552 targets for 127 known miRNAs and 69 targets for 40 predicted novel miRNAs were bioinformatically identified. Functional annotation and GO (Gene Ontology) analysis indicated that the putative miRNA targets showed significant enrichment in GO terms that were related to plant organ formation and morphogenesis. Some of enriched GO terms were detected for the targets directly involved in plant male reproduction development. These findings provided new insights into the functions of miRNA-mediated regulatory networks in broccoli pollen development.

Robust Selection Algorithm (RSA) for Multi-Omic Biomarker Discovery; Integration with Functional Network Analysis to Identify miRNA Regulated Pathways in Multiple Cancers.

Science.gov (United States)

Sehgal, Vasudha; Seviour, Elena G; Moss, Tyler J; Mills, Gordon B; Azencott, Robert; Ram, Prahlad T

2015-01-01

MicroRNAs (miRNAs) play a crucial role in the maintenance of cellular homeostasis by regulating the expression of their target genes. As such, the dysregulation of miRNA expression has been frequently linked to cancer. With rapidly accumulating molecular data linked to patient outcome, the need for identification of robust multi-omic molecular markers is critical in order to provide clinical impact. While previous bioinformatic tools have been developed to identify potential biomarkers in cancer, these methods do not allow for rapid classification of oncogenes versus tumor suppressors taking into account robust differential expression, cutoffs, p-values and non-normality of the data. Here, we propose a methodology, Robust Selection Algorithm (RSA) that addresses these important problems in big data omics analysis. The robustness of the survival analysis is ensured by identification of optimal cutoff values of omics expression, strengthened by p-value computed through intensive random resampling taking into account any non-normality in the data and integration into multi-omic functional networks. Here we have analyzed pan-cancer miRNA patient data to identify functional pathways involved in cancer progression that are associated with selected miRNA identified by RSA. Our approach demonstrates the way in which existing survival analysis techniques can be integrated with a functional network analysis framework to efficiently identify promising biomarkers and novel therapeutic candidates across diseases.

The miRNA Pull Out Assay as a Method to Validate the miR-28-5p Targets Identified in Other Tumor Contexts in Prostate Cancer

DEFF Research Database (Denmark)

Rizzo, Milena; Berti, Gabriele; Russo, Francesco

2017-01-01

targets in the pull out sample. We showed that E2F6, TEX-261, MAPK1, MPL, N4BP1, and RAP1B but not BAG1, OTUB1, MAD2L1, and p21 were significantly enriched, suggesting that not all the miR-28-5p targets are regulated by this miRNA in PCa. We then verified whether the miR-28-5p-interacting targets were...

In silico analysis and expression profiling of miRNAs targeting genes of steviol glycosides biosynthetic pathway and their relationship with steviol glycosides content in different tissues of Stevia rebaudiana.

Science.gov (United States)

Saifi, Monica; Nasrullah, Nazima; Ahmad, Malik Mobeen; Ali, Athar; Khan, Jawaid A; Abdin, M Z

2015-09-01

miRNAs are emerging as potential regulators of the gene expression. Their proven promising role in regulating biosynthetic pathways related gene networks may hold the key to understand the genetic regulation of these pathways which may assist in selection and manipulation to get high performing plant genotypes with better secondary metabolites yields and increased biomass. miRNAs associated with genes of steviol glycosides biosynthetic pathway, however, have not been identified so far. In this study miRNAs targeting genes of steviol glycosides biosynthetic pathway were identified for the first time whose precursors were potentially generated from ESTs and nucleotide sequences of Stevia rebaudiana. Thereafter, stem-loop coupled real time PCR based expressions of these miRNAs in different tissues of Stevia rebaudiana were investigated and their relationship pattern was analysed with the expression levels of their target mRNAs as well as steviol glycoside contents. All the miRNAs investigated showed differential expressions in all the three tissues studied, viz. leaves, flowers and stems. Out of the eleven miRNAs validated, the expression levels of nine miRNAs (miR319a, miR319b, miR319c, miR319d, miR319e, miR319f, miR319h, miRstv_7, miRstv_9) were found to be inversely related, while expression levels of the two, i.e. miR319g and miRstv_11 on the contrary, showed direct relation with the expression levels of their target mRNAs and steviol glycoside contents in the leaves, flowers and stems. This study provides a platform for better understanding of the steviol glycosides biosynthetic pathway and these miRNAs can further be employed to manipulate the biosynthesis of these metabolites to enhance their contents and yield in S. rebaudiana. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

High-Throughput Sequencing of Small RNA Transcriptomes in Maize Kernel Identifies miRNAs Involved in Embryo and Endosperm Development.

Science.gov (United States)

Xing, Lijuan; Zhu, Ming; Zhang, Min; Li, Wenzong; Jiang, Haiyang; Zou, Junjie; Wang, Lei; Xu, Miaoyun

2017-12-14

Maize kernel development is a complex biological process that involves the temporal and spatial expression of many genes and fine gene regulation at a transcriptional and post-transcriptional level, and microRNAs (miRNAs) play vital roles during this process. To gain insight into miRNA-mediated regulation of maize kernel development, a deep-sequencing technique was used to investigate the dynamic expression of miRNAs in the embryo and endosperm at three developmental stages in B73. By miRNA transcriptomic analysis, we characterized 132 known miRNAs and six novel miRNAs in developing maize kernel, among which, 15 and 14 miRNAs were commonly differentially expressed between the embryo and endosperm at 9 days after pollination (DAP), 15 DAP and 20 DAP respectively. Conserved miRNA families such as miR159, miR160, miR166, miR390, miR319, miR528 and miR529 were highly expressed in developing embryos; miR164, miR171, miR393 and miR2118 were highly expressed in developing endosperm. Genes targeted by those highly expressed miRNAs were found to be largely related to a regulation category, including the transcription, macromolecule biosynthetic and metabolic process in the embryo as well as the vitamin biosynthetic and metabolic process in the endosperm. Quantitative reverse transcription-PCR (qRT-PCR) analysis showed that these miRNAs displayed a negative correlation with the levels of their corresponding target genes. Importantly, our findings revealed that members of the miR169 family were highly and dynamically expressed in the developing kernel, which will help to exploit new players functioning in maize kernel development.

Methylation of miRNA genes and oncogenesis.

Science.gov (United States)

Loginov, V I; Rykov, S V; Fridman, M V; Braga, E A

2015-02-01

Interaction between microRNA (miRNA) and messenger RNA of target genes at the posttranscriptional level provides fine-tuned dynamic regulation of cell signaling pathways. Each miRNA can be involved in regulating hundreds of protein-coding genes, and, conversely, a number of different miRNAs usually target a structural gene. Epigenetic gene inactivation associated with methylation of promoter CpG-islands is common to both protein-coding genes and miRNA genes. Here, data on functions of miRNAs in development of tumor-cell phenotype are reviewed. Genomic organization of promoter CpG-islands of the miRNA genes located in inter- and intragenic areas is discussed. The literature and our own results on frequency of CpG-island methylation in miRNA genes from tumors are summarized, and data regarding a link between such modification and changed activity of miRNA genes and, consequently, protein-coding target genes are presented. Moreover, the impact of miRNA gene methylation on key oncogenetic processes as well as affected signaling pathways is discussed.

Bioinformatics of cardiovascular miRNA biology.

Science.gov (United States)

Kunz, Meik; Xiao, Ke; Liang, Chunguang; Viereck, Janika; Pachel, Christina; Frantz, Stefan; Thum, Thomas; Dandekar, Thomas

2015-12-01

MicroRNAs (miRNAs) are small ~22 nucleotide non-coding RNAs and are highly conserved among species. Moreover, miRNAs regulate gene expression of a large number of genes associated with important biological functions and signaling pathways. Recently, several miRNAs have been found to be associated with cardiovascular diseases. Thus, investigating the complex regulatory effect of miRNAs may lead to a better understanding of their functional role in the heart. To achieve this, bioinformatics approaches have to be coupled with validation and screening experiments to understand the complex interactions of miRNAs with the genome. This will boost the subsequent development of diagnostic markers and our understanding of the physiological and therapeutic role of miRNAs in cardiac remodeling. In this review, we focus on and explain different bioinformatics strategies and algorithms for the identification and analysis of miRNAs and their regulatory elements to better understand cardiac miRNA biology. Starting with the biogenesis of miRNAs, we present approaches such as LocARNA and miRBase for combining sequence and structure analysis including phylogenetic comparisons as well as detailed analysis of RNA folding patterns, functional target prediction, signaling pathway as well as functional analysis. We also show how far bioinformatics helps to tackle the unprecedented level of complexity and systemic effects by miRNA, underlining the strong therapeutic potential of miRNA and miRNA target structures in cardiovascular disease. In addition, we discuss drawbacks and limitations of bioinformatics algorithms and the necessity of experimental approaches for miRNA target identification. This article is part of a Special Issue entitled 'Non-coding RNAs'. Copyright © 2014 Elsevier Ltd. All rights reserved.

Excess fertilizer responsive miRNAs revealed in Linum usitatissimum L.

Science.gov (United States)

Melnikova, Nataliya V; Dmitriev, Alexey A; Belenikin, Maxim S; Speranskaya, Anna S; Krinitsina, Anastasia A; Rachinskaia, Olga A; Lakunina, Valentina A; Krasnov, George S; Snezhkina, Anastasiya V; Sadritdinova, Asiya F; Uroshlev, Leonid A; Koroban, Nadezda V; Samatadze, Tatiana E; Amosova, Alexandra V; Zelenin, Alexander V; Muravenko, Olga V; Bolsheva, Nadezhda L; Kudryavtseva, Anna V

2015-02-01

Effective fertilizer application is necessary to increase crop yields and reduce risk of plant overdosing. It is known that expression level of microRNAs (miRNAs) alters in plants under different nutrient concentrations in soil. The aim of our study was to identify and characterize miRNAs with expression alterations under excessive fertilizer in agriculturally important crop - flax (Linum usitatissimum L.). We have sequenced small RNAs in flax grown under normal and excessive fertilizer using Illumina GAIIx. Over 14 million raw reads was obtained for two small RNA libraries. 84 conserved miRNAs from 20 families were identified. Differential expression was revealed for several flax miRNAs under excessive fertilizer according to high-throughput sequencing data. For 6 miRNA families (miR395, miR169, miR408, miR399, miR398 and miR168) expression level alterations were evaluated on the extended sampling using qPCR. Statistically significant up-regulation was revealed for miR395 under excessive fertilizer. It is known that target genes of miR395 are involved in sulfate uptake and assimilation. However, according to our data alterations of the expression level of miR395 could be associated not only with excess sulfur application, but also with redundancy of other macro- and micronutrients. Furthermore expression level was evaluated for miRNAs and their predicted targets. The negative correlation between miR399 expression and expression of its predicted target ubiquitin-conjugating enzyme E2 gene was shown in flax for the first time. So we suggested miR399 involvement in phosphate regulation in L. usitatissimum. Revealed in our study expression alterations contribute to miRNA role in flax response to excessive fertilizer. Copyright © 2014 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.

A tale of two sequences: microRNA-target chimeric reads.

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Broughton, James P; Pasquinelli, Amy E

2016-04-04

In animals, a functional interaction between a microRNA (miRNA) and its target RNA requires only partial base pairing. The limited number of base pair interactions required for miRNA targeting provides miRNAs with broad regulatory potential and also makes target prediction challenging. Computational approaches to target prediction have focused on identifying miRNA target sites based on known sequence features that are important for canonical targeting and may miss non-canonical targets. Current state-of-the-art experimental approaches, such as CLIP-seq (cross-linking immunoprecipitation with sequencing), PAR-CLIP (photoactivatable-ribonucleoside-enhanced CLIP), and iCLIP (individual-nucleotide resolution CLIP), require inference of which miRNA is bound at each site. Recently, the development of methods to ligate miRNAs to their target RNAs during the preparation of sequencing libraries has provided a new tool for the identification of miRNA target sites. The chimeric, or hybrid, miRNA-target reads that are produced by these methods unambiguously identify the miRNA bound at a specific target site. The information provided by these chimeric reads has revealed extensive non-canonical interactions between miRNAs and their target mRNAs, and identified many novel interactions between miRNAs and noncoding RNAs.

Post-transcriptional generation of miRNA variants by multiple nucleotidyl transferases contributes to miRNA transcriptome complexity.

Science.gov (United States)

Wyman, Stacia K; Knouf, Emily C; Parkin, Rachael K; Fritz, Brian R; Lin, Daniel W; Dennis, Lucas M; Krouse, Michael A; Webster, Philippa J; Tewari, Muneesh

2011-09-01

Modification of microRNA sequences by the 3' addition of nucleotides to generate so-called "isomiRs" adds to the complexity of miRNA function, with recent reports showing that 3' modifications can influence miRNA stability and efficiency of target repression. Here, we show that the 3' modification of miRNAs is a physiological and common post-transcriptional event that shows selectivity for specific miRNAs and is observed across species ranging from C. elegans to human. The modifications result predominantly from adenylation and uridylation and are seen across tissue types, disease states, and developmental stages. To quantitatively profile 3' nucleotide additions, we developed and validated a novel assay based on NanoString Technologies' nCounter platform. For certain miRNAs, the frequency of modification was altered by processes such as cell differentiation, indicating that 3' modification is a biologically regulated process. To investigate the mechanism of 3' nucleotide additions, we used RNA interference to screen a panel of eight candidate miRNA nucleotidyl transferases for 3' miRNA modification activity in human cells. Multiple enzymes, including MTPAP, PAPD4, PAPD5, ZCCHC6, ZCCHC11, and TUT1, were found to govern 3' nucleotide addition to miRNAs in a miRNA-specific manner. Three of these enzymes-MTPAP, ZCCHC6, and TUT1-have not previously been known to modify miRNAs. Collectively, our results indicate that 3' modification observed in next-generation small RNA sequencing data is a biologically relevant process, and identify enzymatic mechanisms that may lead to new approaches for modulating miRNA activity in vivo.

Identification and characterization of miRNAs and targets in flax (Linum usitatissimum) under saline, alkaline, and saline-alkaline stresses.

Science.gov (United States)

Yu, Ying; Wu, Guangwen; Yuan, Hongmei; Cheng, Lili; Zhao, Dongsheng; Huang, Wengong; Zhang, Shuquan; Zhang, Liguo; Chen, Hongyu; Zhang, Jian; Guan, Fengzhi

2016-05-27

MicroRNAs (miRNAs) play a critical role in responses to biotic and abiotic stress and have been characterized in a large number of plant species. Although flax (Linum usitatissimum L.) is one of the most important fiber and oil crops worldwide, no reports have been published describing flax miRNAs (Lus-miRNAs) induced in response to saline, alkaline, and saline-alkaline stresses. In this work, combined small RNA and degradome deep sequencing was used to analyze flax libraries constructed after alkaline-salt stress (AS2), neutral salt stress (NSS), alkaline stress (AS), and the non-stressed control (CK). From the CK, AS, AS2, and NSS libraries, a total of 118, 119, 122, and 120 known Lus-miRNAs and 233, 213, 211, and 212 novel Lus-miRNAs were isolated, respectively. After assessment of differential expression profiles, 17 known Lus-miRNAs and 36 novel Lus-miRNAs were selected and used to predict putative target genes. Gene ontology term enrichment analysis revealed target genes that were involved in responses to stimuli, including signaling and catalytic activity. Eight Lus-miRNAs were selected for analysis using qRT-PCR to confirm the accuracy and reliability of the miRNA-seq results. The qRT-PCR results showed that changes in stress-induced expression profiles of these miRNAs mirrored expression trends observed using miRNA-seq. Degradome sequencing and transcriptome profiling showed that expression of 29 miRNA-target pairs displayed inverse expression patterns under saline, alkaline, and saline-alkaline stresses. From the target prediction analysis, the miR398a-targeted gene codes for a copper/zinc superoxide dismutase, and the miR530 has been shown to explicitly target WRKY family transcription factors, which suggesting that these two micRNAs and their targets may significant involve in the saline, alkaline, and saline-alkaline stress response in flax. Identification and characterization of flax miRNAs, their target genes, functional annotations, and gene

High-throughput sequencing, characterization and detection of new and conserved cucumber miRNAs.

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Germán Martínez

Full Text Available Micro RNAS (miRNAs are a class of endogenous small non coding RNAs involved in the post-transcriptional regulation of gene expression. In plants, a great number of conserved and specific miRNAs, mainly arising from model species, have been identified to date. However less is known about the diversity of these regulatory RNAs in vegetal species with agricultural and/or horticultural importance. Here we report a combined approach of bioinformatics prediction, high-throughput sequencing data and molecular methods to analyze miRNAs populations in cucumber (Cucumis sativus plants. A set of 19 conserved and 6 known but non-conserved miRNA families were found in our cucumber small RNA dataset. We also identified 7 (3 with their miRNA* strand not previously described miRNAs, candidates to be cucumber-specific. To validate their description these new C. sativus miRNAs were detected by northern blot hybridization. Additionally, potential targets for most conserved and new miRNAs were identified in cucumber genome.In summary, in this study we have identified, by first time, conserved, known non-conserved and new miRNAs arising from an agronomically important species such as C. sativus. The detection of this complex population of regulatory small RNAs suggests that similarly to that observe in other plant species, cucumber miRNAs may possibly play an important role in diverse biological and metabolic processes.

Generation of miRNA sponge constructs

NARCIS (Netherlands)

Kluiver, Joost; Slezak-Prochazka, Izabella; Smigielska-Czepiel, Katarzyna; Halsema, Nancy; Kroesen, Bart-Jan; van den Berg, Anke

2012-01-01

MicroRNA (miRNA) sponges are RNA molecules with repeated miRNA antisense sequences that can sequester miRNAs from their endogenous targets and thus serve as a decoy. Stably expressed miRNA sponges are especially valuable for long-term loss-of-function studies and can be used in vitro and in vivo. We

miRNAs in Normal and Malignant Hematopoiesis

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Ryutaro Kotaki

2017-07-01

Full Text Available Lineage specification is primarily regulated at the transcriptional level and lineage-specific transcription factors determine cell fates. MicroRNAs (miRNAs are 18–24 nucleotide-long non-coding RNAs that post-transcriptionally decrease the translation of target mRNAs and are essential for many cellular functions. miRNAs also regulate lineage specification during hematopoiesis. This review highlights the roles of miRNAs in B-cell development and malignancies, and discusses how miRNA expression profiles correlate with disease prognoses and phenotypes. We also discuss the potential for miRNAs as therapeutic targets and diagnostic tools for B-cell malignancies.

Identification and characterization of miRNAs in ripening fruit of Lycium barbarum L. using high-throughput sequencing

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Shaohua eZeng

2015-09-01

Full Text Available MicroRNAs (miRNAs are master regulators of gene activity documented to play central roles in fruit ripening in model plant species, yet little is known of their roles in Lycium barbarum L. fruits. In this study, miRNA levels in L. barbarum fruit samples at four developmental stages, were assayed using Illumina HiSeqTM2000. This revealed the presence of 50 novel miRNAs and 38 known miRNAs in L. barbarum fruits. Of the novel miRNAs, 36 were specific to L. barbarum fruits compared with L. chinense. A number of stage-specific miRNAs were identified and GO terms were assigned to 194 unigenes targeted by miRNAs. The majority of GO terms of unigenes targeted by differentially expressed miRNAs are ‘intracellular organelle’, ‘binding’, ‘metabolic process’, ‘pigmentation’, and ‘biological regulation’. Enriched KEGG analysis indicated that nucleotide excision repair and ubiquitin mediated proteolysis were over-represented during the initial stage of ripening, with ABC transporters and sulfur metabolism pathways active during the middle stages and ABC transporters and spliceosome enriched in the final stages of ripening. Several miRNAs and their targets serving as potential regulators in L. barbarum fruit ripening were identified using quantitative reverse transcription polymerase chain reaction. The miRNA-target interactions were predicted for L. barbarum ripening regulators including miR156/157 with LbCNR and LbWRKY8, and miR171 with LbGRAS. Additionally, regulatory interactions potentially controlling fruit quality and nutritional value via sugar and secondary metabolite accumulation were identified. These include miR156 targeting of fructokinase and 1-deoxy-D-xylulose-5-phosphate synthase and miR164 targeting of beta-fructofuranosidase. In sum, valuable information revealed by small RNA sequencing in this study will provide a solid foundation for uncovering the miRNA-mediated mechanism of fruit ripening and quality in this

Where we stand, where we are moving: Surveying computational techniques for identifying miRNA genes and uncovering their regulatory role

KAUST Repository

Kleftogiannis, Dimitrios A.; Korfiati, Aigli; Theofilatos, Konstantinos A.; Likothanassis, Spiridon D.; Tsakalidis, Athanasios K.; Mavroudi, Seferina P.

2013-01-01

Traditional biology was forced to restate some of its principles when the microRNA (miRNA) genes and their regulatory role were firstly discovered. Typically, miRNAs are small non-coding RNA molecules which have the ability to bind to the 3'untraslated region (UTR) of their mRNA target genes for cleavage or translational repression. Existing experimental techniques for their identification and the prediction of the target genes share some important limitations such as low coverage, time consuming experiments and high cost reagents. Hence, many computational methods have been proposed for these tasks to overcome these limitations. Recently, many researchers emphasized on the development of computational approaches to predict the participation of miRNA genes in regulatory networks and to analyze their transcription mechanisms. All these approaches have certain advantages and disadvantages which are going to be described in the present survey. Our work is differentiated from existing review papers by updating the methodologies list and emphasizing on the computational issues that arise from the miRNA data analysis. Furthermore, in the present survey, the various miRNA data analysis steps are treated as an integrated procedure whose aims and scope is to uncover the regulatory role and mechanisms of the miRNA genes. This integrated view of the miRNA data analysis steps may be extremely useful for all researchers even if they work on just a single step. © 2013 Elsevier Inc.

Where we stand, where we are moving: Surveying computational techniques for identifying miRNA genes and uncovering their regulatory role

KAUST Repository

Kleftogiannis, Dimitrios A.

2013-06-01

Traditional biology was forced to restate some of its principles when the microRNA (miRNA) genes and their regulatory role were firstly discovered. Typically, miRNAs are small non-coding RNA molecules which have the ability to bind to the 3\\'untraslated region (UTR) of their mRNA target genes for cleavage or translational repression. Existing experimental techniques for their identification and the prediction of the target genes share some important limitations such as low coverage, time consuming experiments and high cost reagents. Hence, many computational methods have been proposed for these tasks to overcome these limitations. Recently, many researchers emphasized on the development of computational approaches to predict the participation of miRNA genes in regulatory networks and to analyze their transcription mechanisms. All these approaches have certain advantages and disadvantages which are going to be described in the present survey. Our work is differentiated from existing review papers by updating the methodologies list and emphasizing on the computational issues that arise from the miRNA data analysis. Furthermore, in the present survey, the various miRNA data analysis steps are treated as an integrated procedure whose aims and scope is to uncover the regulatory role and mechanisms of the miRNA genes. This integrated view of the miRNA data analysis steps may be extremely useful for all researchers even if they work on just a single step. © 2013 Elsevier Inc.

Identification of miRNAs associated with recurrence of stage II colorectal cancer

DEFF Research Database (Denmark)

Christensen, Lise Lotte; Tobiasen, Heidi; Schepeler, Troels

2011-01-01

Colorectal cancer (CRC) is one of the leading causes of cancer deaths. Twenty-five percent of the patients radically treated for a stage II CRC (no lymph node or distant metastasis) later develop recurrence and dies from the disease. MicroRNAs (miRNAs) are aberrantly expressed or mutated in human...... target prediction and transcript profiling. Initially, miRNA over-expression in HCT116 cells was followed by transcriptional profiling of transfected cells using GeneChip Human Exon 1.0 ST Arrays. Three in silico predicted miRNA targets showing differential mRNA expression upon miRNA up-regulation were...... cancers, and function either as tumour suppressors or oncogenes. Additionally, they also appear to have both diagnostic and prognostic significance. The aim of the present study was to identify miRNAs associated with recurrence of stage II CRC, followed up by an investigation of how these potential...

Deep Sequencing Analysis of miRNA Expression in Breast Muscle of Fast-Growing and Slow-Growing Broilers

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Hongjia Ouyang

2015-07-01

Full Text Available Growth performance is an important economic trait in chicken. MicroRNAs (miRNAs have been shown to play important roles in various biological processes, but their functions in chicken growth are not yet clear. To investigate the function of miRNAs in chicken growth, breast muscle tissues of the two-tail samples (highest and lowest body weight from Recessive White Rock (WRR and Xinghua Chickens (XH were performed on high throughput small RNA deep sequencing. In this study, a total of 921 miRNAs were identified, including 733 known mature miRNAs and 188 novel miRNAs. There were 200, 279, 257 and 297 differentially expressed miRNAs in the comparisons of WRRh vs. WRRl, WRRh vs. XHh, WRRl vs. XHl, and XHh vs. XHl group, respectively. A total of 22 highly differentially expressed miRNAs (fold change > 2 or < 0.5; p-value < 0.05; q-value < 0.01, which also have abundant expression (read counts > 1000 were found in our comparisons. As far as two analyses (WRRh vs. WRRl, and XHh vs. XHl are concerned, we found 80 common differentially expressed miRNAs, while 110 miRNAs were found in WRRh vs. XHh and WRRl vs. XHl. Furthermore, 26 common miRNAs were identified among all four comparisons. Four differentially expressed miRNAs (miR-223, miR-16, miR-205a and miR-222b-5p were validated by quantitative real-time RT-PCR (qRT-PCR. Regulatory networks of interactions among miRNAs and their targets were constructed using integrative miRNA target-prediction and network-analysis. Growth hormone receptor (GHR was confirmed as a target of miR-146b-3p by dual-luciferase assay and qPCR, indicating that miR-34c, miR-223, miR-146b-3p, miR-21 and miR-205a are key growth-related target genes in the network. These miRNAs are proposed as candidate miRNAs for future studies concerning miRNA-target function on regulation of chicken growth.

Hypothesis: Do miRNAs Targeting the Leucine-Rich Repeat Kinase 2 Gene (LRRK2) Influence Parkinson's Disease Susceptibility?

Science.gov (United States)

Yılmaz, Şenay Görücü; Geyik, Sırma; Neyal, Ayşe Münife; Soko, Nyarai D; Bozkurt, Hakan; Dandara, Collet

2016-04-01

Parkinson's disease (PD) is a frequently occurring neurodegenerative motor disorder adversely impacting global health. There is a paucity of biomarkers and diagnostics that can forecast susceptibility to PD. A new research frontier for PD pathophysiology is the study of variations in microRNA (miRNA) expression whereby miRNAs serve as "upstream regulators" of gene expression in relation to functioning of the dopamine neuronal pathways. Leucine-Rich Repeat Kinase 2 (LRRK2) is a frequently studied gene in PD. Little is known about the ways in which expression of miRNAs targeting LRKK2 impact PD susceptibility. In a sample of 204 unrelated subjects (102 persons with PD and 102 healthy controls), we report here candidate miRNA expression in whole blood samples as measured by real-time PCR (hsa-miR-4671-3p, hsa-miR-335-3p, hsa-miR-561-3p, hsa-miR-579-3p, and hsa-miR-3143) that target LRRK2. Using step-wise logistic regression, and controlling for covariates such as age, gender, PD disease severity, concomitant medications, and co-morbidity, we found that the combination of has-miR-335-3p, has-miR-561-3p, and has-miR-579-3p account for 50% of the variation in regards to PD susceptibility (p<0.0001). Notably, the hsa-miR-561-3p expression was the most robust predictor of PD in both univariate and multivariate analyses (p<0.001). Moreover, the biological direction (polarity) of the association was plausible in that the candidate miRNAs displayed a diminished expression in patients. This is consistent with the hypothesis that decreased levels of miRNAs targeting LRRK2 might result in a gain of function for LRRK2, and by extension, loss of neuronal viability. To the best of our knowledge, this is the first clinical association study of the above candidate miRNAs' expression in PD using peripheral samples. These observations may guide future clinical diagnostics research on PD.

Genome-wide identification and characterization of miRNAs in the hypocotyl and cotyledon of cauliflower (Brassica oleracea L. var. botrytis) seedlings.

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Geng, Meijuan; Li, Hui; Jin, Chuan; Liu, Qian; Chen, Chengbin; Song, Wenqin; Wang, Chunguo

2014-02-01

MicroRNAs (miRNAs) are a class of small endogenous, non-coding RNAs that have key regulatory functions in plant growth, development, and other biological processes. Hypocotyl and cotyledon are the two major tissues of cauliflower (Brassica oleracea L. var. botrytis) seedlings. Tissue culture experiments have indicated that the regenerative abilities of these two tissues are significantly different. However, the characterization of miRNAs and their roles in regulating organ development in cauliflower remain unexplored. In the present study, two small RNA libraries were sequenced by Solexa sequencing technology. 99 known miRNAs belonging to 28 miRNA families were identified, in which 6 miRNA families were detected only in Brassicaceae. A total of 162 new miRNA sequences with single nucleotide substitutions corresponding to the known miRNAs, and 32 potentially novel miRNAs were also first discovered. Comparative analysis indicated that 42 of 99 known miRNAs and 17 of 32 novel miRNAs exhibited significantly differential expression between hypocotyl and cotyledon, and the differential expression of several miRNAs was further validated by stem-loop RT-PCR. In addition, 235 targets for 89 known miRNAs and 198 targets for 24 novel miRNAs were predicted, and their functions were further discussed. The expression patterns of several representative targets were also confirmed by qRT-PCR analysis. The results identified that the transcriptional expression patterns of miRNAs were negatively correlated with their targets. These findings gave new insights into the characteristics of miRNAs in cauliflower, and provided important clues to elucidate the roles of miRNAs in the tissue differentiation and development of cauliflower.

De novo sequencing of circulating miRNAs identifies novel markers predicting clinical outcome of locally advanced breast cancer

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Wu Xiwei

2012-03-01

Full Text Available Abstract Background MicroRNAs (miRNAs have been recently detected in the circulation of cancer patients, where they are associated with clinical parameters. Discovery profiling of circulating small RNAs has not been reported in breast cancer (BC, and was carried out in this study to identify blood-based small RNA markers of BC clinical outcome. Methods The pre-treatment sera of 42 stage II-III locally advanced and inflammatory BC patients who received neoadjuvant chemotherapy (NCT followed by surgical tumor resection were analyzed for marker identification by deep sequencing all circulating small RNAs. An independent validation cohort of 26 stage II-III BC patients was used to assess the power of identified miRNA markers. Results More than 800 miRNA species were detected in the circulation, and observed patterns showed association with histopathological profiles of BC. Groups of circulating miRNAs differentially associated with ER/PR/HER2 status and inflammatory BC were identified. The relative levels of selected miRNAs measured by PCR showed consistency with their abundance determined by deep sequencing. Two circulating miRNAs, miR-375 and miR-122, exhibited strong correlations with clinical outcomes, including NCT response and relapse with metastatic disease. In the validation cohort, higher levels of circulating miR-122 specifically predicted metastatic recurrence in stage II-III BC patients. Conclusions Our study indicates that certain miRNAs can serve as potential blood-based biomarkers for NCT response, and that miR-122 prevalence in the circulation predicts BC metastasis in early-stage patients. These results may allow optimized chemotherapy treatments and preventive anti-metastasis interventions in future clinical applications.

Expression profiles of miRNAs from bovine mammary glands in response to Streptococcus agalactiae-induced mastitis.

Science.gov (United States)

Pu, Junhua; Li, Rui; Zhang, Chenglong; Chen, Dan; Liao, Xiangxiang; Zhu, Yihui; Geng, Xiaohan; Ji, Dejun; Mao, Yongjiang; Gong, Yunchen; Yang, Zhangping

2017-08-01

This study aimed to describe the expression profiles of microRNAs (miRNAs) from mammary gland tissues collected from dairy cows with Streptococcus agalactiae-induced mastitis and to identify differentially expressed miRNAs related to mastitis. The mammary glands of Chinese Holstein cows were challenged with Streptococcus agalactiae to induce mastitis. Small RNAs were isolated from the mammary tissues of the test and control groups and then sequenced using the Solexa sequencing technology to construct two small RNA libraries. Potential target genes of these differentially expressed miRNAs were predicted using the RNAhybrid software, and KEGG pathways associated with these genes were analysed. A total of 18 555 913 and 20 847 000 effective reads were obtained from the test and control groups, respectively. In total, 373 known and 399 novel miRNAs were detected in the test group, and 358 known and 232 novel miRNAs were uncovered in the control group. A total of 35 differentially expressed miRNAs were identified in the test group compared to the control group, including 10 up-regulated miRNAs and 25 down-regulated miRNAs. Of these miRNAs, miR-223 exhibited the highest degree of up-regulation with an approximately 3-fold increase in expression, whereas miR-26a exhibited the most decreased expression level (more than 2-fold). The RNAhybrid software predicted 18 801 genes as potential targets of these 35 miRNAs. Furthermore, several immune response and signal transduction pathways, including the RIG-I-like receptor signalling pathway, cytosolic DNA sensing pathway and Notch signal pathway, were enriched in these predicted targets. In summary, this study provided experimental evidence for the mechanism underlying the regulation of bovine mastitis by miRNAs and showed that miRNAs might be involved in signal pathways during S. agalactiae-induced mastitis.

Integrative Analysis of miRNA and mRNA Profiles in Response to Ethylene in Rose Petals during Flower Opening

Science.gov (United States)

Pei, Haixia; Ma, Nan; Chen, Jiwei; Zheng, Yi; Tian, Ji; Li, Jing; Zhang, Shuai; Fei, Zhangjun; Gao, Junping

2013-01-01

MicroRNAs play an important role in plant development and plant responses to various biotic and abiotic stimuli. As one of the most important ornamental crops, rose (Rosa hybrida) possesses several specific morphological and physiological features, including recurrent flowering, highly divergent flower shapes, colors and volatiles. Ethylene plays an important role in regulating petal cell expansion during rose flower opening. Here, we report the population and expression profiles of miRNAs in rose petals during flower opening and in response to ethylene based on high throughput sequencing. We identified a total of 33 conserved miRNAs, as well as 47 putative novel miRNAs were identified from rose petals. The conserved and novel targets to those miRNAs were predicted using the rose floral transcriptome database. Expression profiling revealed that expression of 28 known (84.8% of known miRNAs) and 39 novel (83.0% of novel miRNAs) miRNAs was substantially changed in rose petals during the earlier opening period. We also found that 28 known and 22 novel miRNAs showed expression changes in response to ethylene treatment. Furthermore, we performed integrative analysis of expression profiles of miRNAs and their targets. We found that ethylene-caused expression changes of five miRNAs (miR156, miR164, miR166, miR5139 and rhy-miRC1) were inversely correlated to those of their seven target genes. These results indicate that these miRNA/target modules might be regulated by ethylene and were involved in ethylene-regulated petal growth. PMID:23696879

EBV-encoded miRNAs target ATM-mediated response in nasopharyngeal carcinoma.

Science.gov (United States)

Lung, Raymond W-M; Hau, Pok-Man; Yu, Ken H-O; Yip, Kevin Y; Tong, Joanna H-M; Chak, Wing-Po; Chan, Anthony W-H; Lam, Ka-Hei; Lo, Angela Kwok-Fung; Tin, Edith K-Y; Chau, Shuk-Ling; Pang, Jesse C-S; Kwan, Johnny S-H; Busson, Pierre; Young, Lawrence S; Yap, Lee-Fah; Tsao, Sai-Wah; To, Ka-Fai; Lo, Kwok-Wai

2018-04-01

Nasopharyngeal carcinoma (NPC) is a highly invasive epithelial malignancy that is prevalent in southern China and Southeast Asia. It is consistently associated with latent Epstein-Barr virus (EBV) infection. In NPC, miR-BARTs, the EBV-encoded miRNAs derived from BamH1-A rightward transcripts, are abundantly expressed and contribute to cancer development by targeting various cellular and viral genes. In this study, we establish a comprehensive transcriptional profile of EBV-encoded miRNAs in a panel of NPC patient-derived xenografts and an EBV-positive NPC cell line by small RNA sequencing. Among the 40 miR-BARTs, predominant expression of 22 miRNAs was consistently detected in these tumors. Among the abundantly expressed EBV-miRNAs, BART5-5p, BART7-3p, BART9-3p, and BART14-3p could negatively regulate the expression of a key DNA double-strand break (DSB) repair gene, ataxia telangiectasia mutated (ATM), by binding to multiple sites on its 3'-UTR. Notably, the expression of these four miR-BARTs represented more than 10% of all EBV-encoded miRNAs in tumor cells, while downregulation of ATM expression was commonly detected in all of our tested sequenced samples. In addition, downregulation of ATM was also observed in primary NPC tissues in both qRT-PCR (16 NP and 45 NPC cases) and immunohistochemical staining (35 NP and 46 NPC cases) analysis. Modulation of ATM expression by BART5-5p, BART7-3p, BART9-3p, and BART14-3p was demonstrated in the transient transfection assays. These findings suggest that EBV uses miRNA machinery as a key mechanism to control the ATM signaling pathway in NPC cells. By suppressing these endogenous miR-BARTs in EBV-positive NPC cells, we further demonstrated the novel function of miR-BARTs in inhibiting Zta-induced lytic reactivation. These findings imply that the four viral miRNAs work co-operatively to modulate ATM activity in response to DNA damage and to maintain viral latency, contributing to the tumorigenesis of NPC. © 2017 The Authors

EBV‐encoded miRNAs target ATM‐mediated response in nasopharyngeal carcinoma

Science.gov (United States)

Lung, Raymond W‐M; Hau, Pok‐Man; Yu, Ken H‐O; Yip, Kevin Y; Tong, Joanna H‐M; Chak, Wing‐Po; Chan, Anthony W‐H; Lam, Ka‐Hei; Lo, Angela Kwok‐Fung; Tin, Edith K‐Y; Chau, Shuk‐Ling; Pang, Jesse C‐S; Kwan, Johnny S‐H; Busson, Pierre; Young, Lawrence S; Yap, Lee‐Fah; Tsao, Sai‐Wah

2018-01-01

Abstract Nasopharyngeal carcinoma (NPC) is a highly invasive epithelial malignancy that is prevalent in southern China and Southeast Asia. It is consistently associated with latent Epstein–Barr virus (EBV) infection. In NPC, miR‐BARTs, the EBV‐encoded miRNAs derived from BamH1‐A rightward transcripts, are abundantly expressed and contribute to cancer development by targeting various cellular and viral genes. In this study, we establish a comprehensive transcriptional profile of EBV‐encoded miRNAs in a panel of NPC patient‐derived xenografts and an EBV‐positive NPC cell line by small RNA sequencing. Among the 40 miR‐BARTs, predominant expression of 22 miRNAs was consistently detected in these tumors. Among the abundantly expressed EBV‐miRNAs, BART5‐5p, BART7‐3p, BART9‐3p, and BART14‐3p could negatively regulate the expression of a key DNA double‐strand break (DSB) repair gene, ataxia telangiectasia mutated (ATM), by binding to multiple sites on its 3'‐UTR. Notably, the expression of these four miR‐BARTs represented more than 10% of all EBV‐encoded miRNAs in tumor cells, while downregulation of ATM expression was commonly detected in all of our tested sequenced samples. In addition, downregulation of ATM was also observed in primary NPC tissues in both qRT‐PCR (16 NP and 45 NPC cases) and immunohistochemical staining (35 NP and 46 NPC cases) analysis. Modulation of ATM expression by BART5‐5p, BART7‐3p, BART9‐3p, and BART14‐3p was demonstrated in the transient transfection assays. These findings suggest that EBV uses miRNA machinery as a key mechanism to control the ATM signaling pathway in NPC cells. By suppressing these endogenous miR‐BARTs in EBV‐positive NPC cells, we further demonstrated the novel function of miR‐BARTs in inhibiting Zta‐induced lytic reactivation. These findings imply that the four viral miRNAs work co‐operatively to modulate ATM activity in response to DNA damage and to maintain viral latency

Integrated analysis of miRNA and mRNA expression in childhood medulloblastoma compared with neural stem cells.

Directory of Open Access Journals (Sweden)

Laura A Genovesi

Full Text Available Medulloblastoma (MB is the most common malignant brain tumor in children and a leading cause of cancer-related mortality and morbidity. Several molecular sub-types of MB have been identified, suggesting they may arise from distinct cells of origin. Data from animal models indicate that some MB sub-types arise from multipotent cerebellar neural stem cells (NSCs. Hence, microRNA (miRNA expression profiles of primary MB samples were compared to CD133+ NSCs, aiming to identify deregulated miRNAs involved in MB pathogenesis. Expression profiling of 662 miRNAs in primary MB specimens, MB cell lines, and human CD133+ NSCs and CD133- neural progenitor cells was performed by qRT-PCR. Clustering analysis identified two distinct sub-types of MB primary specimens, reminiscent of sub-types obtained from their mRNA profiles. 21 significantly up-regulated and 12 significantly down-regulated miRNAs were identified in MB primary specimens relative to CD133+ NSCs (p<0.01. The majority of up-regulated miRNAs mapped to chromosomal regions 14q32 and 17q. Integration of the predicted targets of deregulated miRNAs with mRNA expression data from the same specimens revealed enrichment of pathways regulating neuronal migration, nervous system development and cell proliferation. Transient over-expression of a down-regulated miRNA, miR-935, resulted in significant down-regulation of three of the seven predicted miR-935 target genes at the mRNA level in a MB cell line, confirming the validity of this approach. This study represents the first integrated analysis of MB miRNA and mRNA expression profiles and is the first to compare MB miRNA expression profiles to those of CD133+ NSCs. We identified several differentially expressed miRNAs that potentially target networks of genes and signaling pathways that may be involved in the transformation of normal NSCs to brain tumor stem cells. Based on this integrative approach, our data provide an important platform for future

Genome-wide analysis of miRNA and mRNA transcriptomes during amelogenesis.

Science.gov (United States)

Yin, Kaifeng; Hacia, Joseph G; Zhong, Zhe; Paine, Michael L

2014-11-19

In the rodent incisor during amelogenesis, as ameloblast cells transition from secretory stage to maturation stage, their morphology and transcriptome profiles change dramatically. Prior whole genome transcriptome analysis has given a broad picture of the molecular activities dominating both stages of amelogenesis, but this type of analysis has not included miRNA transcript profiling. In this study, we set out to document which miRNAs and corresponding target genes change significantly as ameloblasts transition from secretory- to maturation-stage amelogenesis. Total RNA samples from both secretory- and maturation-stage rat enamel organs were subjected to genome-wide miRNA and mRNA transcript profiling. We identified 59 miRNAs that were differentially expressed at the maturation stage relative to the secretory stage of enamel development (False Discovery Rate (FDR)<0.05, fold change (FC)≥1.8). In parallel, transcriptome profiling experiments identified 1,729 mRNA transcripts that were differentially expressed in the maturation stage compared to the secretory stage (FDR<0.05, FC≥1.8). Based on bioinformatics analyses, 5.8% (629 total) of these differentially expressed genes (DEGS) were highlighted as being the potential targets of 59 miRNAs that were differentially expressed in the opposite direction, in the same tissue samples. Although the number of predicted target DEGs was not higher than baseline expectations generated by examination of stably expressed miRNAs, Gene Ontology (GO) analysis showed that these 629 DEGS were enriched for ion transport, pH regulation, calcium handling, endocytotic, and apoptotic activities. Seven differentially expressed miRNAs (miR-21, miR-31, miR-488, miR-153, miR-135b, miR-135a and miR298) in secretory- and/or maturation-stage enamel organs were confirmed by in situ hybridization. Further, we used luciferase reporter assays to provide evidence that two of these differentially expressed miRNAs, miR-153 and miR-31, are potential

The miRNA biogenesis in marine bivalves

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Umberto Rosani

2016-03-01

Full Text Available Small non-coding RNAs include powerful regulators of gene expression, transposon mobility and virus activity. Among the various categories, mature microRNAs (miRNAs guide the translational repression and decay of several targeted mRNAs. The biogenesis of miRNAs depends on few gene products, essentially conserved from basal to higher metazoans, whose protein domains allow specific interactions with dsRNA. Here, we report the identification of key genes responsible of the miRNA biogenesis in 32 bivalves, with particular attention to the aquaculture species Mytilus galloprovincialis and Crassostrea gigas. In detail, we have identified and phylogenetically compared eight evolutionary conserved proteins: DROSHA, DGCR8, EXP5, RAN, DICER TARBP2, AGO and PIWI. In mussels, we recognized several other proteins participating in the miRNA biogenesis or in the subsequent RNA silencing. According to digital expression analysis, these genes display low and not inducible expression levels in adult mussels and oysters whereas they are considerably expressed during development. As miRNAs play an important role also in the antiviral responses, knowledge on their production and regulative effects can shed light on essential molecular processes and provide new hints for disease prevention in bivalves.

ARMOUR - A Rice miRNA: mRNA Interaction Resource.

Science.gov (United States)

Sanan-Mishra, Neeti; Tripathi, Anita; Goswami, Kavita; Shukla, Rohit N; Vasudevan, Madavan; Goswami, Hitesh

2018-01-01

ARMOUR was developed as A Rice miRNA:mRNA interaction resource. This informative and interactive database includes the experimentally validated expression profiles of miRNAs under different developmental and abiotic stress conditions across seven Indian rice cultivars. This comprehensive database covers 689 known and 1664 predicted novel miRNAs and their expression profiles in more than 38 different tissues or conditions along with their predicted/known target transcripts. The understanding of miRNA:mRNA interactome in regulation of functional cellular machinery is supported by the sequence information of the mature and hairpin structures. ARMOUR provides flexibility to users in querying the database using multiple ways like known gene identifiers, gene ontology identifiers, KEGG identifiers and also allows on the fly fold change analysis and sequence search query with inbuilt BLAST algorithm. ARMOUR database provides a cohesive platform for novel and mature miRNAs and their expression in different experimental conditions and allows searching for their interacting mRNA targets, GO annotation and their involvement in various biological pathways. The ARMOUR database includes a provision for adding more experimental data from users, with an aim to develop it as a platform for sharing and comparing experimental data contributed by research groups working on rice.

Synergic Functions of miRNAs Determine Neuronal Fate of Adult Neural Stem Cells

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Meritxell Pons-Espinal

2017-04-01

Full Text Available Summary: Adult neurogenesis requires the precise control of neuronal versus astrocyte lineage determination in neural stem cells. While microRNAs (miRNAs are critically involved in this step during development, their actions in adult hippocampal neural stem cells (aNSCs has been unclear. As entry point to address that question we chose DICER, an endoribonuclease essential for miRNA biogenesis and other RNAi-related processes. By specific ablation of Dicer in aNSCs in vivo and in vitro, we demonstrate that miRNAs are required for the generation of new neurons, but not astrocytes, in the adult murine hippocampus. Moreover, we identify 11 miRNAs, of which 9 have not been previously characterized in neurogenesis, that determine neurogenic lineage fate choice of aNSCs at the expense of astrogliogenesis. Finally, we propose that the 11 miRNAs sustain adult hippocampal neurogenesis through synergistic modulation of 26 putative targets from different pathways. : In this article, the authors demonstrate that Dicer-dependent miRNAs are required for the generation of new neurons, but not astrocytes, in the adult hippocampus in vivo and in vitro. The authors identify a new set of 11 miRNAs that synergistically converge on multiple targets in different pathways to sustain neurogenic lineage fate commitment in aNSCs. Keywords: mouse, hippocampus, neural stem cells, fate choice, adult neurogenesis, astrogliogenesis, DICER, microRNAs, synergy

Functional screening identifies miRNAs influencing apoptosis and proliferation in colorectal cancer

DEFF Research Database (Denmark)

Christensen, Lise Lotte; Holm, Anja; Rantala, Juha

2014-01-01

MicroRNAs (miRNAs) play a critical role in many biological processes and are aberrantly expressed in human cancers. Particular miRNAs function either as tumor suppressors or oncogenes and appear to have diagnostic and prognostic significance. Although numerous miRNAs are dys-regulated in colorect...

RBiomirGS: an all-in-one miRNA gene set analysis solution featuring target mRNA mapping and expression profile integration

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Jing Zhang

2018-01-01

Full Text Available Background With the continuous discovery of microRNA’s (miRNA association with a wide range of biological and cellular processes, expression profile-based functional characterization of such post-transcriptional regulation is crucial for revealing its significance behind particular phenotypes. Profound advancement in bioinformatics has been made to enable in depth investigation of miRNA’s role in regulating cellular and molecular events, resulting in a huge quantity of software packages covering different aspects of miRNA functional analysis. Therefore, an all-in-one software solution is in demand for a comprehensive yet highly efficient workflow. Here we present RBiomirGS, an R package for a miRNA gene set (GS analysis. Methods The package utilizes multiple databases for target mRNA mapping, estimates miRNA effect on the target mRNAs through miRNA expression profile and conducts a logistic regression-based GS enrichment. Additionally, human ortholog Entrez ID conversion functionality is included for target mRNAs. Results By incorporating all the core steps into one package, RBiomirGS eliminates the need for switching between different software packages. The modular structure of RBiomirGS enables various access points to the analysis, with which users can choose the most relevant functionalities for their workflow. Conclusions With RBiomirGS, users are able to assess the functional significance of the miRNA expression profile under the corresponding experimental condition by minimal input and intervention. Accordingly, RBiomirGS encompasses an all-in-one solution for miRNA GS analysis. RBiomirGS is available on GitHub (http://github.com/jzhangc/RBiomirGS. More information including instruction and examples can be found on website (http://kenstoreylab.com/?page_id=2865.

Integrative testis transcriptome analysis reveals differentially expressed miRNAs and their mRNA targets during early puberty in Atlantic salmon.

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Skaftnesmo, K O; Edvardsen, R B; Furmanek, T; Crespo, D; Andersson, E; Kleppe, L; Taranger, G L; Bogerd, J; Schulz, R W; Wargelius, A

2017-10-18

Our understanding of the molecular mechanisms implementing pubertal maturation of the testis in vertebrates is incomplete. This topic is relevant in Atlantic salmon aquaculture, since precocious male puberty negatively impacts animal welfare and growth. We hypothesize that certain miRNAs modulate mRNAs relevant for the initiation of puberty. To explore which miRNAs regulate mRNAs during initiation of puberty in salmon, we performed an integrated transcriptome analysis (miRNA and mRNA-seq) of salmon testis at three stages of development: an immature, long-term quiescent stage, a prepubertal stage just before, and a pubertal stage just after the onset of single cell proliferation activity in the testis. Differentially expressed miRNAs clustered into 5 distinct expression profiles related to the immature, prepubertal and pubertal salmon testis. Potential mRNA targets of these miRNAs were predicted with miRmap and filtered for mRNAs displaying negatively correlated expression patterns. In summary, this analysis revealed miRNAs previously known to be regulated in immature vertebrate testis (miR-101, miR-137, miR-92b, miR-18a, miR-20a), but also miRNAs first reported here as regulated in the testis (miR-new289, miR-30c, miR-724, miR-26b, miR-new271, miR-217, miR-216a, miR-135a, miR-new194 and the novel predicted n268). By KEGG enrichment analysis, progesterone signaling and cell cycle pathway genes were found regulated by these differentially expressed miRNAs. During the transition into puberty we found differential expression of miRNAs previously associated (let7a/b/c), or newly associated (miR-15c, miR-2184, miR-145 and the novel predicted n7a and b) with this stage. KEGG enrichment analysis revealed that mRNAs of the Wnt, Hedgehog and Apelin signaling pathways were potential regulated targets during the transition into puberty. Likewise, several regulated miRNAs in the pubertal stage had earlier been associated (miR-20a, miR-25, miR-181a, miR-202, let7c/d/a, miR-125b

Adverse Intrauterine Environment and Cardiac miRNA Expression

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Mitchell C. Lock

2017-12-01

Full Text Available Placental insufficiency, high altitude pregnancies, maternal obesity/diabetes, maternal undernutrition and stress can result in a poor setting for growth of the developing fetus. These adverse intrauterine environments result in physiological changes to the developing heart that impact how the heart will function in postnatal life. The intrauterine environment plays a key role in the complex interplay between genes and the epigenetic mechanisms that regulate their expression. In this review we describe how an adverse intrauterine environment can influence the expression of miRNAs (a sub-set of non-coding RNAs and how these changes may impact heart development. Potential consequences of altered miRNA expression in the fetal heart include; Hypoxia inducible factor (HIF activation, dysregulation of angiogenesis, mitochondrial abnormalities and altered glucose and fatty acid transport/metabolism. It is important to understand how miRNAs are altered in these adverse environments to identify key pathways that can be targeted using miRNA mimics or inhibitors to condition an improved developmental response.

ARMOUR – A Rice miRNA: mRNA Interaction Resource

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Neeti Sanan-Mishra

2018-05-01

Full Text Available ARMOUR was developed as ARice miRNA:mRNA interaction resource. This informative and interactive database includes the experimentally validated expression profiles of miRNAs under different developmental and abiotic stress conditions across seven Indian rice cultivars. This comprehensive database covers 689 known and 1664 predicted novel miRNAs and their expression profiles in more than 38 different tissues or conditions along with their predicted/known target transcripts. The understanding of miRNA:mRNA interactome in regulation of functional cellular machinery is supported by the sequence information of the mature and hairpin structures. ARMOUR provides flexibility to users in querying the database using multiple ways like known gene identifiers, gene ontology identifiers, KEGG identifiers and also allows on the fly fold change analysis and sequence search query with inbuilt BLAST algorithm. ARMOUR database provides a cohesive platform for novel and mature miRNAs and their expression in different experimental conditions and allows searching for their interacting mRNA targets, GO annotation and their involvement in various biological pathways. The ARMOUR database includes a provision for adding more experimental data from users, with an aim to develop it as a platform for sharing and comparing experimental data contributed by research groups working on rice.

Post-transcriptional generation of miRNA variants by multiple nucleotidyl transferases contributes to miRNA transcriptome complexity

OpenAIRE

Wyman, Stacia K.; Knouf, Emily C.; Parkin, Rachael K.; Fritz, Brian R.; Lin, Daniel W.; Dennis, Lucas M.; Krouse, Michael A.; Webster, Philippa J.; Tewari, Muneesh

2011-01-01

Modification of microRNA sequences by the 3′ addition of nucleotides to generate so-called “isomiRs” adds to the complexity of miRNA function, with recent reports showing that 3′ modifications can influence miRNA stability and efficiency of target repression. Here, we show that the 3′ modification of miRNAs is a physiological and common post-transcriptional event that shows selectivity for specific miRNAs and is observed across species ranging from C. elegans to human. The modifications resul...

Embryonic miRNA profiles of normal and ectopic pregnancies.

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Francisco Dominguez

Full Text Available Our objective was to investigate the miRNA profile of embryonic tissues in ectopic pregnancies (EPs and controlled abortions (voluntary termination of pregnancy; VTOP. Twenty-three patients suffering from tubal EP and twenty-nine patients with a normal ongoing pregnancy scheduled for a VTOP were recruited. Embryonic tissue samples were analyzed by miRNA microarray and further validated by real time PCR. Microarray studies showed that four miRNAs were differentially downregulated (hsa-mir-196b, hsa-mir-30a, hsa-mir-873, and hsa-mir-337-3p and three upregulated (hsa-mir-1288, hsa-mir-451, and hsa-mir-223 in EP compared to control tissue samples. Hsa-miR-196, hsa-miR-223, and hsa-miR-451 were further validated by real time PCR in a wider population of EP and control samples. We also performed a computational analysis to identify the gene targets and pathways which might be modulated by these three differentially expressed miRNAs. The most significant pathways found were the mucin type O-glycan biosynthesis and the ECM-receptor-interaction pathways. We also checked that the dysregulation of these three miRNAs was able to alter the expression of the gene targets in the embryonic tissues included in these pathways such as GALNT13 and ITGA2 genes. In conclusion, analysis of miRNAs in ectopic and eutopic embryonic tissues shows different expression patterns that could modify pathways which are critical for correct implantation, providing new insights into the understanding of ectopic implantation in humans.

Analysis of miRNAs Involved in Mouse Brain Damage upon Enterovirus 71 Infection.

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Yang, Xiaoxia; Xie, Jing; Jia, Leili; Liu, Nan; Liang, Yuan; Wu, Fuli; Liang, Beibei; Li, Yongrui; Wang, Jinyan; Sheng, Chunyu; Li, Hao; Liu, Hongbo; Ma, Qiuxia; Yang, Chaojie; Du, Xinying; Qiu, Shaofu; Song, Hongbin

2017-01-01

Enterovirus 71 (EV71) infects the central nervous system (CNS) and causes brainstem encephalitis in children. MiRNAs have been found to play various functions in EV71 infection in human cell lines. To identify potential miRNAs involved in the inflammatory injury in CNS, our study, for the first time, performed a miRNA microarray assay in vivo using EV71 infected mice brains. Twenty differentially expressed miRNAs were identified (four up- and 16 down-regulated) and confirmed by qRT-PCR. The target genes of these miRNAs were analyzed using KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis, revealing that the miRNAs were mainly involved in the regulation of inflammation and neural system function. MiR-150-5p, -3082-5p, -3473a, -468-3p, -669n, -721, -709, and -5107-5p that regulate MAPK and chemokine signaling were all down-regulated, which might result in increased cytokine production. In addition, miR-3473a could also regulate focal adhesion and leukocyte trans-endothelial migration, suggesting a role in virus-induced blood-brain barrier disruption. The miRNAs and pathways identified in this study could help to understand the intricate interactions between EV71 and the brain injury, offering new insight for the future research of the molecular mechanism of EV71 induced brainstem encephalitis.

miRNA targeted signaling pathway in the early stage of denervated fast and slow muscle atrophy

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Gang Li

2016-01-01

Full Text Available Denervation often results in skeletal muscle atrophy. Different mechanisms seem to be involved in the determination between denervated slow and fast skeletal muscle atrophy. At the epigenetic level, miRNAs are thought to be highly involved in the pathophysiological progress of denervated muscles. We used miRNA microarrays to determine miRNA expression profiles from a typical slow muscle (soleus muscle and a typical fast muscle (tibialis anterior muscle at an early denervation stage in a rat model. Results showed that miR-206, miR-195, miR-23a, and miR-30e might be key factors in the transformation process from slow to fast muscle in denervated slow muscles. Additionally, certain miRNA molecules (miR-214, miR-221, miR-222, miR-152, miR-320, and Let-7e could be key regulatory factors in the denervated atrophy process involved in fast muscle. Analysis of signaling pathway networks revealed the miRNA molecules that were responsible for regulating certain signaling pathways, which were the final targets (e.g., p38 MAPK pathway; Pax3/Pax7 regulates Utrophin and follistatin by HDAC4; IGF1/PI3K/Akt/mTOR pathway regulates atrogin-1 and MuRF1 expression via FoxO phosphorylation. Our results provide a better understanding of the mechanisms of denervated skeletal muscle pathophysiology.

Expression Profile of Stress-responsive Arabidopsis thaliana miRNAs and their Target Genes in Response to Inoculation with Pectobacterium carotovorum subsp. carotovorum.

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Djami-Tchatchou, A T; Ntushelo, K

2017-01-01

Pectobacterium carotovorum subsp. carotovorum (Pcc) is a soft rot bacterium which upon entry into the plant macerates plant tissues by producing plant cell wall degrading enzymes. It has a wide host range which includes carrot, potato, tomato, leafy greens, squash and other cucurbits, onion, green peppers and cassava. During plant-microbe interactions, one of the ways of plant response to pathogen infection is through the small RNA silencing mechanism. Under pathogen attack the plant utilizes microRNAs to regulate gene expression by means of mediating gene silencing at transcriptional and post-transcriptional level. This study aims to assess for the first time, the expression profile of some stress-responsive miRNA and differential expression pattern of their target genes in Arabidopsis thaliana inoculated with Pcc. Leaves of five weeks old Arabidopsis thaliana plants were infected with Pcc and the quantitative real time-PCR, was used to investigate after 0, 24, 48 and 72 h post infection, the expression profiling of the stress-responsive miRNAs which include: miR156, miR159, miR169, miR393, miR396 miR398, miR399 and miR408 along with their target genes which include: Squamosa promoter-binding-like protein, myb domain protein 101, nuclear factor Y subunit A8, concanavalin A-like lectin protein kinase, growth regulating factor 4, copper superoxide dismutase, ubiquitin-protein ligase and plantacyanin respectively. The findings showed that the overexpression of 6 miRNAs at 24, 48 and 72 h after infection resulted in the repression of their target genes and the expression of 2 miRNAs didn't affect their target genes. These results provide the first indication of the miRNAs role in response to the infection of Pcc in A. thaliana and open new vistas for a better understanding of miRNA regulation of plant response to Pcc.

Dynamics of miRNA biogenesis and nuclear transport

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Kotipalli Aneesh

2016-12-01

Full Text Available MicroRNAs (miRNAs are short noncoding RNA sequences ~22 nucleotides in length that play an important role in gene regulation-transcription and translation. The processing of these miRNAs takes place in both the nucleus and the cytoplasm while the final maturation occurs in the cytoplasm. Some mature miRNAs with nuclear localisation signals (NLS are transported back to the nucleus and some remain in the cytoplasm. The functional roles of these miRNAs are seen in both the nucleus and the cytoplasm. In the nucleus, miRNAs regulate gene expression by binding to the targeted promoter sequences and affect either the transcriptional gene silencing (TGS or transcriptional gene activation (TGA. In the cytoplasm, targeted mRNAs are translationally repressed or cleaved based on the complementarity between the two sequences at the seed region of miRNA and mRNA. The selective transport of mature miRNAs to the nucleus follows the classical nuclear import mechanism. The classical nuclear import mechanism is a highly regulated process, involving exportins and importins. The nuclear pore complex (NPC regulates all these transport events like a gate keeper. The half-life of miRNAs is rather low, so within a short time miRNAs perform their function. Temporal studies of miRNA biogenesis are, therefore, useful. We have carried out simulation studies for important miRNA biogenesis steps and also classical nuclear import mechanism using ordinary differential equation (ODE solver in the Octave software.

HomoTarget: a new algorithm for prediction of microRNA targets in Homo sapiens.

Science.gov (United States)

Ahmadi, Hamed; Ahmadi, Ali; Azimzadeh-Jamalkandi, Sadegh; Shoorehdeli, Mahdi Aliyari; Salehzadeh-Yazdi, Ali; Bidkhori, Gholamreza; Masoudi-Nejad, Ali

2013-02-01

MiRNAs play an essential role in the networks of gene regulation by inhibiting the translation of target mRNAs. Several computational approaches have been proposed for the prediction of miRNA target-genes. Reports reveal a large fraction of under-predicted or falsely predicted target genes. Thus, there is an imperative need to develop a computational method by which the target mRNAs of existing miRNAs can be correctly identified. In this study, combined pattern recognition neural network (PRNN) and principle component analysis (PCA) architecture has been proposed in order to model the complicated relationship between miRNAs and their target mRNAs in humans. The results of several types of intelligent classifiers and our proposed model were compared, showing that our algorithm outperformed them with higher sensitivity and specificity. Using the recent release of the mirBase database to find potential targets of miRNAs, this model incorporated twelve structural, thermodynamic and positional features of miRNA:mRNA binding sites to select target candidates. Copyright © 2012 Elsevier Inc. All rights reserved.

Computational Identification of MicroRNAs and Their Targets from Finger Millet (Eleusine coracana).

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Usha, S; Jyothi, M N; Suchithra, B; Dixit, Rekha; Rai, D V; Nagesh Babu, R

2017-03-01

MicroRNAs are endogenous small RNAs regulating intrinsic normal growth and development of plant. Discovering miRNAs, their targets and further inferring their functions had become routine process to comprehend the normal biological processes of miRNAs and their roles in plant development. In this study, we used homology-based analysis with available expressed sequence tag of finger millet (Eleusine coracana) to predict conserved miRNAs. Three potent miRNAs targeting 88 genes were identified. The newly identified miRNAs were found to be homologous with miR166 and miR1310. The targets recognized were transcription factors and enzymes, and GO analysis showed these miRNAs played varied roles in gene regulation. The identification of miRNAs and their targets is anticipated to hasten the pace of key epigenetic regulators in plant development.

Dual miRNA targeting restricts host range and attenuates neurovirulence of flaviviruses.

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Konstantin A Tsetsarkin

2015-04-01

Full Text Available Mosquito-borne flaviviruses are among the most significant arboviral pathogens worldwide. Vaccinations and mosquito population control programs remain the most reliable means for flavivirus disease prevention, and live attenuated viruses remain one of the most attractive flavivirus vaccine platforms. Some live attenuated viruses are capable of infecting principle mosquito vectors, as demonstrated in the laboratory, which in combination with their intrinsic genetic instability could potentially lead to a vaccine virus reversion back to wild-type in nature, followed by introduction and dissemination of potentially dangerous viral strains into new geographic locations. To mitigate this risk we developed a microRNA-targeting approach that selectively restricts replication of flavivirus in the mosquito host. Introduction of sequences complementary to a mosquito-specific mir-184 and mir-275 miRNAs individually or in combination into the 3'NCR and/or ORF region resulted in selective restriction of dengue type 4 virus (DEN4 replication in mosquito cell lines and adult Aedes mosquitos. Moreover a combined targeting of DEN4 genome with mosquito-specific and vertebrate CNS-specific mir-124 miRNA can silence viral replication in two evolutionally distant biological systems: mosquitoes and mouse brains. Thus, this approach can reinforce the safety of newly developed or existing vaccines for use in humans and could provide an additional level of biosafety for laboratories using viruses with altered pathogenic or transmissibility characteristics.

CpG preconditioning regulates miRNA expression that modulates genomic reprogramming associated with neuroprotection against ischemic injury

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Vartanian, Keri B; Mitchell, Hugh D; Stevens, Susan L; Conrad, Valerie K; McDermott, Jason E; Stenzel-Poore, Mary P

2015-01-01

Cytosine-phosphate-guanine (CpG) preconditioning reprograms the genomic response to stroke to protect the brain against ischemic injury. The mechanisms underlying genomic reprogramming are incompletely understood. MicroRNAs (miRNAs) regulate gene expression; however, their role in modulating gene responses produced by CpG preconditioning is unknown. We evaluated brain miRNA expression in response to CpG preconditioning before and after stroke using microarray. Importantly, we have data from previous gene microarrays under the same conditions, which allowed integration of miRNA and gene expression data to specifically identify regulated miRNA gene targets. CpG preconditioning did not significantly alter miRNA expression before stroke, indicating that miRNA regulation is not critical for the initiation of preconditioning-induced neuroprotection. However, after stroke, differentially regulated miRNAs between CpG- and saline-treated animals associated with the upregulation of several neuroprotective genes, implicating these miRNAs in genomic reprogramming that increases neuroprotection. Statistical analysis revealed that the miRNA targets were enriched in the gene population regulated in the setting of stroke, implying that miRNAs likely orchestrate this gene expression. These data suggest that miRNAs regulate endogenous responses to stroke and that manipulation of these miRNAs may have the potential to acutely activate novel neuroprotective processes that reduce damage. PMID:25388675

miRNA genes of an invasive vector mosquito, Aedes albopictus.

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Jinbao Gu

Full Text Available Aedes albopictus, a vector of Dengue and Chikungunya viruses, is a robust invasive species in both tropical and temperate environments. MicroRNAs (miRNAs regulate gene expression and biological processes including embryonic development, innate immunity and infection. While a number of miRNAs have been discovered in some mosquitoes, no comprehensive effort has been made to characterize them from different developmental stages from a single species. Systematic analysis of miRNAs in Ae. albopictus will improve our understanding of its basic biology and inform novel strategies to prevent virus transmission. Between 10-14 million Illumina sequencing reads per sample were obtained from embryos, larvae, pupae, adult males, sugar-fed and blood-fed adult females. A total of 119 miRNA genes represented by 215 miRNA or miRNA star (miRNA* sequences were identified, 15 of which are novel. Eleven, two, and two of the newly-discovered miRNA genes appear specific to Aedes, Culicinae, and Culicidae, respectively. A number of miRNAs accumulate predominantly in one or two developmental stages and the large number that showed differences in abundance following a blood meal likely are important in blood-induced mosquito biology. Gene Ontology (GO analysis of the targets of all Ae. albopictus miRNAs provides a useful starting point for the study of their functions in mosquitoes. This study is the first systematic analysis of miRNAs based on deep-sequencing of small RNA samples of all developmental stages of a mosquito species. A number of miRNAs are related to specific physiological states, most notably, pre- and post-blood feeding. The distribution of lineage-specific miRNAs is consistent with mosquito phylogeny and the presence of a number of Aedes-specific miRNAs likely reflects the divergence between the Aedes and Culex genera.

Identification of Differentially Expressed miRNAs between White and Black Hair Follicles by RNA-Sequencing in the Goat (Capra hircus)

Science.gov (United States)

Wu, Zhenyang; Fu, Yuhua; Cao, Jianhua; Yu, Mei; Tang, Xiaohui; Zhao, Shuhong

2014-01-01

MicroRNAs (miRNAs) play a key role in many biological processes by regulating gene expression at the post-transcriptional level. A number of miRNAs have been identified from livestock species. However, compared with other animals, such as pigs and cows, the number of miRNAs identified in goats is quite low, particularly in hair follicles. In this study, to investigate the functional roles of miRNAs in goat hair follicles of goats with different coat colors, we sequenced miRNAs from two hair follicles samples (white and black) using Solexa sequencing. A total of 35,604,016 reads were obtained, which included 30,878,637 clean reads (86.73%). MiRDeep2 software identified 214 miRNAs. Among them, 205 were conserved among species and nine were novel miRNAs. Furthermore, DESeq software identified six differentially expressed miRNAs. Quantitative PCR confirmed differential expression of two miRNAs, miR-10b and miR-211. KEGG pathways were analyzed using the DAVID website for the predicted target genes of the differentially expressed miRNAs. Several signaling pathways including Notch and MAPK pathways may affect the process of coat color formation. Our study showed that the identified miRNAs might play an essential role in black and white follicle formation in goats. PMID:24879525

Identification of Differentially Expressed miRNAs between White and Black Hair Follicles by RNA-Sequencing in the Goat (Capra hircus

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Zhenyang Wu

2014-05-01

Full Text Available MicroRNAs (miRNAs play a key role in many biological processes by regulating gene expression at the post-transcriptional level. A number of miRNAs have been identified from livestock species. However, compared with other animals, such as pigs and cows, the number of miRNAs identified in goats is quite low, particularly in hair follicles. In this study, to investigate the functional roles of miRNAs in goat hair follicles of goats with different coat colors, we sequenced miRNAs from two hair follicles samples (white and black using Solexa sequencing. A total of 35,604,016 reads were obtained, which included 30,878,637 clean reads (86.73%. MiRDeep2 software identified 214 miRNAs. Among them, 205 were conserved among species and nine were novel miRNAs. Furthermore, DESeq software identified six differentially expressed miRNAs. Quantitative PCR confirmed differential expression of two miRNAs, miR-10b and miR-211. KEGG pathways were analyzed using the DAVID website for the predicted target genes of the differentially expressed miRNAs. Several signaling pathways including Notch and MAPK pathways may affect the process of coat color formation. Our study showed that the identified miRNAs might play an essential role in black and white follicle formation in goats.

IIKmTA: Inter and Intra Kingdom miRNA-Target Analyzer.

Science.gov (United States)

Mal, Chittabrata; Aftabuddin, Md; Kundu, Sudip

2018-03-16

Growing evidences suggest that microRNAs (miRNAs) can efficiently regulate gene expression at intracellular and extracellular levels. It has been previously reported that plant/food-derived miRNAs are highly enriched in human serum or serum from phytophagous animals, and they are responsible for regulating mammalian gene expression. Thus, miRNAs could function as active signaling molecules, which carry information across distinct species or even kingdoms. However, the mode of miRNA shuttling among various organisms is still a mystery to unravel. The intra and inter kingdom miRNA transfer has boosted up the hypothesis about the potential impact of plant or animal miRNAs on each other. To our knowledge, the software for analyzing cross-kingdom miRNA-targets is lacking. We have developed a web-tool "IIKmTA: Inter and Intra Kingdom miRNA-Target Analyzer" utilizing a database; the data of which have been collected from another web server. Here, user can analyze the targeting potential of (i) plant miRNAs on animal UTRs (Untranslated regions), and vice versa (i.e., inter kingdom), (ii) plant miRNAs on plant UTRs and animal miRNAs on animal UTRs (i.e., intra kingdom). Further, user can analyze (i) miRNAs to targets, (ii) targets to miRNAs, and (iii) miRNA sets targeting sets of targets. For a wide variety of animal and plant species, IIKmTA can identify the miRNA binding sites in the probable target UTRs. Moreover, GC% and AU% of miRNAs will be calculated. All the results can be saved as .csv file. Recent researches identified miRNAs in plants and human secretions and their role in regulating the human genes. Such findings indicate the therapeutic role of secretory miRNAs of such plants which exhibits medicinal value and in near future many diseases may be treated by consumption of these plant miRNAs through food. Using our newly developed database and analyzing tool, one can easily determine the different relationships between miRNAs and their targets across kingdoms

Exploring the miRNA regulatory network using evolutionary correlations.

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Benedikt Obermayer

2014-10-01

Full Text Available Post-transcriptional regulation by miRNAs is a widespread and highly conserved phenomenon in metazoans, with several hundreds to thousands of conserved binding sites for each miRNA, and up to two thirds of all genes under miRNA regulation. At the same time, the effect of miRNA regulation on mRNA and protein levels is usually quite modest and associated phenotypes are often weak or subtle. This has given rise to the notion that the highly interconnected miRNA regulatory network exerts its function less through any individual link and more via collective effects that lead to a functional interdependence of network links. We present a Bayesian framework to quantify conservation of miRNA target sites using vertebrate whole-genome alignments. The increased statistical power of our phylogenetic model allows detection of evolutionary correlation in the conservation patterns of site pairs. Such correlations could result from collective functions in the regulatory network. For instance, co-conservation of target site pairs supports a selective benefit of combinatorial regulation by multiple miRNAs. We find that some miRNA families are under pronounced co-targeting constraints, indicating a high connectivity in the regulatory network, while others appear to function in a more isolated way. By analyzing coordinated targeting of different curated gene sets, we observe distinct evolutionary signatures for protein complexes and signaling pathways that could reflect differences in control strategies. Our method is easily scalable to analyze upcoming larger data sets, and readily adaptable to detect high-level selective constraints between other genomic loci. We thus provide a proof-of-principle method to understand regulatory networks from an evolutionary perspective.

High throughput sequencing of small RNA component of leaves and inflorescence revealed conserved and novel miRNAs as well as phasiRNA loci in chickpea.

Science.gov (United States)

Srivastava, Sangeeta; Zheng, Yun; Kudapa, Himabindu; Jagadeeswaran, Guru; Hivrale, Vandana; Varshney, Rajeev K; Sunkar, Ramanjulu

2015-06-01

Among legumes, chickpea (Cicer arietinum L.) is the second most important crop after soybean. MicroRNAs (miRNAs) play important roles by regulating target gene expression important for plant development and tolerance to stress conditions. Additionally, recently discovered phased siRNAs (phasiRNAs), a new class of small RNAs, are abundantly produced in legumes. Nevertheless, little is known about these regulatory molecules in chickpea. The small RNA population was sequenced from leaves and flowers of chickpea to identify conserved and novel miRNAs as well as phasiRNAs/phasiRNA loci. Bioinformatics analysis revealed 157 miRNA loci for the 96 highly conserved and known miRNA homologs belonging to 38 miRNA families in chickpea. Furthermore, 20 novel miRNAs belonging to 17 miRNA families were identified. Sequence analysis revealed approximately 60 phasiRNA loci. Potential target genes likely to be regulated by these miRNAs were predicted and some were confirmed by modified 5' RACE assay. Predicted targets are mostly transcription factors that might be important for developmental processes, and others include superoxide dismutases, plantacyanin, laccases and F-box proteins that could participate in stress responses and protein degradation. Overall, this study provides an inventory of miRNA-target gene interactions for chickpea, useful for the comparative analysis of small RNAs among legumes. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Evolutionary relationships between miRNA genes and their activity.

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Zhu, Yan; Skogerbø, Geir; Ning, Qianqian; Wang, Zhen; Li, Biqing; Yang, Shuang; Sun, Hong; Li, Yixue

2012-12-22

The emergence of vertebrates is characterized by a strong increase in miRNA families. MicroRNAs interact broadly with many transcripts, and the evolution of such a system is intriguing. However, evolutionary questions concerning the origin of miRNA genes and their subsequent evolution remain unexplained. In order to systematically understand the evolutionary relationship between miRNAs gene and their function, we classified human known miRNAs into eight groups based on their evolutionary ages estimated by maximum parsimony method. New miRNA genes with new functional sequences accumulated more dynamically in vertebrates than that observed in Drosophila. Different levels of evolutionary selection were observed over miRNA gene sequences with different time of origin. Most genic miRNAs differ from their host genes in time of origin, there is no particular relationship between the age of a miRNA and the age of its host genes, genic miRNAs are mostly younger than the corresponding host genes. MicroRNAs originated over different time-scales are often predicted/verified to target the same or overlapping sets of genes, opening the possibility of substantial functional redundancy among miRNAs of different ages. Higher degree of tissue specificity and lower expression level was found in young miRNAs. Our data showed that compared with protein coding genes, miRNA genes are more dynamic in terms of emergence and decay. Evolution patterns are quite different between miRNAs of different ages. MicroRNAs activity is under tight control with well-regulated expression increased and targeting decreased over time. Our work calls attention to the study of miRNA activity with a consideration of their origin time.

miRNA signature and Dicer requirement during human endometrial stromal decidualization in vitro.

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Carlos Estella

Full Text Available Decidualization is a morphological and biochemical transformation of endometrial stromal fibroblast into differentiated decidual cells, which is critical for embryo implantation and pregnancy establishment. The complex regulatory networks have been elucidated at both the transcriptome and the proteome levels, however very little is known about the post-transcriptional regulation of this process. miRNAs regulate multiple physiological pathways and their de-regulation is associated with human disorders including gynaecological conditions such as endometriosis and preeclampsia. In this study we profile the miRNAs expression throughout human endometrial stromal (hESCs decidualization and analyze the requirement of the miRNA biogenesis enzyme Dicer during this process. A total of 26 miRNAs were upregulated and 17 miRNAs downregulated in decidualized hESCs compared to non-decidualized hESCs. Three miRNAs families, miR-181, miR-183 and miR-200, are down-regulated during the decidualization process. Using miRNAs target prediction algorithms we have identified the potential targets and pathways regulated by these miRNAs. The knockdown of Dicer has a minor effect on hESCs during in vitro decidualization. We have analyzed a battery of decidualization markers such as cell morphology, Prolactin, IGFBP-1, MPIF-1 and TIMP-3 secretion as well as HOXA10, COX2, SP1, C/EBPß and FOXO1 expression in decidualized hESCs with decreased Dicer function. We found decreased levels of HOXA10 and altered intracellular organization of actin filaments in Dicer knockdown decidualized hESCs compared to control. Our results provide the miRNA signature of hESC during the decidualization process in vitro. We also provide the first functional characterization of Dicer during human endometrial decidualization although surprisingly we found that Dicer plays a minor role regulating this process suggesting that alternative biogenesis miRNAs pathways must be involved in human

Small RNA profiling reveals important roles for miRNAs in Arabidopsis response to Bacillus velezensis FZB42.

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Xie, Shanshan; Jiang, Haiyang; Xu, Zhilan; Xu, Qianqian; Cheng, Beijiu

2017-09-20

Bacillus velezensis FZB42 (previously classified as Bacillus amyloliquefaciens FZB42) has been confirmed to successfully colonize plant roots and enhance defense response against pathogen infection. This study indicated that FZB42 inoculation enhanced Arabidopsis defense response against Pseudomonas syringae DC3000 through inducing the expression of PR1, PDF1.2 and stomata closure. To further clarify the induced defense response at miRNA level, sRNA libraries from Arabidopsis roots inoculated with FZB42 and control were constructed and sequenced. The reads of 21nt and 24nt in length were the most abundant groups in FZB42-treated library and control library, respectively. 234 known miRNAs and 16 novel miRNAs were identified. Among them, 11 known miRNAs and 4 novel miRNAs were differentially expressed after FZB42 inoculation. Moreover cis-elements (TC-rich repeats, TCA-element and CGTCA-motif) associated with plant defense were also found in the promoters of these miRNAs. Additionally, 141 mRNAs were predicted as potential targets of these differentially expressed miRNAs. GO annotations of the target genes indicated their potential roles in polyamine biosynthetic process and intracellular protein transport biological process, which may contribute to increased defense response. Our findings indicated that Bacillus velezensis FZB42 inoculation altered the expression of Arabidopsis miRNAs and their target genes, which were associated with defense response. Copyright © 2017 Elsevier B.V. All rights reserved.

Integrated analysis of miRNA and mRNA expression profiles in tilapia gonads at an early stage of sex differentiation.

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Tao, Wenjing; Sun, Lina; Shi, Hongjuan; Cheng, Yunying; Jiang, Dongneng; Fu, Beide; Conte, Matthew A; Gammerdinger, William J; Kocher, Thomas D; Wang, Deshou

2016-05-04

MicroRNAs (miRNAs) represent a second regulatory network that has important effects on gene expression and protein translation during biological process. However, the possible role of miRNAs in the early stages of fish sex differentiation is not well understood. In this study, we carried an integrated analysis of miRNA and mRNA expression profiles to explore their possibly regulatory patterns at the critical stage of sex differentiation in tilapia. We identified 279 pre-miRNA genes in tilapia genome, which were highly conserved in other fish species. Based on small RNA library sequencing, we identified 635 mature miRNAs in tilapia gonads, in which 62 and 49 miRNAs showed higher expression in XX and XY gonads, respectively. The predicted targets of these sex-biased miRNAs (e.g., miR-9, miR-21, miR-30a, miR-96, miR-200b, miR-212 and miR-7977) included genes encoding key enzymes in steroidogenic pathways (Cyp11a1, Hsd3b, Cyp19a1a, Hsd11b) and key molecules involved in vertebrate sex differentiation (Foxl2, Amh, Star1, Sf1, Dmrt1, and Gsdf). These genes also showed sex-biased expression in tilapia gonads at 5 dah. Some miRNAs (e.g., miR-96 and miR-737) targeted multiple genes involved in steroid synthesis, suggesting a complex miRNA regulatory network during early sex differentiation in this fish. The sequence and expression patterns of most miRNAs in tilapia are conserved in fishes, indicating the basic functions of vertebrate miRNAs might share a common evolutionary origin. This comprehensive analysis of miRNA and mRNA at the early stage of molecular sex differentiation in tilapia XX and XY gonads lead to the discovery of differentially expressed miRNAs and their putative targets, which will facilitate studies of the regulatory network of molecular sex determination and differentiation in fishes.

Parallel mRNA, proteomics and miRNA expression analysis in cell line models of the intestine.

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O'Sullivan, Finbarr; Keenan, Joanne; Aherne, Sinead; O'Neill, Fiona; Clarke, Colin; Henry, Michael; Meleady, Paula; Breen, Laura; Barron, Niall; Clynes, Martin; Horgan, Karina; Doolan, Padraig; Murphy, Richard

2017-11-07

To identify miRNA-regulated proteins differentially expressed between Caco2 and HT-29: two principal cell line models of the intestine. Exponentially growing Caco-2 and HT-29 cells were harvested and prepared for mRNA, miRNA and proteomic profiling. mRNA microarray profiling analysis was carried out using the Affymetrix GeneChip Human Gene 1.0 ST array. miRNA microarray profiling analysis was carried out using the Affymetrix Genechip miRNA 3.0 array. Quantitative Label-free LC-MS/MS proteomic analysis was performed using a Dionex Ultimate 3000 RSLCnano system coupled to a hybrid linear ion trap/Orbitrap mass spectrometer. Peptide identities were validated in Proteome Discoverer 2.1 and were subsequently imported into Progenesis QI software for further analysis. Hierarchical cluster analysis for all three parallel datasets (miRNA, proteomics, mRNA) was conducted in the R software environment using the Euclidean distance measure and Ward's clustering algorithm. The prediction of miRNA and oppositely correlated protein/mRNA interactions was performed using TargetScan 6.1. GO biological process, molecular function and cellular component enrichment analysis was carried out for the DE miRNA, protein and mRNA lists via the Pathway Studio 11.3 Web interface using their Mammalian database. Differential expression (DE) profiling comparing the intestinal cell lines HT-29 and Caco-2 identified 1795 Genes, 168 Proteins and 160 miRNAs as DE between the two cell lines. At the gene level, 1084 genes were upregulated and 711 were downregulated in the Caco-2 cell line relative to the HT-29 cell line. At the protein level, 57 proteins were found to be upregulated and 111 downregulated in the Caco-2 cell line relative to the HT-29 cell line. Finally, at the miRNAs level, 104 were upregulated and 56 downregulated in the Caco-2 cell line relative to the HT-29 cell line. Gene ontology (GO) analysis of the DE mRNA identified cell adhesion, migration and ECM organization, cellular lipid

Base Composition Characteristics of Mammalian miRNAs

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Bin Wang

2013-01-01

Full Text Available MicroRNAs (miRNAs are short RNA sequences that repress protein synthesis by either inhibiting the translation of messenger RNA (mRNA or increasing mRNA degradation. Endogenous miRNAs have been found in various organisms, including animals, plants, and viruses. Mammalian miRNAs are evolutionarily conserved, are scattered throughout chromosomes, and play an important role in the immune response and the onset of cancer. For this study, the author explored the base composition characteristics of miRNA genes from the six mammalian species that contain the largest number of known miRNAs. It was found that mammalian miRNAs are evolutionarily conserved and GU-rich. Interestingly, in the miRNA sequences investigated, A residues are clearly the most frequent occupants of positions 2 and 3 of the 5′ end of miRNAs. Unlike G and U residues that may pair with C/U and A/G, respectively, A residues can only pair with U residues of target mRNAs, which may augment the recognition specificity of the 5′ seed region.

Simultaneous inhibition of multiple oncogenic miRNAs by a multi-potent microRNA sponge.

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Jung, Jaeyun; Yeom, Chanjoo; Choi, Yeon-Sook; Kim, Sinae; Lee, EunJi; Park, Min Ji; Kang, Sang Wook; Kim, Sung Bae; Chang, Suhwan

2015-08-21

The roles of oncogenic miRNAs are widely recognized in many cancers. Inhibition of single miRNA using antagomiR can efficiently knock-down a specific miRNA. However, the effect is transient and often results in subtle phenotype, as there are other miRNAs contribute to tumorigenesis. Here we report a multi-potent miRNA sponge inhibiting multiple miRNAs simultaneously. As a model system, we targeted miR-21, miR-155 and miR-221/222, known as oncogenic miRNAs in multiple tumors including breast and pancreatic cancers. To achieve efficient knockdown, we generated perfect and bulged-matched miRNA binding sites (MBS) and introduced multiple copies of MBS, ranging from one to five, in the multi-potent miRNA sponge. Luciferase reporter assay showed the multi-potent miRNA sponge efficiently inhibited 4 miRNAs in breast and pancreatic cancer cells. Furthermore, a stable and inducible version of the multi-potent miRNA sponge cell line showed the miRNA sponge efficiently reduces the level of 4 target miRNAs and increase target protein level of these oncogenic miRNAs. Finally, we showed the miRNA sponge sensitize cells to cancer drug and attenuate cell migratory activity. Altogether, our study demonstrates the multi-potent miRNA sponge is a useful tool to examine the functional impact of simultaneous inhibition of multiple miRNAs and proposes a therapeutic potential.

Establishment of lipofection for studying miRNA function in human adipocytes.

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Enlund, Eveliina; Fischer, Simon; Handrick, René; Otte, Kerstin; Debatin, Klaus-Michael; Wabitsch, Martin; Fischer-Posovszky, Pamela

2014-01-01

miRNA dysregulation has recently been linked to human obesity and its related complications such as type 2 diabetes. In order to study miRNA function in human adipocytes, we aimed for the modulation of mature miRNA concentration in these cells. Adipocytes, however, tend to be resistant to transfection and there is often a need to resort to viral transduction or electroporation. Our objective therefore was to identify an efficient, non-viral transfection reagent capable of delivering small RNAs into these cells. To achieve this, we compared the efficiencies of three transfection agents, Lipofectamine 2000, ScreenFect A and BPEI 1.2 k in delivering fluorescent-labelled siRNA into human Simpson-Golabi-Behmel syndrome (SGBS) preadipocytes and adipocytes. Downregulation of a specific target gene in response to miRNA mimic overexpression was assayed in SGBS cells and also in ex vivo differentiated primary human adipocytes. Our results demonstrated that while all three transfection agents were able to internalize the oligos, only lipofection resulted in the efficient downregulation of a specific target gene both in SGBS cells and in primary human adipocytes. Lipofectamine 2000 outperformed ScreenFect A in preadipocytes, but in adipocytes the two reagents gave comparable results making ScreenFect A a notable new alternative for the gold standard Lipofectamine 2000.

Analysis of physiological and miRNA responses to Pi deficiency in alfalfa (Medicago sativa L.).

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Li, Zhenyi; Xu, Hongyu; Li, Yue; Wan, Xiufu; Ma, Zhao; Cao, Jing; Li, Zhensong; He, Feng; Wang, Yufei; Wan, Liqiang; Tong, Zongyong; Li, Xianglin

2018-03-01

The induction of miR399 and miR398 and the inhibition of miR156, miR159, miR160, miR171, miR2111, and miR2643 were observed under Pi deficiency in alfalfa. The miRNA-mediated genes involved in basic metabolic process, root and shoot development, stress response and Pi uptake. Inorganic phosphate (Pi) deficiency is known to be a limiting factor in plant development and growth. However, the underlying miRNAs associated with the Pi deficiency-responsive mechanism in alfalfa are unclear. To elucidate the molecular mechanism at the miRNA level, we constructed four small RNA (sRNA) libraries from the roots and shoots of alfalfa grown under normal or Pi-deficient conditions. In the present study, alfalfa plants showed reductions in biomass, photosynthesis, and Pi content and increases in their root-to-shoot ratio and citric, malic, and succinic acid contents under Pi limitation. Sequencing results identified 47 and 44 differentially expressed miRNAs in the roots and shoots, respectively. Furthermore, 909 potential target genes were predicted, and some targets were validated by RLM-RACE assays. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed prominent enrichment in signal transducer activity, binding and basic metabolic pathways for carbohydrates, fatty acids and amino acids; cellular response to hormone stimulus and response to auxin pathways were also enriched. qPCR results verified that the differentially expressed miRNA profile was consistent with sequencing results, and putative target genes exhibited opposite expression patterns. In this study, the miRNAs associated with the response to Pi limitation in alfalfa were identified. In addition, there was an enrichment of miRNA-targeted genes involved in biological regulatory processes such as basic metabolic pathways, root and shoot development, stress response, Pi transportation and citric acid secretion.

Delineating miRNA profile induced by chewing tobacco in oral keratinocytes

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Mohd Younis Bhat

2017-10-01

Full Text Available The major established etiologic risk factor for oral cancer is tobacco (chewed, smoked and snuffed forms. Chewing form of tobacco is predominantly used in India making it the leading cause of oral cancer. Despite being one of the leading causes of oral cancer, the molecular alterations induced by chewing tobacco remains largely unclear. Carcinogenic effect of chewing tobacco is through chronic and not acute exposure. To understand the molecular alterations induced by chewing tobacco, we developed a cell line model where non-neoplastic oral keratinocytes were chronically exposed to chewing tobacco for a period of 6 months. This resulted in increased cellular proliferation and invasive ability of normal oral keratinocytes. Using this cellular model we studied the differential expression of miRNAs associated with chewing tobacco and the altered signaling pathways through which the aberrantly expressed miRNAs affect tumorigenesis. miRNA sequencing  was carried out using Illumina HiSeq 2500 platform  which resulted in the identification of 427 annotated miRNAs of which 10 were significantly dysregulated (≥ 4 fold; p-value ≤ 0.05 in tobacco exposed cells compared to untreated parental cells. To study the altered signaling in oral keratinocytes chronically exposed to chewing tobacco, we employed quantitative proteomics to characterize the dysregulated proteins. Integration of miRNA sequencing data with proteomic data resulted in identification of 36 proven protein targets which (≥1.5 fold; p-value ≤ 0.05 showed expression correlation with the 10 significantly dysregulated miRNAs. Pathway analysis of the dysregulated targets revealed enrichment of interferon signaling and mRNA processing related pathways in the chewing tobacco exposed cells. In addition, we also identified 6 novel miRNA in oral keratinocytes chronically exposed to chewing tobacco extract. Our study provides a framework to understand the oncogenic transformation induced by

Aberration of miRNAs Expression in Leukocytes from Sporadic Amyotrophic Lateral Sclerosis.

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Chen, YongPing; Wei, QianQian; Chen, XuePing; Li, ChunYu; Cao, Bei; Ou, RuWei; Hadano, Shinji; Shang, Hui-Fang

2016-01-01

Accumulating evidence indicates that miRNAs play an important role in the development of amyotrophic lateral sclerosis (ALS). Most of previous studies on miRNA dysregulation in ALS focused on the alterative expression in ALS animal model or in limited samples from European patients with ALS. In the present study, the miRNA expression profiles were investigated in Chinese ALS patients to explore leukocytes miRNAs as a potential biomarker for the diagnosis of ALS. We analyzed the expression profiles of 1733 human mature miRNAs using microarray technology in leukocytes obtained from 5 patients with sporadic ALS (SALS) and 5 healthy controls. An independent group of 83 SALS patients, 24 Parkinson's disease (PD) patients and 61 controls was used for validation by real-time polymerase chain reaction assay. Area under the receiver operating characteristic curve (AUC) was used to evaluate diagnostic accuracy. In addition, target genes and signaling information of validated differential expression miRNAs were predicted using Bioinformatics. Eleven miRNAs, including four over-expressed and seven under-expressed miRNAs detected in SALS patients compared to healthy controls were selected for validation. Four under-expressed microRNAs, including hsa-miR-183, hsa-miR-193b, hsa-miR-451, and hsa-miR-3935, were confirmed in validation stage by comparison of 83 SALS patients and 61 HCs. Moreover, we identified a miRNA panel (hsa-miR-183, hsa-miR-193b, hsa-miR-451, and hsa-miR-3935) having a high diagnostic accuracy of SALS (AUC 0.857 for the validation group). However, only hsa-miR-183 was significantly lower in SALS patients than that in PD patients and in HCs, while no differences were found between PD patients and HCs. By bioinformatics analysis, we obtained a large number of target genes and signaling information that are linked to neurodegeneration. This study provided evidence of abnormal miRNA expression patterns in the peripheral blood leukocytes of SALS patients. Leukocytes

Aberration of miRNAs Expression in leukocytes from sporadic amyotrophic lateral sclerosis

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Yongping Chen

2016-08-01

Full Text Available Background: Accumulating evidence indicates that miRNAs play an important role in the development of amyotrophic lateral sclerosis (ALS. Most of previous studies on miRNA dysregulation in ALS focused on the alterative expression in ALS animal model or in limited samples from European patients with ALS. In the present study, the miRNA expression profiles were investigated in Chinese ALS patients to explore leukocytes miRNAs as a potential biomarker for the diagnosis of ALS.Methods: We analyzed the expression profiles of 1733 human mature miRNAs using microarray technology in leukocytes obtained from 5 patients with sporadic ALS (SALS and 5 healthy controls. An independent group of 83 SALS patients, 24 Parkinson’s disease (PD patients and 61 controls was used for validation by real-time polymerase chain reaction assay. Area under the receiver operating characteristic curve (AUC was used to evaluate diagnostic accuracy. In addition, target genes and signaling information of validated differential expression miRNAs were predicted using Bioinformatics.Results: Eleven miRNAs, including four over-expressed and seven under-expressed miRNAs detected in SALS patients compared to healthy controls were selected for validation. Four under-expressed microRNAs, including hsa-miR-183, hsa-miR-193b, hsa-miR-451 and hsa-miR-3935, were confirmed in validation stage by comparison of 83 SALS patients and 61 HCs. Moreover, we identified a miRNA panel (hsa-miR-183, hsa-miR-193b, hsa-miR-451 and hsa-miR-3935 having a high diagnostic accuracy of SALS (AUC 0.857 for the validation group. However, only hsa-miR-183 was significantly lower in SALS patients than that in PD patients and in HCs, while no differences were found between PD patients and HCs. By bioinformatics analysis, we obtained a large number of target genes and signaling information that are linked to neurodegeneration. Conclusion: This study provided evidence of abnormal miRNA expression patterns in the

MiRNA-548ah, a Potential Molecule Associated with Transition from Immune Tolerance to Immune Activation of Chronic Hepatitis B

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Tong-Jing Xing

2014-08-01

Full Text Available Objective: The present study aims to identify the differently expressed microRNA (miRNA molecules and target genes of miRNA in the immune tolerance (IT and immune activation (IA stages of chronic hepatitis B (CHB. Methods: miRNA expression profiles of peripheral blood mononuclear cells (PBMCs at the IT and IA stages of CHB were screened using miRNA microarrays and authenticated using a quantitative real-time polymerase chain reaction (RT-PCR. Gene ontology (GO and the Kyoto encyclopedia of genes and genomes (KEGG were used to analyze the significant functions and pathways of possible target genes of miRNAs. Assays of the gain and loss of function of the miRNAs were performed to verify the target genes in THP-1 cell lines. The luciferase reporter test was used on 293T cells as direct targets. Results: Significantly upregulated miR-548 and miR-4804 were observed in the miRNA microarrays and confirmed by RT-PCR in PBMCs at the IT and IA stages of CHB. GO and KEGG analysis revealed that MiR-548 and miR-4804 could be involved in numerous signaling pathways and protein binding activity. IFNγR1 was predicted as a target gene and validated as the direct gene of MiR-548. Significant negative correlation was found between the miR-548ah and mRNA levels of IFN-γR1 in CHB patients. Conclusions: The abnormal expression profiles of miRNA in PBMCs could be closely associated with immune activation of chronic HBV infection. miR-548, by targeting IFN-γR1, may represent a mechanism that can facilitate viral pathogenesis and help determine new therapeutic molecular targets.

A bioinformatics-based update on microRNAs and their targets in rainbow trout (Oncorhynchus mykiss).

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Yang, Liandong; He, Shunping

2014-01-01

MicroRNAs (miRNAs) participate in various vitally biological processes via controlling target genes activity and thousands of miRNAs have been identified in many species to date, including 18,698 known animal miRNA in miRBase. However, there are only limited studies reported in rainbow trout (Oncorhynchus mykiss) especially via the computational-based approaches. In present study, we systematically investigated the miRNAs in rainbow trout using a well-developed comparative genome-based homologue search. A total of 196 potential miRNAs, belonging to 124 miRNA families, were identified, most of which were firstly reported in rainbow trout. The length of miRNAs ranged from 17 to 24 nt with an average of 20 nt while the length of their precursors varied from 47 to 152 nt with an average of 85 nt. The identified miRNAs were not evenly distributed in each miRNA family, with only one member per family for a majority, and multiple members were also identified for several families. Nucleotide U was dominant in the pre-miRNAs with a percentage of 30.04%. The rainbow trout pre-miRNAs had relatively high negative minimal folding free energy (MFE) and adjusted MFE (AMFE). Not only the mature miRNAs but their precursor sequences are conserved among the living organisms. About 2466 O. mykiss genes were predicted as potential targets for 189 miRNAs. Gene Ontology (GO) analysis showed that nearly 2093, 2107, and 2081 target genes are involved in cellular component, molecular function, and biological processes respectively. KEGG pathway enrichment analysis illuminated that these miRNAs targets might regulate 105 metabolic pathways, including those of purine metabolism, nitrogen metabolism, and oxidative phosphorylation. This study has provided an update on rainbow trout miRNAs and their targets, which represents a foundation for future studies. © 2013.

Comparative miRNAs analysis of Two contrasting broccoli inbred lines with divergent head-forming capacity under temperature stress.

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Chen, Chi-Chien; Fu, Shih-Feng; Norikazu, Monma; Yang, Yau-Wen; Liu, Yu-Ju; Ikeo, Kazuho; Gojobori, Takashi; Huang, Hao-Jen

2015-12-01

MicroRNAs (miRNAs) play a vital role in growth, development, and stress response at the post-transcriptional level. Broccoli (Brassica oleracea L. var italic) is an important vegetable crop, and the yield and quality of broccoli are decreased by heat stress. The broccoli inbred lines that are capable of producing head at high temperature in summer are unique varieties in Taiwan. However, knowledge of miRNAomes during the broccoli head formation under heat stress is limited. In this study, molecular characterization of two nearly isogenic lines with contrasting head-forming capacity was investigated. Head-forming capacity was better for heat-tolerant (HT) than heat-sensitive (HS) broccoli under heat stress. By deep sequencing and computational analysis, 20 known miRNAs showed significant differential expression between HT and HS genotypes. According to the criteria for annotation of new miRNAs, 24 novel miRNA sequences with differential expression between the two genotypes were identified. To gain insight into functional significance, 213 unique potential targets of these 44 differentially expressed miRNAs were predicted. These targets were implicated in shoot apical development, phase change, response to temperature stimulus, hormone and energy metabolism. The head-forming capacity of the unique HT line was related to autonomous regulation of Bo-FT genes and less expression level of heat shock protein genes as compared to HS. For the genotypic comparison, a set of miRNAs and their targets had consistent expression patterns in various HT genotypes. This large-scale characterization of broccoli miRNAs and their potential targets is to unravel the regulatory roles of miRNAs underlying heat-tolerant head-forming capacity.

Over-expression of the miRNA cluster at chromosome 14q32 in the alcoholic brain correlates with suppression of predicted target mRNA required for oligodendrocyte proliferation.

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Manzardo, A M; Gunewardena, S; Butler, M G

2013-09-10

We examined miRNA expression from RNA isolated from the frontal cortex (Broadman area 9) of 9 alcoholics (6 males, 3 females, mean age 48 years) and 9 matched controls using both the Affymetrix GeneChip miRNA 2.0 and Human Exon 1.0 ST Arrays to further characterize genetic influences in alcoholism and the effects of alcohol consumption on predicted target mRNA expression. A total of 12 human miRNAs were significantly up-regulated in alcohol dependent subjects (fold change≥1.5, false discovery rate (FDR)≤0.3; p<0.05) compared with controls including a cluster of 4 miRNAs (e.g., miR-377, miR-379) from the maternally expressed 14q32 chromosome region. The status of the up-regulated miRNAs was supported using the high-throughput method of exon microarrays showing decreased predicted mRNA gene target expression as anticipated from the same RNA aliquot. Predicted mRNA targets were involved in cellular adhesion (e.g., THBS2), tissue differentiation (e.g., CHN2), neuronal migration (e.g., NDE1), myelination (e.g., UGT8, CNP) and oligodendrocyte proliferation (e.g., ENPP2, SEMA4D1). Our data support an association of alcoholism with up-regulation of a cluster of miRNAs located in the genomic imprinted domain on chromosome 14q32 with their predicted gene targets involved with oligodendrocyte growth, differentiation and signaling. Copyright © 2013 Elsevier B.V. All rights reserved.

Disruption of Claudin-1 Expression by miRNA-182 Alters the Susceptibility to Viral Infectivity in HCV Cell Models

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Sarah E. Riad

2018-03-01

Full Text Available HCV entry involves a complex interplay between viral and host molecules. During post-binding interactions, the viral E2 complexes with CD81 receptor for delivery to the tight junction proteins CLDN1 and OCLN, which aid in viral internalization. Targeting HCV entry receptors represents an appealing approach to inhibit viral infectivity. This study aimed at investigating the impact of targeting CLDN1 by microRNAs on HCV infectivity. miR-155 was previously shown to target the 3′UTR of CLDN1 mRNA. Therefore, miR-155 was used as a control in this study. In-silico analysis and luciferase reporter assay were utilized to identify potential targeting miRNAs. The impact of the identified miRNAs on CLDN1 mRNA and protein expression was examined by qRT-PCR, indirect immunofluorescence and western blotting, respectively. The role of the selected miRNAs on HCV infectivity was assessed by measuring the viral load following the ectopic expression of the selected miRNAs. miR-182 was identified in-silico and by experimental validation to target CLDN1. Both miR-155 and miR-182 inhibited CLDN1 mRNA and protein expression in infected Huh7 cells. Ectopic expression of miR-155 increased, while miR-182 reduced the viral load. In conclusion, despite repressing CLDN1, the impact of miR-155 and miR-182 on HCV infectivity is contradictory. Ectopic miR-182 expression is suggested as an upstream regulator of the entry factor CLDN1, harnessing HCV infection.

The miRNAome of globe artichoke: conserved and novel micro RNAs and target analysis

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De Paola Domenico

2012-01-01

Full Text Available Abstract Background Plant microRNAs (miRNAs are involved in post-transcriptional regulatory mechanisms of several processes, including the response to biotic and abiotic stress, often contributing to the adaptive response of the plant to adverse conditions. In addition to conserved miRNAs, found in a wide range of plant species a number of novel species-specific miRNAs, displaying lower levels of expression can be found. Due to low abundance, non conserved miRNAs are difficult to identify and isolate using conventional approaches. Conversely, deep-sequencing of small RNA (sRNA libraries can detect even poorly expressed miRNAs. No miRNAs from globe artichoke have been described to date. We analyzed the miRNAome from artichoke by deep sequencing four sRNA libraries obtained from NaCl stressed and control leaves and roots. Results Conserved and novel miRNAs were discovered using accepted criteria. The expression level of selected miRNAs was monitored by quantitative real-time PCR. Targets were predicted and validated for their cleavage site. A total of 122 artichoke miRNAs were identified, 98 (25 families of which were conserved with other plant species, and 24 were novel. Some miRNAs were differentially expressed according to tissue or condition, magnitude of variation after salt stress being more pronounced in roots. Target function was predicted by comparison to Arabidopsis proteins; the 43 targets (23 for novel miRNAs identified included transcription factors and other genes, most of which involved in the response to various stresses. An unusual cleaved transcript was detected for miR393 target, transport inhibitor response 1. Conclusions The miRNAome from artichoke, including novel miRNAs, was unveiled, providing useful information on the expression in different organs and conditions. New target genes were identified. We suggest that the generation of secondary short-interfering RNAs from miR393 target can be a general rule in the plant

Effect of β-hydroxy-β-methylbutyrate on miRNA expression in differentiating equine satellite cells exposed to hydrogen peroxide.

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Chodkowska, Karolina A; Ciecierska, Anna; Majchrzak, Kinga; Ostaszewski, Piotr; Sadkowski, Tomasz

2018-01-01

Skeletal muscle injury activates satellite cells to initiate processes of proliferation, differentiation, and hypertrophy in order to regenerate muscle fibers. The number of microRNAs and their target genes are engaged in satellite cell activation. β-Hydroxy-β-methylbutyrate (HMB) is known to prevent exercise-induced muscle damage. The purpose of this study was to evaluate the effect of HMB on miRNA and relevant target gene expression in differentiating equine satellite cells exposed to H 2 O 2 . We hypothesized that HMB may regulate satellite cell activity, proliferation, and differentiation, hence attenuate the pathological processes induced during an in vitro model of H 2 O 2 -related injury by changing the expression of miRNAs. Equine satellite cells (ESC) were isolated from the samples of skeletal muscle collected from young horses. ESC were treated with HMB (24 h) and then exposed to H 2 O 2 (1 h). For the microRNA and gene expression assessment microarrays, technique was used. Identified miRNAs and genes were validated using real-time qPCR. Cell viability, oxidative stress, and cell damage were measured using colorimetric method and flow cytometry. Analysis of miRNA and gene profile in differentiating ESC pre-incubated with HMB and then exposed to H 2 O 2 revealed difference in the expression of 27 miRNAs and 4740 genes, of which 344 were potential target genes for identified miRNAs. Special attention was focused on differentially expressed miRNAs and their target genes involved in processes related to skeletal muscle injury. Western blot analysis showed protein protection in HMB-pre-treated group compared to control. The viability test confirmed that HMB enhanced cell survival after the hydrogen peroxide exposition. Our results suggest that ESC pre-incubated with HMB and exposed to H 2 O 2 could affect expression on miRNA levels responsible for skeletal muscle development, cell proliferation and differentiation, and activation of tissue repair after

Towards an understanding of miRNA regulation

DEFF Research Database (Denmark)

Jensen, Trine Ilsø

miRNAs are well-known regulators of gene expression. They function post-transcriptionally by binding to complementary sites within the 3´UTR of target mRNAs, which mediates translational repression and destabilization. However, miRNA expression itself is also subjected to regulation. Here, we...... report a new method to investigate and potentially characterize the pri-miRNA transcript. Overexpression of a transdominant Drosha mutant, which is unable to cleave its substrate, enables stabilization of the pri-miRNA transcript. Drosha mutant immunoprecipitation from the nuclear compartment...... is performed followed by high-throughput sequencing (nuclear Drosha Mt2 RIPseq). This method allows for the detection of global pri-miRNA signature and also provides a method to potentially identify new Drosha substrates. Furthermore, data on the identification of a novel endogenous circular RNA sponge (ciRS-7...

Comparative analysis of miRNAs of two rapeseed genotypes in response to acetohydroxyacid synthase-inhibiting herbicides by high-throughput sequencing.

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Maolong Hu

Full Text Available Acetohydroxyacid synthase (AHAS, also called acetolactate synthase, is a key enzyme involved in the first step of the biosynthesis of the branched-chain amino acids valine, isoleucine and leucine. Acetohydroxyacid synthase-inhibiting herbicides (AHAS herbicides are five chemical families of herbicides that inhibit AHAS enzymes, including imidazolinones (IMI, sulfonylureas (SU, pyrimidinylthiobenzoates, triazolinones and triazolopyrimidines. Five AHAS genes have been identified in rapeseed, but little information is available regarding the role of miRNAs in response to AHAS herbicides. In this study, an AHAS herbicides tolerant genotype and a sensitive genotype were used for miRNA comparative analysis. A total of 20 small RNA libraries were obtained of these two genotypes at three time points (0h, 24 h and 48 h after spraying SU and IMI herbicides with two replicates. We identified 940 conserved miRNAs and 1515 novel candidate miRNAs in Brassica napus using high-throughput sequencing methods combined with computing analysis. A total of 3284 genes were predicted to be targets of these miRNAs, and their functions were shown using GO, KOG and KEGG annotations. The differentiation expression results of miRNAs showed almost twice as many differentiated miRNAs were found in tolerant genotype M342 (309 miRNAs after SU herbicide application than in sensitive genotype N131 (164 miRNAs. In additiond 177 and 296 miRNAs defined as differentiated in sensitive genotype and tolerant genotype in response to SU herbicides. The miR398 family was observed to be associated with AHAS herbicide tolerance because their expression increased in the tolerant genotype but decreased in the sensitive genotype. Moreover, 50 novel miRNAs from 39 precursors were predicted. There were 8 conserved miRNAs, 4 novel miRNAs and 3 target genes were validated by quantitative real-time PCR experiment. This study not only provides novel insights into the miRNA content of AHAS herbicides

Computational identification of 18 micrornas and their targets in three species of rose

International Nuclear Information System (INIS)

Baloch, I.A.; Barozai, M.Y.K.; Achakzai, A.K.K.

2015-01-01

MicroRNAs (miRNAs) are non-protein coding, small endogenous RNAs. Their length ranges from 18-26 nucleotides (nt). The miRNAs convergence property becomes a rational approach for the hunt of novel miRNAs in other organisms by homology search. As presently very little miRNAs are reported for rose species, so this study deals with the identification of miRNAs in different species of rose. Consequently 18 miRNA belonging to 17 miRNA families were identified in 3 species of rose (Rosa hybrid, Rosa chinensis and Rosa virginiana). All of the identified miRNA families (miR156, 160, 164, 166, 398, 482, 831, 837, 838, 841, 847, 3436, 3627, 6135, 6285, 6287 and 6288) are being reported for the first time in rose. Precursors of the identified miRNAs form stable minimum free energy (MFE) stem-loop structures and the mature miRNAs are found in the stem portions of their corresponding precursors. 11 putative targets of the miRNAs have also been identified. The identified targets are various proteins including transcription factors. Identification of 18 miRNAs will be supportive to explore the gene regulation phenomenon in various species of roses and it will be a good contribution for understanding the post transcriptional gene regulation in various stages of the life cycles of roses. (author)

Detection and analysis of apoptosis- and autophagy-related miRNAs of mouse vascular endothelial cells in chronic intermittent hypoxia model.

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Liu, Kai-Xiong; Chen, Gong-Ping; Lin, Ping-Li; Huang, Jian-Chai; Lin, Xin; Qi, Jia-Chao; Lin, Qi-Chang

2018-01-15

Endothelial dysfunction is the main pathogenic mechanism of cardiovascular complications induced by obstructive sleep apnea/hyponea syndrome (OSAHS). Chronic intermittent hypoxia (CIH) is the primary factor of OSAHS-associated endothelial dysfunction. The hypoxia inducible factor (HIF) pathway regulates the expression of downstream target genes and mediates cell apoptosis caused by CIH-induced endothelial injury. miRNAs play extensive and important negative regulatory roles in this process at the post-transcriptional level. However, the regulatory mechanism of miRNAs in CIH tissue models remains unclear. The present study established a mouse aortic endothelial cell model of CIH in an attempt to screen out specific miRNAs by using miRNA chip analysis. It was found that 14 miRNAs were differentially expressed. Of them, 6 were significantly different and verified by quantitative real-time PCR (Q-PCR), of which four were up-regulated and two were down-regulated markedly. To gain an unbiased global perspective on subsequent regulation by altered miRNAs, we established signaling networks by GO to predict the target genes of the 6 miRNAs. It was found that the 6 identified miRNAs were apoptosis- or autophagy-related target genes. Down-regulation of miR-193 inhibits CIH induced endothelial injury and apoptosis- or autophagy-related protein expression. In conclusion, our results showed that CIH could induce differential expression of miRNAs, and alteration in the miRNA expression pattern was associated with the expression of apoptosis- or autophagy-related genes. Copyright © 2017 Elsevier Inc. All rights reserved.

MicroRNA target finding by comparative genomics.

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Friedman, Robin C; Burge, Christopher B

2014-01-01

MicroRNAs (miRNAs) have been implicated in virtually every metazoan biological process, exerting a widespread impact on gene expression. MicroRNA repression is conferred by relatively short "seed match" sequences, although the degree of repression varies widely for individual target sites. The factors controlling whether, and to what extent, a target site is repressed are not fully understood. As an alternative to target prediction based on sequence alone, comparative genomics has emerged as an invaluable tool for identifying miRNA targets that are conserved by natural selection, and hence likely effective and important. Here we present a general method for quantifying conservation of miRNA seed match sites, separating it from background conservation, controlling for various biases, and predicting miRNA targets. This method is useful not only for generating predictions but also as a tool for empirically evaluating the importance of various target prediction criteria.

miRNA regulation of LDL-cholesterol metabolism.

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Goedeke, Leigh; Wagschal, Alexandre; Fernández-Hernando, Carlos; Näär, Anders M

2016-12-01

In the past decade, microRNAs (miRNAs) have emerged as key regulators of circulating levels of lipoproteins. Specifically, recent work has uncovered the role of miRNAs in controlling the levels of atherogenic low-density lipoprotein LDL (LDL)-cholesterol by post-transcriptionally regulating genes involved in very low-density lipoprotein (VLDL) secretion, cholesterol biosynthesis, and hepatic LDL receptor (LDLR) expression. Interestingly, several of these miRNAs are located in genomic loci associated with abnormal levels of circulating lipids in humans. These findings reinforce the interest of targeting this subset of non-coding RNAs as potential therapeutic avenues for regulating plasma cholesterol and triglyceride (TAG) levels. In this review, we will discuss how these new miRNAs represent potential pre-disposition factors for cardiovascular disease (CVD), and putative therapeutic targets in patients with cardiometabolic disorders. This article is part of a Special Issue entitled: MicroRNAs and lipid/energy metabolism and related diseases edited by Carlos Fernández-Hernando and Yajaira Suárez. Copyright © 2016 Elsevier B.V. All rights reserved.

miRNA-34b is directly involved in the aging of macrophages.

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Liang, Wei; Gao, Sheng; Liang, Liu; Huang, Xianing; Hu, Nan; Lu, Xiaoling; Zhao, Yongxiang

2017-08-01

MicroRNAs (miRNAs) are a class of short noncoding RNA that play important regulatory roles in living organisms. These RNA molecules are implicated in the development and progression of malignant diseases such as cancer and are closely associated with cell aging. Findings demonstrating that microRNA is associated with aging in macrophages have nevertheless rarely been reported. This study's objective was to investigate if miRNA-34 is linked to aging process of macrophages. We built a cell aging model in mouse RAW264.7 macrophages using D-galactose and determined the expression levels of miRNA-34a, miRNA-34b, and miRNA-34c in aging and normal macrophages by fluorescence quantitative polymerase chain reaction (q-PCR). We predicted a target gene of miRNA-34 using biological information techniques and constructed the recombinant plasmid pGL3-E2f3 for the putative target gene E2f3. The expression level of miRNA-34b was 5.23 times higher in aging macrophages than in normal macrophages. The luciferase activity decreased by nearly 50 % in cells transfected with miRNA-34b mimics, while no significant decrease in luciferase activity was noted in cells transfected with the miRNA-34b inhibitor or unrelated sequences. Our findings provide the groundwork for further research into the molecular mechanisms whereby miRNA-34b regulates the aging of macrophages. miRNA-34b is associated with the aging of RAW264.7 macrophages, and E2f3 is a target gene of miRNA-34b.

Therapeutic modulation of miRNA for the treatment of proinflammatory lung diseases.

LENUS (Irish Health Repository)

Hassan, Tidi

2012-03-01

miRNAs are short, nonprotein coding RNAs that regulate target gene expression principally by causing translational repression and\\/or mRNA degradation. miRNAs are involved in most mammalian biological processes and have pivotal roles in controlling the expression of factors involved in basal and stimulus-induced signaling pathways. Considering their central role in the regulation of gene expression, miRNAs represent therapeutic drug targets. Here we describe how miRNAs are involved in the regulation of aspects of innate immunity and inflammation, what happens when this goes awry, such as in the chronic inflammatory lung diseases cystic fibrosis and asthma, and discuss the current state-of-the-art miRNA-targeted therapeutics.

Viruses and miRNAs: More Friends than Foes.

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Bruscella, Patrice; Bottini, Silvia; Baudesson, Camille; Pawlotsky, Jean-Michel; Feray, Cyrille; Trabucchi, Michele

2017-01-01

There is evidence that eukaryotic miRNAs (hereafter called host miRNAs) play a role in the replication and propagation of viruses. Expression or targeting of host miRNAs can be involved in cellular antiviral responses. Most times host miRNAs play a role in viral life-cycles and promote infection through complex regulatory pathways. miRNAs can also be encoded by a viral genome and be expressed in the host cell. Viral miRNAs can share common sequences with host miRNAs or have totally different sequences. They can regulate a variety of biological processes involved in viral infection, including apoptosis, evasion of the immune response, or modulation of viral life-cycle phases. Overall, virus/miRNA pathway interaction is defined by a plethora of complex mechanisms, though not yet fully understood. This article review summarizes recent advances and novel biological concepts related to the understanding of miRNA expression, control and function during viral infections. The article also discusses potential therapeutic applications of this particular host-pathogen interaction.

Establishment of lipofection for studying miRNA function in human adipocytes.

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Eveliina Enlund

Full Text Available miRNA dysregulation has recently been linked to human obesity and its related complications such as type 2 diabetes. In order to study miRNA function in human adipocytes, we aimed for the modulation of mature miRNA concentration in these cells. Adipocytes, however, tend to be resistant to transfection and there is often a need to resort to viral transduction or electroporation. Our objective therefore was to identify an efficient, non-viral transfection reagent capable of delivering small RNAs into these cells. To achieve this, we compared the efficiencies of three transfection agents, Lipofectamine 2000, ScreenFect A and BPEI 1.2 k in delivering fluorescent-labelled siRNA into human Simpson-Golabi-Behmel syndrome (SGBS preadipocytes and adipocytes. Downregulation of a specific target gene in response to miRNA mimic overexpression was assayed in SGBS cells and also in ex vivo differentiated primary human adipocytes. Our results demonstrated that while all three transfection agents were able to internalize the oligos, only lipofection resulted in the efficient downregulation of a specific target gene both in SGBS cells and in primary human adipocytes. Lipofectamine 2000 outperformed ScreenFect A in preadipocytes, but in adipocytes the two reagents gave comparable results making ScreenFect A a notable new alternative for the gold standard Lipofectamine 2000.

Identification of drought-responsive miRNAs and physiological characterization of tea plant (Camellia sinensis L.) under drought stress.

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Guo, Yuqiong; Zhao, Shanshan; Zhu, Chen; Chang, Xiaojun; Yue, Chuan; Wang, Zhong; Lin, Yuling; Lai, Zhongxiong

2017-11-21

Drought stress is one of the major natural challenges in the main tea-producing regions of China. The tea plant (Camellia sinensis) is a traditional beverage plant whose growth status directly affects tea quality. Recent studies have revealed that microRNAs (miRNAs) play key functions in plant growth and development. Although some miRNAs have been identified in C. sinensis, little is known about their roles in the drought stress response of tea plants. Physiological characterization of Camellia sinensis 'Tieguanyin' under drought stress showed that the malondialdehyde concentration and electrical conductivity of leaves of drought-stressed plants increased when the chlorophyll concentration decreased under severe drought stress. We sequenced four small-RNA (sRNA) libraries constructed from leaves of plants subjected to four different treatments, normal water supply (CK); mild drought stress (T1); moderate drought stress (T2) and severe drought stress (T3). A total of 299 known mature miRNA sequences and 46 novel miRNAs were identified. Gene Ontology enrichment analysis revealed that most of the differentially expressed-miRNA target genes were related to regulation of transcription. Kyoto Encyclopedia of Genes and Genomes analysis revealed that the most highly enriched pathways under drought stress were D-alanine metabolism, sulfur metabolism, and mineral absorption pathways. Real-time quantitative PCR (qPCR) was used to validate the expression patterns of 21 miRNAs (2 up-regulated and 19 down-regulated under drought stress). The observed co-regulation of the miR166 family and their targets ATHB-14-like and ATHB-15-like indicate the presence of negative feedback regulation in miRNA pathways. Analyses of drought-responsive miRNAs in tea plants showed that most of differentially expressed-miRNA target genes were related to regulation of transcription. The results of study revealed that the expressions of phase-specific miRNAs vary with morphological, physiological, and

[miRNA profile of the human dental pulp cells during odontoblast differentiation induced by BMP-2].

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Bao, Li-Rong; Zhao, Wen-Qing; Lin, Tian; Lu, Yan-Ling; Wu, Yu

2017-10-01

To screen and verify the differentially expressed microRNAs (miRNAs) during the differentiation of human dental pulp cells (hDPCs) to odontoblasts induced by BMP-2. The isolated hDPCs were cultured in vitro and induced by BMP-2. The levels of ALP, DMP-1 and DSPP were quantified by quantitative real-time polymerase chain reaction (qRT-PCR). The potential characteristics of hDPCs were investigated by miRNA microarray and highly expressed miRNAs were selected with bio-information software for predicting target genes and their biological functions. Then the results were validated using qRT-PCR analysis for the selected miRNAs. Statistical analysis was performed using SPSS 18.0 software package. The expression of ALP, DSPP, and DMP-1 showed significantly higher levels in BMP-2 induced groups compared to the control group(Pfunction(33%), while the function of other 0.2% genes remained unknown. This study identified differential expression of miRNAs in BMP-2-induced odontoblastic differentiation of hDPCs, thus contributing to further investigations of regulatory mechanisms and biological effect of target genes in BMP-2-induced odontoblastic differentiation of hDPCs.

Sensitive and label-free detection of miRNA-145 by triplex formation.

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Aviñó, Anna; Huertas, César S; Lechuga, Laura M; Eritja, Ramon

2016-01-01

The development of new strategies for detecting microRNAs (miRNAs) has become a crucial step in the diagnostic field. miRNA profiles depend greatly on the sample and the analytical platform employed, leading sometimes to contradictory results. In this work, we study the use of modified parallel tail-clamps to detect a miRNA sequence involved in tumor suppression by triplex formation. Thermal denaturing curves and circular dichroism (CD) measurements have been performed to confirm that parallel clamps carrying 8-aminoguanine form the most stable triplex structures with their target miRNA. The modified tail-clamps have been tested as bioreceptors in a surface plasmon resonance (SPR) biosensor for the detection of miRNA-145. The detection limit was improved 2.4 times demonstrating that a stable triplex structure is formed between target miRNA and 8-aminoguanine tail-clamp bioreceptor. This new approach is an essential step toward the label-free and reliable detection of miRNA signatures for diagnostic purposes.

An integrated computational validation approach for potential novel miRNA prediction

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Pooja Viswam

2017-12-01

Full Text Available MicroRNAs (miRNAs are short, non-coding RNAs between 17bp-24bp length that regulate gene expression by targeting mRNA molecules. The regulatory functions of miRNAs are known to be majorly associated with disease phenotypes such as cancer, cell signaling, cell division, growth and other metabolisms. Novel miRNAs are defined as sequences which does not have any similarity with the existing known sequences and void of any experimental evidences. In recent decades, the advent of next-generation sequencing allows us to capture the small RNA molecules form the cells and developing methods to estimate their expression levels. Several computational algorithms are available to predict the novel miRNAs from the deep sequencing data. In this work, we integrated three novel miRNA prediction programs miRDeep, miRanalyzer and miRPRo to compare and validate their prediction efficiency. The dicer cleavage sites, alignment density, seed conservation, minimum free energy, AU-GC percentage, secondary loop scores, false discovery rates and confidence scores will be considered for comparison and evaluation. Efficiency to identify isomiRs and base pair mismatches in a strand specific manner will also be considered for the computational validation. Further, the criteria and parameters for the identification of the best possible novel miRNA with minimal false positive rates were deduced.

The regulatory effect of miRNAs is a heritable genetic trait in humans

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Geeleher Paul

2012-08-01

Full Text Available Abstract Background microRNAs (miRNAs have been shown to regulate the expression of a large number of genes and play key roles in many biological processes. Several previous studies have quantified the inhibitory effect of a miRNA indirectly by considering the expression levels of genes that are predicted to be targeted by the miRNA and this approach has been shown to be robust to the choice of prediction algorithm. Given a gene expression dataset, Cheng et al. defined the regulatory effect score (RE-score of a miRNA as the difference in the gene expression rank of targets of the miRNA compared to non-targeted genes. Results Using microarray data from parent-offspring trios from the International HapMap project, we show that the RE-score of most miRNAs is correlated between parents and offspring and, thus, inter-individual variation in RE-score has a genetic component in humans. Indeed, the mean RE-score across miRNAs is correlated between parents and offspring, suggesting genetic differences in the overall efficiency of the miRNA biogenesis pathway between individuals. To explore the genetics of this quantitative trait further, we carried out a genome-wide association study of the mean RE-score separately in two HapMap populations (CEU and YRI. No genome-wide significant associations were discovered; however, a SNP rs17409624, in an intron of DROSHA, was significantly associated with mean RE-score in the CEU population following permutation-based control for multiple testing based on all SNPs mapped to the canonical miRNA biogenesis pathway; of 244 individual miRNA RE-scores assessed in the CEU, 214 were associated (p p = 0.04 with mean RE-score in the YRI population. Interestingly, the same SNP was associated with 17 (8.5% of all expressed miRNA expression levels in the CEU. We also show here that the expression of the targets of most miRNAs is more highly correlated with global changes in miRNA regulatory effect than with the expression of

In vivo delivery of miRNAs for cancer therapy: Challenges and strategies⋆

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Chen, Yunching; Gao, Dong-Yu; Huang, Leaf

2016-01-01

MicroRNAs (miRNAs), small non-coding RNAs, can regulate post-transcriptional gene expressions and silence a broad set of target genes. miRNAs, aberrantly expressed in cancer cells, play an important role in modulating gene expressions, thereby regulating downstream signaling pathways and affecting cancer formation and progression. Oncogenes or tumor suppressor genes regulated by miRNAs mediate cell cycle progression, metabolism, cell death, angiogenesis, metastasis and immunosuppression in cancer. Recently, miRNAs have emerged as therapeutic targets or tools and biomarkers for diagnosis and therapy monitoring in cancer. Since miRNAs can regulate multiple cancer-related genes simultaneously, using miRNAs as a therapeutic approach plays an important role in cancer therapy. However, one of the major challenges of miRNA-based cancer therapy is to achieve specific, efficient and safe systemic delivery of therapeutic miRNAs In vivo. This review discusses the key challenges to the development of the carriers for miRNA-based therapy and explores current strategies to systemically deliver miRNAs to cancer without induction of toxicity. PMID:24859533

smRNAome profiling to identify conserved and novel microRNAs in Stevia rebaudiana Bertoni

Science.gov (United States)

2012-01-01

Background MicroRNAs (miRNAs) constitute a family of small RNA (sRNA) population that regulates the gene expression and plays an important role in plant development, metabolism, signal transduction and stress response. Extensive studies on miRNAs have been performed in different plants such as Arabidopsis thaliana, Oryza sativa etc. and volume of the miRNA database, mirBASE, has been increasing on day to day basis. Stevia rebaudiana Bertoni is an important perennial herb which accumulates high concentrations of diterpene steviol glycosides which contributes to its high indexed sweetening property with no calorific value. Several studies have been carried out for understanding molecular mechanism involved in biosynthesis of these glycosides, however, information about miRNAs has been lacking in S. rebaudiana. Deep sequencing of small RNAs combined with transcriptomic data is a powerful tool for identifying conserved and novel miRNAs irrespective of availability of genome sequence data. Results To identify miRNAs in S. rebaudiana, sRNA library was constructed and sequenced using Illumina genome analyzer II. A total of 30,472,534 reads representing 2,509,190 distinct sequences were obtained from sRNA library. Based on sequence similarity, we identified 100 miRNAs belonging to 34 highly conserved families. Also, we identified 12 novel miRNAs whose precursors were potentially generated from stevia EST and nucleotide sequences. All novel sequences have not been earlier described in other plant species. Putative target genes were predicted for most conserved and novel miRNAs. The predicted targets are mainly mRNA encoding enzymes regulating essential plant metabolic and signaling pathways. Conclusions This study led to the identification of 34 highly conserved miRNA families and 12 novel potential miRNAs indicating that specific miRNAs exist in stevia species. Our results provided information on stevia miRNAs and their targets building a foundation for future studies to

No miRNA were found in Plasmodium and the ones identified in erythrocytes could not be correlated with infection

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Feng Le

2008-03-01

Full Text Available Abstract Background The transcriptional regulation of Plasmodium during its complex life cycle requires sequential activation and/or repression of different genetic programmes. MicroRNAs (miRNAs are a highly conserved class of non-coding RNAs that are important in regulating diverse cellular functions by sequence-specific inhibition of gene expression. What is know about double-stranded RNA-mediated gene silencing (RNAi and posttranscriptional gene silencing (PTGS in Plasmodium parasites entice us to speculate whether miRNAs can also function in Plasmodium-infected RBCs. Results Of 132 small RNA sequences, no Plasmodium-specific miRNAs have been found. However, a human miRNA, miR-451, was highly expressed, comprising approximately one third of the total identified miRNAs. Further analysis of miR-451 expression and malaria infection showed no association between the accumulation of miR-451 in Plasmodium falciparum-iRBCs, the life cycle stage of P. falciparum in the erythrocyte, or of P. berghei in mice. Moreover, treatment with an antisense oligonucleotide to miR-451 had no significant effect on the growth of the erythrocytic-stage P. falciparum. Methods Short RNAs from a mixed-stage of P. falciparum-iRBC were separated in a denaturing polyacrylamide gel and cloned into T vectors to create a cDNA library. Individual clones were then sequenced and further analysed by bioinformatics prediction to discover probable miRNAs in P. falciparum-iRBC. The association between miR-451 expression and the parasite were analysed by Northern blotting and antisense oligonucleotide (ASO of miR-451. Conclusion These results contribute to eliminate the probability of miRNAs in P. falciparum. The absence of miRNA in P. falciparum could be correlated with absence of argonaute/dicer genes. In addition, the miR-451 accumulation in Plasmodium-infected RBCs is independent of parasite infection. Its accumulation might be only the residual of erythroid differentiation or a

microRNA analysis of Taenia crassiceps cysticerci under praziquantel treatment and genome-wide identification of Taenia solium miRNAs.

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Pérez, Matías Gastón; Macchiaroli, Natalia; Lichtenstein, Gabriel; Conti, Gabriela; Asurmendi, Sebastián; Milone, Diego Humberto; Stegmayer, Georgina; Kamenetzky, Laura; Cucher, Marcela; Rosenzvit, Mara Cecilia

2017-09-01

MicroRNAs (miRNAs) are small non-coding RNAs that have emerged as important regulators of gene expression and perform critical functions in development and disease. In spite of the increased interest in miRNAs from helminth parasites, no information is available on miRNAs from Taenia solium, the causative agent of cysticercosis, a neglected disease affecting millions of people worldwide. Here we performed a comprehensive analysis of miRNAs from Taenia crassiceps, a laboratory model for T. solium studies, and identified miRNAs in the T. solium genome. Moreover, we analysed the effect of praziquantel, one of the two main drugs used for cysticercosis treatment, on the miRNA expression profile of T. crassiceps cysticerci. Using small RNA-seq and two independent algorithms for miRNA prediction, as well as northern blot validation, we found transcriptional evidence of 39 miRNA loci in T. crassiceps. Since miRNAs were mapped to the T. solium genome, these miRNAs are considered common to both parasites. The miRNA expression profile of T. crassiceps was biased to the same set of highly expressed miRNAs reported in other cestodes. We found a significant altered expression of miR-7b under praziquantel treatment. In addition, we searched for miRNAs predicted to target genes related to drug response. We performed a detailed target prediction for miR-7b and found genes related to drug action. We report an initial approach to study the effect of sub-lethal drug treatment on miRNA expression in a cestode parasite, which provides a platform for further studies of miRNA involvement in drug effects. The results of our work could be applied to drug development and provide basic knowledge of cysticercosis and other neglected helminth infections. Copyright © 2017 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

Identification of targets of miR-200b by a SILAC-based quantitative proteomic approach

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Arivusudar Marimuthu

2014-09-01

Full Text Available miRNAs regulate gene expression by binding to cognate mRNAs causing mRNA degradation or translational repression. Mass spectrometry-based proteomic analysis is being widely used to identify miRNA targets. The miR-200b miRNA cluster is often overexpressed in multiple cancer types, but the identity of the targets remains elusive. Using SILAC-based analysis, we examined the effects of overexpression of a miR-200b mimic or a control miRNA in fibrosarcoma cells. We identified around 300 potential targets of miR-200b based on a change in the expression of protein levels. We validated a subset of potential targets at the transcript level using quantitative PCR.

High throughput deep degradome sequencing reveals microRNAs and their targets in response to drought stress in mulberry (Morus alba).

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Li, Ruixue; Chen, Dandan; Wang, Taichu; Wan, Yizhen; Li, Rongfang; Fang, Rongjun; Wang, Yuting; Hu, Fei; Zhou, Hong; Li, Long; Zhao, Weiguo

2017-01-01

MicroRNAs (miRNAs) play important regulatory roles by targeting mRNAs for cleavage or translational repression. Identification of miRNA targets is essential to better understanding the roles of miRNAs. miRNA targets have not been well characterized in mulberry (Morus alba). To anatomize miRNA guided gene regulation under drought stress, transcriptome-wide high throughput degradome sequencing was used in this study to directly detect drought stress responsive miRNA targets in mulberry. A drought library (DL) and a contrast library (CL) were constructed to capture the cleaved mRNAs for sequencing. In CL, 409 target genes of 30 conserved miRNA families and 990 target genes of 199 novel miRNAs were identified. In DL, 373 target genes of 30 conserved miRNA families and 950 target genes of 195 novel miRNAs were identified. Of the conserved miRNA families in DL, mno-miR156, mno-miR172, and mno-miR396 had the highest number of targets with 54, 52 and 41 transcripts, respectively, indicating that these three miRNA families and their target genes might play important functions in response to drought stress in mulberry. Additionally, we found that many of the target genes were transcription factors. By analyzing the miRNA-target molecular network, we found that the DL independent networks consisted of 838 miRNA-mRNA pairs (63.34%). The expression patterns of 11 target genes and 12 correspondent miRNAs were detected using qRT-PCR. Six miRNA targets were further verified by RNA ligase-mediated 5' rapid amplification of cDNA ends (RLM-5' RACE). Gene Ontology (GO) annotations and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that these target transcripts were implicated in a broad range of biological processes and various metabolic pathways. This is the first study to comprehensively characterize target genes and their associated miRNAs in response to drought stress by degradome sequencing in mulberry. This study provides a framework for understanding

MiRNA Profiles in Lymphoblastoid Cell Lines of Finnish Prostate Cancer Families.

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Daniel Fischer

Full Text Available Heritable factors are evidently involved in prostate cancer (PrCa carcinogenesis, but currently, genetic markers are not routinely used in screening or diagnostics of the disease. More precise information is needed for making treatment decisions to distinguish aggressive cases from indolent disease, for which heritable factors could be a useful tool. The genetic makeup of PrCa has only recently begun to be unravelled through large-scale genome-wide association studies (GWAS. The thus far identified Single Nucleotide Polymorphisms (SNPs explain, however, only a fraction of familial clustering. Moreover, the known risk SNPs are not associated with the clinical outcome of the disease, such as aggressive or metastasised disease, and therefore cannot be used to predict the prognosis. Annotating the SNPs with deep clinical data together with miRNA expression profiles can improve the understanding of the underlying mechanisms of different phenotypes of prostate cancer.In this study microRNA (miRNA profiles were studied as potential biomarkers to predict the disease outcome. The study subjects were from Finnish high risk prostate cancer families. To identify potential biomarkers we combined a novel non-parametrical test with an importance measure provided from a Random Forest classifier. This combination delivered a set of nine miRNAs that was able to separate cases from controls. The detected miRNA expression profiles could predict the development of the disease years before the actual PrCa diagnosis or detect the existence of other cancers in the studied individuals. Furthermore, using an expression Quantitative Trait Loci (eQTL analysis, regulatory SNPs for miRNA miR-483-3p that were also directly associated with PrCa were found.Based on our findings, we suggest that blood-based miRNA expression profiling can be used in the diagnosis and maybe even prognosis of the disease. In the future, miRNA profiling could possibly be used in targeted screening

Clinico-Pathological Association of Delineated miRNAs in Uveal Melanoma with Monosomy 3/Disomy 3 Chromosomal Aberrations.

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Nalini Venkatesan

Full Text Available To correlate the differentially expressed miRNAs with clinico-pathological features in uveal melanoma (UM tumors harbouring chromosomal 3 aberrations among South Asian Indian cohort.Based on chromosomal 3 aberration, UM (n = 86 were grouped into monosomy 3 (M3; n = 51 and disomy 3 (D3; n = 35 by chromogenic in-situ hybridisation (CISH. The clinico-pathological features were recorded. miRNA profiling was performed in formalin fixed paraffin embedded (FFPE UM samples (n = 6 using Agilent, Human miRNA microarray, 8x15KV3 arrays. The association between miRNAs and clinico-pathological features were studied using univariate and multivariate analysis. miRNA-gene targets were predicted using Target-scan and MiRanda database. Significantly dys-regulated miRNAs were validated in FFPE UM (n = 86 and mRNAs were validated in frozen UM (n = 10 by qRT-PCR. Metastasis free-survival and miRNA expressions were analysed by Kaplen-Meier analysis in UM tissues (n = 52.Unsupervised analysis revealed 585 differentially expressed miRNAs while supervised analysis demonstrated 82 miRNAs (FDR; Q = 0.0. Differential expression of 8 miRNAs: miR-214, miR-149*, miR-143, miR-146b, miR-199a, let7b, miR-1238 and miR-134 were studied. Gene target prediction revealed SMAD4, WISP1, HIPK1, HDAC8 and C-KIT as the post-transcriptional regulators of miR-146b, miR-199a, miR-1238 and miR-134. Five miRNAs (miR-214, miR146b, miR-143, miR-199a and miR-134 were found to be differentially expressed in M3/ D3 UM tumors. In UM patients with liver metastasis, miR-149* and miR-134 expressions were strongly correlated.UM can be stratified using miRNAs from FFPE sections. miRNAs predicting liver metastasis and survival have been identified. Mechanistic linkage of de-regulated miRNA/mRNA expressions provide new insights on their role in UM progression and aggressiveness.

Role of Viral miRNAs and Epigenetic Modifications in Epstein-Barr Virus-Associated Gastric Carcinogenesis.

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Giudice, Aldo; D'Arena, Giovanni; Crispo, Anna; Tecce, Mario Felice; Nocerino, Flavia; Grimaldi, Maria; Rotondo, Emanuela; D'Ursi, Anna Maria; Scrima, Mario; Galdiero, Massimiliano; Ciliberto, Gennaro; Capunzo, Mario; Franci, Gianluigi; Barbieri, Antonio; Bimonte, Sabrina; Montella, Maurizio

2016-01-01

MicroRNAs are short (21-23 nucleotides), noncoding RNAs that typically silence posttranscriptional gene expression through interaction with target messenger RNAs. Currently, miRNAs have been identified in almost all studied multicellular eukaryotes in the plant and animal kingdoms. Additionally, recent studies reported that miRNAs can also be encoded by certain single-cell eukaryotes and by viruses. The vast majority of viral miRNAs are encoded by the herpesviruses family. These DNA viruses including Epstein-Barr virus encode their own miRNAs and/or manipulate the expression of cellular miRNAs to facilitate respective infection cycles. Modulation of the control pathways of miRNAs expression is often involved in the promotion of tumorigenesis through a specific cascade of transduction signals. Notably, latent infection with Epstein-Barr virus is considered liable of causing several types of malignancies, including the majority of gastric carcinoma cases detected worldwide. In this review, we describe the role of the Epstein-Barr virus in gastric carcinogenesis, summarizing the functions of the Epstein-Barr virus-encoded viral proteins and related epigenetic alterations as well as the roles of Epstein-Barr virus-encoded and virally modulated cellular miRNAs.

Genetic variation in IL-16 miRNA target site and time to prostate cancer diagnosis in African American men

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Hughes, Lucinda; Ruth, Karen; Rebbeck, Timothy R.; Giri, Veda N.

2013-01-01

Background Men with a family history of prostate cancer and African American men are at high risk for prostate cancer and in need of personalized risk estimates to inform screening decisions. This study evaluated genetic variants in genes encoding microRNA (miRNA) binding sites for informing of time to prostate cancer diagnosis among ethnically-diverse, high-risk men undergoing prostate cancer screening. Methods The Prostate Cancer Risk Assessment Program (PRAP) is a longitudinal screening program for high-risk men. Eligibility includes men ages 35-69 with a family history of prostate cancer or African descent. Participants with ≥ 1 follow-up visit were included in the analyses (n=477). Genetic variants in regions encoding miRNA binding sites in four target genes (ALOX15, IL-16, IL-18, and RAF1) previously implicated in prostate cancer development were evaluated. Genotyping methods included Taqman® SNP Genotyping Assay (Applied Biosystems) or pyrosequencing. Cox models were used to assess time to prostate cancer diagnosis by risk genotype. Results Among 256 African Americans with ≥ one follow-up visit, the TT genotype at rs1131445 in IL-16 was significantly associated with earlier time to prostate cancer diagnosis vs. the CC/CT genotypes (p=0.013), with a suggestive association after correction for false-discovery (p=0.065). Hazard ratio after controlling for age and PSA for TT vs. CC/CT among African Americans was 3.0 (95% CI 1.26-7.12). No association to time to diagnosis was detected among Caucasians by IL-16 genotype. No association to time to prostate cancer diagnosis was found for the other miRNA target genotypes. Conclusions Genetic variation in IL-16 encoding miRNA target site may be informative of time to prostate cancer diagnosis among African American men enrolled in prostate cancer risk assessment, which may inform individualized prostate cancer screening strategies in the future. PMID:24061634

SKI2 mediates degradation of RISC 5'-cleavage fragments and prevents secondary siRNA production from miRNA targets in Arabidopsis.

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Branscheid, Anja; Marchais, Antonin; Schott, Gregory; Lange, Heike; Gagliardi, Dominique; Andersen, Stig Uggerhøj; Voinnet, Olivier; Brodersen, Peter

2015-12-15

Small regulatory RNAs are fundamental in eukaryotic and prokaryotic gene regulation. In plants, an important element of post-transcriptional control is effected by 20-24 nt microRNAs (miRNAs) and short interfering RNAs (siRNAs) bound to the ARGONAUTE1 (AGO1) protein in an RNA induced silencing complex (RISC). AGO1 may cleave target mRNAs with small RNA complementarity, but the fate of the resulting cleavage fragments remains incompletely understood. Here, we show that SKI2, SKI3 and SKI8, subunits of a cytoplasmic cofactor of the RNA exosome, are required for degradation of RISC 5', but not 3'-cleavage fragments in Arabidopsis. In the absence of SKI2 activity, many miRNA targets produce siRNAs via the RNA-dependent RNA polymerase 6 (RDR6) pathway. These siRNAs are low-abundant, and map close to the cleavage site. In most cases, siRNAs were produced 5' to the cleavage site, but several examples of 3'-spreading were also identified. These observations suggest that siRNAs do not simply derive from RDR6 action on stable 5'-cleavage fragments and hence that SKI2 has a direct role in limiting secondary siRNA production in addition to its function in mediating degradation of 5'-cleavage fragments. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Simultaneous visualization of the subfemtomolar expression of microRNA and microRNA target gene using HILO microscopy† †Electronic supplementary information (ESI) available: The LED device for the sample photobleaching, a schematic presentation of HILO microscopy, fluorescence spectra and hybridization curves of the molecular beacons, the linear correlation between the miRNA fluorescence intensity and the miRNA copy number, a validation of the miRNA adsorption and miRNA target gene expression via RT-qPCR, a validation of RT-qPCR using capillary electrophoresis, the reproducibility of RT-qPCR and Poisson distribution of the miRNA pipetting as well as a complete list of the oligonucleotides used in this study. See DOI: 10.1039/c7sc02701j Click here for additional data file.

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Lin, Yi-Zhen; Ou, Da-Liang; Chang, Hsin-Yuan; Lin, Wei-Yu; Hsu, Chiun

2017-01-01

The family of microRNAs (miRNAs) not only plays an important role in gene regulation but is also useful for the diagnosis of diseases. A reliable method with high sensitivity may allow researchers to detect slight fluctuations in ultra-trace amounts of miRNA. In this study, we propose a sensitive imaging method for the direct probing of miR-10b (miR-10b-3p, also called miR-10b*) and its target (HOXD10 mRNA) in fixed cells based on the specific recognition of molecular beacons combined with highly inclined and laminated optical sheet (HILO) fluorescence microscopy. The designed dye-quencher-labelled molecular beacons offer excellent efficiencies of fluorescence resonance energy transfer that allow us to detect miRNA and the target mRNA simultaneously in hepatocellular carcinoma cells using HILO fluorescence microscopy. Not only can the basal trace amount of miRNA be observed in each individual cell, but the obtained images also indicate that this method is useful for monitoring the fluctuations in ultra-trace amounts of miRNA when the cells are transfected with a miRNA precursor or a miRNA inhibitor (anti-miR). Furthermore, a reasonable causal relation between the miR-10b and HOXD10 expression levels was observed in miR-10b* precursor-transfected cells and miR-10b* inhibitor-transfected cells. The trends of the miRNA alterations obtained using HILO microscopy completely matched the RT-qPCR data and showed remarkable reproducibility (the coefficient of variation [CV] = 0.86%) and sensitivity (<1.0 fM). This proposed imaging method appears to be useful for the simultaneous visualisation of ultra-trace amounts of miRNA and target mRNA and excludes the procedures for RNA extraction and amplification. Therefore, the visualisation of miRNA and the target mRNA should facilitate the exploration of the functions of ultra-trace amounts of miRNA in fixed cells in biological studies and may serve as a powerful tool for diagnoses based on circulating cancer cells. PMID:28989695

Identification of miRNAs Responsive to Botrytis cinerea in Herbaceous Peony (Paeonia lactiflora Pall. by High-Throughput Sequencing

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Daqiu Zhao

2015-09-01

Full Text Available Herbaceous peony (Paeonia lactiflora Pall., one of the world’s most important ornamental plants, is highly susceptible to Botrytis cinerea, and improving resistance to this pathogenic fungus is a problem yet to be solved. MicroRNAs (miRNAs play an essential role in resistance to B. cinerea, but until now, no studies have been reported concerning miRNAs induction in P. lactiflora. Here, we constructed and sequenced two small RNA (sRNA libraries from two B. cinerea-infected P. lactiflora cultivars (“Zifengyu” and “Dafugui” with significantly different levels of resistance to B. cinerea, using the Illumina HiSeq 2000 platform. From the raw reads generated, 4,592,881 and 5,809,796 sRNAs were obtained, and 280 and 306 miRNAs were identified from “Zifengyu” and “Dafugui”, respectively. A total of 237 conserved and 7 novel sequences of miRNAs were differentially expressed between the two cultivars, and we predicted and annotated their potential target genes. Subsequently, 7 differentially expressed candidate miRNAs were screened according to their target genes annotated in KEGG pathways, and the expression patterns of miRNAs and corresponding target genes were elucidated. We found that miR5254, miR165a-3p, miR3897-3p and miR6450a might be involved in the P. lactiflora response to B. cinerea infection. These results provide insight into the molecular mechanisms responsible for resistance to B. cinerea in P. lactiflora.

EGF receptor targeted lipo-oligocation polyplexes for antitumoral siRNA and miRNA delivery

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Müller, Katharina; Klein, Philipp M.; Heissig, Philipp; Roidl, Andreas; Wagner, Ernst

2016-11-01

Antitumoral siRNA and miRNA delivery was demonstrated by epidermal growth factor receptor (EGFR) targeted oligoaminoamide polyplexes. For this purpose, the T-shaped lipo-oligomer 454 was used to complex RNA into a core polyplex, which was subsequently functionalized with the targeting peptide ligand GE11 via a polyethylene glycol (PEG) linker. To this end, free cysteines on the surface of 454 polyplex were coupled with a maleimide-PEG-GE11 reagent (Mal-GE11). Resulting particles with sizes of 120-150 nm showed receptor-mediated uptake into EGFR-positive T24 bladder cancer cells, MDA-MB 231 breast cancer cells and Huh7 liver cancer cells. Furthermore, these formulations led to ligand-dependent gene silencing. RNA interference (RNAi) triggered antitumoral effects were observed for two different therapeutic RNAs, a miRNA-200c mimic or EG5 siRNA. Using polyplexes modified with a ratio of 0.8 molar equivalents of Mal-GE11, treatment of T24 or MDA-MB 231 cancer cells with miR-200c led to the expected decreased proliferation and migration, changes in cell cycle and enhanced sensitivity towards doxorubicin. Delivery of EG5 siRNA into Huh7 cells resulted in antitumoral activity with G2/M arrest, triggered by loss of mitotic spindle separation and formation of mono-astral spindles. These findings demonstrate the potential of GE11 ligand-containing RNAi polyplexes for cancer treatment.

Microarray-based approach identifies microRNAs and their target functional patterns in polycystic kidney disease

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Boehn Susanne NE

2008-12-01

Full Text Available Abstract Background MicroRNAs (miRNAs play key roles in mammalian gene expression and several cellular processes, including differentiation, development, apoptosis and cancer pathomechanisms. Recently the biological importance of primary cilia has been recognized in a number of human genetic diseases. Numerous disorders are related to cilia dysfunction, including polycystic kidney disease (PKD. Although involvement of certain genes and transcriptional networks in PKD development has been shown, not much is known how they are regulated molecularly. Results Given the emerging role of miRNAs in gene expression, we explored the possibilities of miRNA-based regulations in PKD. Here, we analyzed the simultaneous expression changes of miRNAs and mRNAs by microarrays. 935 genes, classified into 24 functional categories, were differentially regulated between PKD and control animals. In parallel, 30 miRNAs were differentially regulated in PKD rats: our results suggest that several miRNAs might be involved in regulating genetic switches in PKD. Furthermore, we describe some newly detected miRNAs, miR-31 and miR-217, in the kidney which have not been reported previously. We determine functionally related gene sets, or pathways to reveal the functional correlation between differentially expressed mRNAs and miRNAs. Conclusion We find that the functional patterns of predicted miRNA targets and differentially expressed mRNAs are similar. Our results suggest an important role of miRNAs in specific pathways underlying PKD.

MIRNA-DISTILLER: a stand-alone application to compile microRNA data from databases

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Jessica K. Rieger

2011-07-01

Full Text Available MicroRNAs (miRNA are small non-coding RNA molecules of ~22 nucleotides which regulate large numbers of genes by binding to seed sequences at the 3’-UTR of target gene transcripts. The target mRNA is then usually degraded or translation is inhibited, although thus resulting in posttranscriptional down regulation of gene expression at the mRNA and/or protein level. Due to the bioinformatic difficulties in predicting functional miRNA binding sites, several publically available databases have been developed that predict miRNA binding sites based on different algorithms. The parallel use of different databases is currently indispensable, but highly uncomfortable and time consuming, especially when working with numerous genes of interest. We have therefore developed a new stand-alone program, termed MIRNA-DISTILLER, which allows to compile miRNA data for given target genes from public databases. Currently implemented are TargetScan, microCosm, and miRDB, which may be queried independently, pairwise, or together to calculate the respective intersections. Data are stored locally for application of further analysis tools including freely definable biological parameter filters, customized output-lists for both miRNAs and target genes, and various graphical facilities. The software, a data example file and a tutorial are freely available at http://www.ikp-stuttgart.de/content/language1/html/10415.asp

MIRNA-DISTILLER: A Stand-Alone Application to Compile microRNA Data from Databases.

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Rieger, Jessica K; Bodan, Denis A; Zanger, Ulrich M

2011-01-01

MicroRNAs (miRNA) are small non-coding RNA molecules of ∼22 nucleotides which regulate large numbers of genes by binding to seed sequences at the 3'-untranslated region of target gene transcripts. The target mRNA is then usually degraded or translation is inhibited, although thus resulting in posttranscriptional down regulation of gene expression at the mRNA and/or protein level. Due to the bioinformatic difficulties in predicting functional miRNA binding sites, several publically available databases have been developed that predict miRNA binding sites based on different algorithms. The parallel use of different databases is currently indispensable, but highly uncomfortable and time consuming, especially when working with numerous genes of interest. We have therefore developed a new stand-alone program, termed MIRNA-DISTILLER, which allows to compile miRNA data for given target genes from public databases. Currently implemented are TargetScan, microCosm, and miRDB, which may be queried independently, pairwise, or together to calculate the respective intersections. Data are stored locally for application of further analysis tools including freely definable biological parameter filters, customized output-lists for both miRNAs and target genes, and various graphical facilities. The software, a data example file and a tutorial are freely available at http://www.ikp-stuttgart.de/content/language1/html/10415.asp.

Genome-wide identification and functional annotation of miRNAs in anti-inflammatory plant and their cross-kingdom regulation in Homo sapiens.

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Sharma, Ankita; Sahu, Sarika; Kumari, Pooja; Gopi, Soundhara Rajan; Malhotra, Rajesh; Biswas, Sagarika

2017-05-01

MicroRNAs (miRNAs) are newly discovered non-coding small (~17-24 nucleotide) RNAs that regulate gene expression of its target mRNA at the post-transcriptional levels. In this study, total 12,593 ESTs of Curcuma longa were taken from database of expressed sequence tags (dbEST) and clustered into 2821 contigs using EGassembler web server. Precursor miRNAs (pre-miRNAs) were predicted from these contigs that folded into stem-loop structure using MFold server. Thirty-four mature C. longa miRNAs (clo-miRNAs) were identified from pre-miRNAs having targets involved in various important functions of plant such as self-defence, growth and development, alkaloid metabolic pathway and ethylene signalling process. Sequence analysis of identified clo-miRNAs indicated that 56% miRNAs belong to ORF and 44% belong to non-ORF region. clo-mir-5 and clo-mir-6 were established as the conserved miRNAs, whereas clo-mir-20 was predicted to be the most stable miRNA. Phylogenetic analysis carried out by molecular evolutionary genetics analysis (MEGA) software indicated close evolutionary relationship of clo-mir-5075 with osa-MIR5075. Further, identified clo-miRNAs were checked for their cross-kingdom regulatory potential. clo-mir-14 was found to regulate various gene transcripts in humans that has been further investigated for its biostability in foetal bovine serum (FBS). The results indicated higher degree of stability of clo-mir-14 (48 h) in FBS. Thus, contribution of this miRNA to the cellular immune response during the inflamed condition of rheumatoid arthritis and adequate stability may make it a good choice for the therapeutic agent in near future.

SETD1A modulates cell cycle progression through a miRNA network that regulates p53 target genes

OpenAIRE

Tajima, Ken; Yae, Toshifumi; Javaid, Sarah; Tam, Oliver; Comaills, Valentine; Morris, Robert; Wittner, Ben S.; Liu, Mingzhu; Engstrom, Amanda; Takahashi, Fumiyuki; Black, Joshua C.; Ramaswamy, Sridhar; Shioda, Toshihiro; Hammell, Molly; Haber, Daniel A.

2015-01-01

Expression of the p53-inducible antiproliferative gene BTG2 is suppressed in many cancers in the absence of inactivating gene mutations, suggesting alternative mechanisms of silencing. Using a shRNA screen targeting 43 histone lysine methyltransferases (KMTs), we show that SETD1A suppresses BTG2 expression through its induction of several BTG2-targeting miRNAs. This indirect but highly specific mechanism, by which a chromatin regulator that mediates transcriptional activating marks can lead t...

miRNAs in inflammatory skin diseases and their clinical implications

DEFF Research Database (Denmark)

Løvendorf, Marianne B; Skov, Lone

2015-01-01

biological processes. The clinical implications of miRNAs are intriguing, both from a diagnostic and a therapeutic perspective. Accordingly, there is emerging evidence for the clinical potential of miRNAs as both biomarkers and possible therapeutic targets in skin diseases. Future studies will hopefully...... incomplete; however, it is known that miRNAs are implicated in various cellular processes of both normal and diseased skin. Some miRNAs appear to be consistently deregulated in several different inflammatory skin diseases, including psoriasis and atopic dermatitis, indicating a common role in fundamental...

Small RNA analysis in Petunia hybrida identifies unusual tissue-specific expression patterns of conserved miRNAs and of a 24mer RNA

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Tedder, Philip; Zubko, Elena; Westhead, David R.; Meyer, Peter

2009-01-01

Two pools of small RNAs were cloned from inflorescences of Petunia hybrida using a 5′-ligation dependent and a 5′-ligation independent approach. The two libraries were integrated into a public website that allows the screening of individual sequences against 359,769 unique clones. The library contains 15 clones with 100% identity and 53 clones with one mismatch to miRNAs described for other plant species. For two conserved miRNAs, miR159 and miR390, we find clear differences in tissue-specific distribution, compared with other species. This shows that evolutionary conservation of miRNA sequences does not necessarily include a conservation of the miRNA expression profile. Almost 60% of all clones in the database are 24-nucleotide clones. In accordance with the role of 24mers in marking repetitive regions, we find them distributed across retroviral and transposable element sequences but other 24mers map to promoter regions and to different transcript regions. For one target region we observe tissue-specific variation of matching 24mers, which demonstrates that, as for 21mers, 24mer concentrations are not necessarily identical in different tissues. Asymmetric distribution of a putative novel miRNA in the two libraries suggests that the cloning method can be selective for the representation of certain small RNAs in a collection. PMID:19369427

Identification of a robust subpathway-based signature for acute myeloid leukemia prognosis using an miRNA integrated strategy.

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Chang, Huijuan; Gao, Qiuying; Ding, Wei; Qing, Xueqin

2018-01-01

Acute myeloid leukemia (AML) is a heterogeneous disease, and survival signatures are urgently needed to better monitor treatment. MiRNAs displayed vital regulatory roles on target genes, which was necessary involved in the complex disease. We therefore examined the expression levels of miRNAs and genes to identify robust signatures for survival benefit analyses. First, we reconstructed subpathway graphs by embedding miRNA components that were derived from low-throughput miRNA-gene interactions. Then, we randomly divided the data sets from The Cancer Genome Atlas (TCGA) into training and testing sets, and further formed 100 subsets based on the training set. Using each subset, we identified survival-related miRNAs and genes, and identified survival subpathways based on the reconstructed subpathway graphs. After statistical analyses of these survival subpathways, the most robust subpathways with the top three ranks were identified, and risk scores were calculated based on these robust subpathways for AML patient prognoses. Among these robust subpathways, three representative subpathways, path: 05200_10 from Pathways in cancer, path: 04110_20 from Cell cycle, and path: 04510_8 from Focal adhesion, were significantly associated with patient survival in the TCGA training and testing sets based on subpathway risk scores. In conclusion, we performed integrated analyses of miRNAs and genes to identify robust prognostic subpathways, and calculated subpathway risk scores to characterize AML patient survival.

Maternal chromium restriction modulates miRNA profiles related to lipid metabolism disorder in mice offspring.

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Zhang, Qian; Xiao, Xinhua; Zheng, Jia; Li, Ming; Yu, Miao; Ping, Fan; Wang, Zhixin; Qi, Cuijuan; Wang, Tong; Wang, Xiaojing

2017-08-01

Increasing evidence shows that maternal nutrition status has a vital effect on offspring susceptibility to obesity. MicroRNAs are related to lipid metabolism processes. This study aimed to evaluate whether maternal chromium restriction could affect miRNA expression involved in lipid metabolism in offspring. Weaning C57BL/6J mice born from mothers fed with normal control diet or chromium-restricted diet were fed for 13 weeks. The adipose miRNA expression profile was analyzed by miRNA array analysis. At 16 weeks old, pups from dams fed with chromium-restricted diet exhibit higher body weight, fat weight, and serum TC, TG levels. Six miRNAs were identified as upregulated in the RC group compared with the CC group, whereas eight miRNAs were lower than the threshold level set in the RC group. In the validated target genes of these differentially expressed miRNA, the MAPK signaling pathway serves an important role in the influence of early life chromium-restricted diet on lipid metabolism through miRNA. Long-term programming on various specific miRNA and MAPK signaling pathway may be involved in maternal chromium restriction in the adipose of female offspring. Impact statement For the first time, our study demonstrates important miRNA differences in the effect of maternal chromium restriction in offspring. These miRNAs may serve as "bridges" between the mother and the offspring by affecting the MAPK pathway.

miRNA delivery for skin wound healing.

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Meng, Zhao; Zhou, Dezhong; Gao, Yongsheng; Zeng, Ming; Wang, Wenxin

2017-12-19

The wound healing has remained a worldwide challenge as one of significant public health problems. Pathological scars and chronic wounds caused by injury, aging or diabetes lead to impaired tissue repair and regeneration. Due to the unique biological wound environment, the wound healing is a highly complicated process, efficient and targeted treatments are still lacking. Hence, research-driven to discover more efficient therapeutics is a highly urgent demand. Recently, the research results have revealed that microRNA (miRNA) is a promising tool in therapeutic and diagnostic fields because miRNA is an essential regulator in cellular physiology and pathology. Therefore, new technologies for wound healing based on miRNA have been developed and miRNA delivery has become a significant research topic in the field of gene delivery. Copyright © 2017. Published by Elsevier B.V.

MicroRNA-221 and -222 Regulate Radiation Sensitivity by Targeting the PTEN Pathway

International Nuclear Information System (INIS)

Zhang Chunzhi; Kang Chunsheng; Wang Ping; Cao Yongzhen; Lv Zhonghong; Yu Shizhu; Wang Guangxiu; Zhang Anling; Jia Zhifan; Han Lei; Yang Chunying; Ishiyama, Hiromichi; Teh, Bin S.; Xu Bo; Pu Peiyu

2011-01-01

Purpose: MicroRNAs (miRNAs) are noncoding RNAs inhibiting expression of numerous target genes by posttranscriptional regulation. miRNA-221 and miRNA-222 (miRNA-221/-222) expression is elevated in radioresistant tumor cell lines; however, it is not known whether and how miRNAs control cellular responses to ionizing irradiation. Methods and Materials: We used bioinformatic analyses, luciferase reporter assay, and genetic knockdown and biochemical assays to characterize the regulation pathways of miRNA-221/-222 in response to radiation treatment. Results: We identified the PTEN gene as a target of miRNA-221/-222. Furthermore, we found that knocking down miRNA-221/-222 by antisense oligonucleotides upregulated PTEN expression. Upregulated PTEN expression suppressed AKT activity and increased radiation-induced apoptosis, resulting in enhancement of radiosensitivity in tumor cells. Conclusions: miRNA-221/-222 control radiation sensitivity by regulating the PTEN/AKT pathway and can be explored as novel targets for radiosensitization.

CID-miRNA: A web server for prediction of novel miRNA precursors in human genome

International Nuclear Information System (INIS)

Tyagi, Sonika; Vaz, Candida; Gupta, Vipin; Bhatia, Rohit; Maheshwari, Sachin; Srinivasan, Ashwin; Bhattacharya, Alok

2008-01-01

microRNAs (miRNA) are a class of non-protein coding functional RNAs that are thought to regulate expression of target genes by direct interaction with mRNAs. miRNAs have been identified through both experimental and computational methods in a variety of eukaryotic organisms. Though these approaches have been partially successful, there is a need to develop more tools for detection of these RNAs as they are also thought to be present in abundance in many genomes. In this report we describe a tool and a web server, named CID-miRNA, for identification of miRNA precursors in a given DNA sequence, utilising secondary structure-based filtering systems and an algorithm based on stochastic context free grammar trained on human miRNAs. CID-miRNA analyses a given sequence using a web interface, for presence of putative miRNA precursors and the generated output lists all the potential regions that can form miRNA-like structures. It can also scan large genomic sequences for the presence of potential miRNA precursors in its stand-alone form. The web server can be accessed at (http://mirna.jnu.ac.in/cidmirna/)

Molecular mechanisms and theranostic potential of miRNAs in drug resistance of gastric cancer.

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Yang, Wanli; Ma, Jiaojiao; Zhou, Wei; Cao, Bo; Zhou, Xin; Yang, Zhiping; Zhang, Hongwei; Zhao, Qingchuan; Fan, Daiming; Hong, Liu

2017-11-01

Systemic chemotherapy is a curative approach to inhibit gastric cancer cells proliferation. Despite the great progress in anti-cancer treatment achieved during the last decades, drug resistance and treatment refractoriness still extensively persists. Recently, accumulating studies have highlighted the role of miRNAs in drug resistance of gastric cancers by modulating some drug resistance-related proteins and genes expression. Pre-clinical reports indicate that miRNAs might serve as ideal biomarkers and potential targets, thus holding great promise for developing targeted therapy and personalized treatment for the patients with gastric cancer. Areas covered: This review provide a comprehensive overview of the current advances of miRNAs and molecular mechanisms underlying miRNA-mediated drug resistance in gastric cancer. We particularly focus on the potential values of drug resistance-related miRNAs as biomarkers and novel targets in gastric cancer therapy and envisage the future research developments of these miRNAs and challenges in translating the new findings into clinical applications. Expert opinion: Although the concrete mechanisms of miRNAs in drug resistance of gastric cancer have not been fully clarified, miRNA may be a promising theranostic approach. Further studies are still needed to facilitate the clinical applications of miRNAs in drug resistant gastric cancer.

Identification and analysis of differential miRNAs in PK-15 cells after foot-and-mouth disease virus infection.

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Ke-Shan Zhang

Full Text Available The alterations of MicroRNAs(miRNAs in host cell after foot-and-mouth disease virus (FMDV infection is still obscure. To increase our understanding of the pathogenesis of FMDV at the post-transcriptional regulation level, Solexa high-throu MicroRNAs (miRNAs play an important role both in the post-transcriptional regulation of gene expression and host-virus interactions. Despite investigations of miRNA expression ghput sequencing and bioinformatic tools were used to identify differentially expressed miRNAs and analyze their functions during FMDV infection of PK-15 cells. Results indicated that 9,165,674 and 9,230,378 clean reads were obtained, with 172 known and 72 novel miRNAs differently expressed in infected and uninfected groups respectively. Some of differently expressed miRNAs were validated using stem-loop real-time quantitative RT-PCR. The GO annotation and KEGG pathway analysis for target genes revealed that differently expressed miRNAs were involved in immune response and cell death pathways.

Elucidating Mechanisms of Molecular Recognition Between Human Argonaute and miRNA Using Computational Approaches

KAUST Repository

Jiang, Hanlun

2016-12-06

MicroRNA (miRNA) and Argonaute (AGO) protein together form the RNA-induced silencing complex (RISC) that plays an essential role in the regulation of gene expression. Elucidating the underlying mechanism of AGO-miRNA recognition is thus of great importance not only for the in-depth understanding of miRNA function but also for inspiring new drugs targeting miRNAs. In this chapter we introduce a combined computational approach of molecular dynamics (MD) simulations, Markov state models (MSMs), and protein-RNA docking to investigate AGO-miRNA recognition. Constructed from MD simulations, MSMs can elucidate the conformational dynamics of AGO at biologically relevant timescales. Protein-RNA docking can then efficiently identify the AGO conformations that are geometrically accessible to miRNA. Using our recent work on human AGO2 as an example, we explain the rationale and the workflow of our method in details. This combined approach holds great promise to complement experiments in unraveling the mechanisms of molecular recognition between large, flexible, and complex biomolecules.

Elucidating Mechanisms of Molecular Recognition Between Human Argonaute and miRNA Using Computational Approaches.

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Jiang, Hanlun; Zhu, Lizhe; Héliou, Amélie; Gao, Xin; Bernauer, Julie; Huang, Xuhui

2017-01-01

MicroRNA (miRNA) and Argonaute (AGO) protein together form the RNA-induced silencing complex (RISC) that plays an essential role in the regulation of gene expression. Elucidating the underlying mechanism of AGO-miRNA recognition is thus of great importance not only for the in-depth understanding of miRNA function but also for inspiring new drugs targeting miRNAs. In this chapter we introduce a combined computational approach of molecular dynamics (MD) simulations, Markov state models (MSMs), and protein-RNA docking to investigate AGO-miRNA recognition. Constructed from MD simulations, MSMs can elucidate the conformational dynamics of AGO at biologically relevant timescales. Protein-RNA docking can then efficiently identify the AGO conformations that are geometrically accessible to miRNA. Using our recent work on human AGO2 as an example, we explain the rationale and the workflow of our method in details. This combined approach holds great promise to complement experiments in unraveling the mechanisms of molecular recognition between large, flexible, and complex biomolecules.

SKI2 mediates degradation of RISC 5′-cleavage fragments and prevents secondary siRNA production from miRNA targets in Arabidopsis

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Branscheid, Anja; Marchais, Antonin; Schott, Gregory; Lange, Heike; Gagliardi, Dominique; Andersen, Stig Uggerhøj; Voinnet, Olivier; Brodersen, Peter

2015-01-01

Small regulatory RNAs are fundamental in eukaryotic and prokaryotic gene regulation. In plants, an important element of post-transcriptional control is effected by 20–24 nt microRNAs (miRNAs) and short interfering RNAs (siRNAs) bound to the ARGONAUTE1 (AGO1) protein in an RNA induced silencing complex (RISC). AGO1 may cleave target mRNAs with small RNA complementarity, but the fate of the resulting cleavage fragments remains incompletely understood. Here, we show that SKI2, SKI3 and SKI8, subunits of a cytoplasmic cofactor of the RNA exosome, are required for degradation of RISC 5′, but not 3′-cleavage fragments in Arabidopsis. In the absence of SKI2 activity, many miRNA targets produce siRNAs via the RNA-dependent RNA polymerase 6 (RDR6) pathway. These siRNAs are low-abundant, and map close to the cleavage site. In most cases, siRNAs were produced 5′ to the cleavage site, but several examples of 3′-spreading were also identified. These observations suggest that siRNAs do not simply derive from RDR6 action on stable 5′-cleavage fragments and hence that SKI2 has a direct role in limiting secondary siRNA production in addition to its function in mediating degradation of 5′-cleavage fragments. PMID:26464441

Capturing microRNA targets using an RNA-induced silencing complex (RISC)-trap approach.

Science.gov (United States)

Cambronne, Xiaolu A; Shen, Rongkun; Auer, Paul L; Goodman, Richard H

2012-12-11

Identifying targets is critical for understanding the biological effects of microRNA (miRNA) expression. The challenge lies in characterizing the cohort of targets for a specific miRNA, especially when targets are being actively down-regulated in miRNA- RNA-induced silencing complex (RISC)-messengerRNA (mRNA) complexes. We have developed a robust and versatile strategy called RISCtrap to stabilize and purify targets from this transient interaction. Its utility was demonstrated by determining specific high-confidence target datasets for miR-124, miR-132, and miR-181 that contained known and previously unknown transcripts. Two previously unknown miR-132 targets identified with RISCtrap, adaptor protein CT10 regulator of kinase 1 (CRK1) and tight junction-associated protein 1 (TJAP1), were shown to be endogenously regulated by miR-132 in adult mouse forebrain. The datasets, moreover, differed in the number of targets and in the types and frequency of microRNA recognition element (MRE) motifs, thus revealing a previously underappreciated level of specificity in the target sets regulated by individual miRNAs.

miRNAtools: Advanced Training Using the miRNA Web of Knowledge.

Science.gov (United States)

Stępień, Ewa Ł; Costa, Marina C; Enguita, Francisco J

2018-02-16

Micro-RNAs (miRNAs) are small non-coding RNAs that act as negative regulators of the genomic output. Their intrinsic importance within cell biology and human disease is well known. Their mechanism of action based on the base pairing binding to their cognate targets have helped the development not only of many computer applications for the prediction of miRNA target recognition but also of specific applications for functional assessment and analysis. Learning about miRNA function requires practical training in the use of specific computer and web-based applications that are complementary to wet-lab studies. In order to guide the learning process about miRNAs, we have created miRNAtools (http://mirnatools.eu), a web repository of miRNA tools and tutorials. This article compiles tools with which miRNAs and their regulatory action can be analyzed and that function to collect and organize information dispersed on the web. The miRNAtools website contains a collection of tutorials that can be used by students and tutors engaged in advanced training courses. The tutorials engage in analyses of the functions of selected miRNAs, starting with their nomenclature and genomic localization and finishing with their involvement in specific cellular functions.

In-Silico Integration Approach to Identify a Key miRNA Regulating a Gene Network in Aggressive Prostate Cancer

Science.gov (United States)

Colaprico, Antonio; Bontempi, Gianluca; Castiglioni, Isabella

2018-01-01

Like other cancer diseases, prostate cancer (PC) is caused by the accumulation of genetic alterations in the cells that drives malignant growth. These alterations are revealed by gene profiling and copy number alteration (CNA) analysis. Moreover, recent evidence suggests that also microRNAs have an important role in PC development. Despite efforts to profile PC, the alterations (gene, CNA, and miRNA) and biological processes that correlate with disease development and progression remain partially elusive. Many gene signatures proposed as diagnostic or prognostic tools in cancer poorly overlap. The identification of co-expressed genes, that are functionally related, can identify a core network of genes associated with PC with a better reproducibility. By combining different approaches, including the integration of mRNA expression profiles, CNAs, and miRNA expression levels, we identified a gene signature of four genes overlapping with other published gene signatures and able to distinguish, in silico, high Gleason-scored PC from normal human tissue, which was further enriched to 19 genes by gene co-expression analysis. From the analysis of miRNAs possibly regulating this network, we found that hsa-miR-153 was highly connected to the genes in the network. Our results identify a four-gene signature with diagnostic and prognostic value in PC and suggest an interesting gene network that could play a key regulatory role in PC development and progression. Furthermore, hsa-miR-153, controlling this network, could be a potential biomarker for theranostics in high Gleason-scored PC. PMID:29562723

miRNA Expression Profile after Status Epilepticus and Hippocampal Neuroprotection by Targeting miR-132

Science.gov (United States)

Jimenez-Mateos, Eva M.; Bray, Isabella; Sanz-Rodriguez, Amaya; Engel, Tobias; McKiernan, Ross C.; Mouri, Genshin; Tanaka, Katsuhiro; Sano, Takanori; Saugstad, Julie A.; Simon, Roger P.; Stallings, Raymond L.; Henshall, David C.

2011-01-01

When an otherwise harmful insult to the brain is preceded by a brief, noninjurious stimulus, the brain becomes tolerant, and the resulting damage is reduced. Epileptic tolerance develops when brief seizures precede an episode of prolonged seizures (status epilepticus). MicroRNAs (miRNAs) are small, noncoding RNAs that function as post-transcriptional regulators of gene expression. We investigated how prior seizure preconditioning affects the miRNA response to status epilepticus evoked by intra-amygdalar kainic acid in mice. The miRNA was extracted from the ipsilateral CA3 subfield 24 hours after focal-onset status epilepticus in animals that had previously received either seizure preconditioning (tolerance) or no preconditioning (injury), and mature miRNA levels were measured using TaqMan low-density arrays. Expression of 21 miRNAs was increased, relative to control, after status epilepticus alone, and expression of 12 miRNAs was decreased. Increased miR-132 levels were matched with increased binding to Argonaute-2, a constituent of the RNA-induced silencing complex. In tolerant animals, expression responses of >40% of the injury-group-detected miRNAs differed, being either unchanged relative to control or down-regulated, and this included miR-132. In vivo microinjection of locked nucleic acid-modified oligonucleotides (antagomirs) against miR-132 depleted hippocampal miR-132 levels and reduced seizure-induced neuronal death. Thus, our data strongly suggest that miRNAs are important regulators of seizure-induced neuronal death. PMID:21945804

Altered expression of miRNAs in the uterus from a letrozole-induced rat PCOS model.

Science.gov (United States)

Li, Chunjin; Chen, Lu; Zhao, Yun; Chen, Shuxiong; Fu, Lulu; Jiang, Yanwen; Gao, Shan; Liu, Zhuo; Wang, Fengge; Zhu, Xiaoling; Rao, Jiahui; Zhang, Jing; Zhou, Xu

2017-01-20

Polycystic ovary syndrome (PCOS) causes female subfertility with ovarian disorders and may be associated with increased rate of early-pregnancy failure. Rat PCOS models were established using letrozole to understand the uterine pathogenesis of PCOS. The differential expression of microRNAs (miRNAs) was observed in rat uterus with PCOS. After estrous cycles were disrupted, significantly abnormal ovarian morphology and hormone level were observed in rats with PCOS. A total of 148 miRNAs differentially expressed were identified in the uterus from the letrozole-induced rat model compared with the control. These miRNAs included 111 upregulated miRNAs and 37 downregulated miRNAs. The differential expression of miR-484, miR-375-3p, miR-324-5p, and miR-223-3p was further confirmed by quantitative reverse transcription polymerase chain reaction. Bioinformatic analysis showed that these four miRNAs were predicted to regulate a large number of genes with different functions. Pathway analysis supported that target genes of miRNAs were involved in insulin secretion and signaling pathways, such as wnt, AMPK, PI3K-Akt, and Ras. These data indicated that miRNAs differentially expressed in rat uterus with PCOS may be associated with PCOS pathogenesis in the uterus. Our findings can help clarify the mechanism of uterine defects in PCOS. Copyright © 2016. Published by Elsevier B.V.

Identification of miRNA targets with stable isotope labeling by amino acids in cell culture

DEFF Research Database (Denmark)

Vinther, Jeppe; Hedegaard, Mads Marquardt; Gardner, Paul Phillip

2006-01-01

miRNAs are small noncoding RNAs that regulate gene expression. We have used stable isotope labeling by amino acids in cell culture (SILAC) to investigate the effect of miRNA-1 on the HeLa cell proteome. Expression of 12 out of 504 investigated proteins was repressed by miRNA-1 transfection...

Psmir: a database of potential associations between small molecules and miRNAs.

Science.gov (United States)

Meng, Fanlin; Wang, Jing; Dai, Enyu; Yang, Feng; Chen, Xiaowen; Wang, Shuyuan; Yu, Xuexin; Liu, Dianming; Jiang, Wei

2016-01-13

miRNAs are key post-transcriptional regulators of many essential biological processes, and their dysregulation has been validated in almost all human cancers. Restoring aberrantly expressed miRNAs might be a novel therapeutics. Recently, many studies have demonstrated that small molecular compounds can affect miRNA expression. Thus, prediction of associations between small molecules and miRNAs is important for investigation of miRNA-targeted drugs. Here, we analyzed 39 miRNA-perturbed gene expression profiles, and then calculated the similarity of transcription responses between miRNA perturbation and drug treatment to predict drug-miRNA associations. At the significance level of 0.05, we obtained 6501 candidate associations between 1295 small molecules and 25 miRNAs, which included 624 FDA approved drugs. Finally, we constructed the Psmir database to store all potential associations and the related materials. In a word, Psmir served as a valuable resource for dissecting the biological significance in small molecules' effects on miRNA expression, which will facilitate developing novel potential therapeutic targets or treatments for human cancers. Psmir is supported by all major browsers, and is freely available at http://www.bio-bigdata.com/Psmir/.

Elsevier Trophoblast Research Award Lecture: origin, evolution and future of placenta miRNAs.

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Morales-Prieto, D M; Ospina-Prieto, S; Schmidt, A; Chaiwangyen, W; Markert, U R

2014-02-01

MicroRNAs (miRNAs) regulate the expression of a large number of genes in plants and animals. Placental miRNAs appeared late in evolution and can be found only in mammals. Nevertheless, these miRNAs are constantly under evolutionary pressure. As a consequence, miRNA sequences and their mRNA targets may differ between species, and some miRNAs can only be found in humans. Their expression can be tissue- or cell-specific and can vary time-dependently. Human placenta tissue exhibits a specific miRNA expression pattern that dynamically changes during pregnancy and is reflected in the maternal plasma. Some placental miRNAs are involved in or associated with major pregnancy disorders, such as preeclampsia, intrauterine growth restriction or preterm delivery and, therefore, have a strong potential for usage as sensitive and specific biomarkers. In this review we summarize current knowledge on the origin of placental miRNAs, their expression in humans with special regard to trophoblast cells, interspecies differences, and their future as biomarkers. It can be concluded that animal models for human reproduction have a different panel of miRNAs and targets, and can only partly reflect or predict the situation in humans. Copyright © 2013. Published by Elsevier Ltd.

miRNA-148a regulates the expression of the estrogen receptor through DNMT1-mediated DNA methylation in breast cancer cells

Science.gov (United States)

Xu, Yurui; Chao, Lin; Wang, Jianyu; Sun, Yonghong

2017-01-01

Breast cancer remains the most prevalent cancer among women worldwide. The expression of estrogen receptor-α (ER-α) is an important marker for prognosis. ER-α status may be positive or negative in breast cancer cells, although the cause of negative or positive status is not yet fully characterized. In the present study, the expression of ER-α and miRNA-148a was assessed in two breast cancer cell lines, HCC1937 and MCF7. An association between ER-α and miRNA-148a expression was identified. It was then demonstrated that DNA methyltransferase 1 (DNMT1) is a target of miRNA-148a, which may suppress the expression of ER-α via DNA methylation. Finally, an miRNA-148a mimic or inhibitor was transfected into MCF7 cells; the miRNA-148a mimic increased ER-α expression whereas the miRNA-148a inhibitor decreased ER-α expression. In conclusion, it was identified that miRNA-148a regulates ER-α expression through DNMT1-mediated DNA methylation in breast cancer cells. This may represent a potential miRNA-based strategy to modulate the expression of ER-α and provide a novel perspective for investigating the role of miRNAs in treating breast cancer. PMID:29085474

A quick reality check for microRNA target prediction.

Science.gov (United States)

Kast, Juergen

2011-04-01

The regulation of protein abundance by microRNA (miRNA)-mediated repression of mRNA translation is a rapidly growing area of interest in biochemical research. In animal cells, the miRNA seed sequence does not perfectly match that of the mRNA it targets, resulting in a large number of possible miRNA targets and varied extents of repression. Several software tools are available for the prediction of miRNA targets, yet the overlap between them is limited. Jovanovic et al. have developed and applied a targeted, quantitative approach to validate predicted miRNA target proteins. Using a proteome database, they have set up and tested selected reaction monitoring assays for approximately 20% of more than 800 predicted let-7 targets, as well as control genes in Caenorhabditis elegans. Their results demonstrate that such assays can be developed quickly and with relative ease, and applied in a high-throughput setup to verify known and identify novel miRNA targets. They also show, however, that the choice of the biological system and material has a noticeable influence on the frequency, extent and direction of the observed changes. Nonetheless, selected reaction monitoring assays, such as those developed by Jovanovic et al., represent an attractive new tool in the study of miRNA function at the organism level.

Identification and profiling of microRNAs and their target genes from developing caprine skeletal Muscle.

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Yanhong Wang

Full Text Available Goat is an important agricultural animal for meat production. Functional studies have demonstrated that microRNAs (miRNAs regulate gene expression at the post-transcriptional level and play an important role in various biological processes. Although studies on miRNAs expression profiles have been performed in various animals, relatively limited information about goat muscle miRNAs has been reported. To investigate the miRNAs involved in regulating different periods of skeletal muscle development, we herein performed a comprehensive research for expression profiles of caprine miRNAs during two developmental stages of skeletal muscles: fetal stage and six month-old stage. As a result, 15,627,457 and 15,593,721 clean reads were obtained from the fetal goat library (FC and the six month old goat library (SMC, respectively. 464 known miRNAs and 83 novel miRNA candidates were identified. Furthermore, by comparing the miRNA profile, 336 differentially expressed miRNAs were identified and then the potential targets of the differentially expressed miRNAs were predicted. To understand the regulatory network of miRNAs during muscle development, the mRNA expression profiles for the two development stages were characterized and 7322 differentially expressed genes (DEGs were identified. Then the potential targets of miRNAs were compared to the DEGs, the intersection of the two gene sets were screened out and called differentially expressed targets (DE-targets, which were involved in 231 pathways. Ten of the 231 pathways that have smallest P-value were shown as network figures. Based on the analysis of pathways and networks, we found that miR-424-5p and miR-29a might have important regulatory effect on muscle development, which needed to be further studied. This study provided the first global view of the miRNAs in caprine muscle tissues. Our results help elucidation of complex regulatory networks between miRNAs and mRNAs and for the study of muscle

Genome-wide identification of alternate bearing-associated microRNAs (miRNAs) in olive (Olea europaea L.)

Science.gov (United States)

2013-01-01

Background Alternate bearing is a widespread phenomenon among crop plants, defined as the tendency of certain fruit trees to produce a high-yield crop one year ("on-year"), followed by a low-yield or even no crop the following year ("off-year"). Several factors may affect the balance between such developmental phase-transition processes. Among them are the microRNA (miRNA), being gene-expression regulators that have been found to be involved as key determinants in several physiological processes. Results Six olive (Olea europaea L. cv. Ayvalik variety) small RNA libraries were constructed from fruits (ripe and unripe) and leaves (”on year” and ”off year” leaves in July and in November, respectively) and sequenced by high-throughput Illumina sequencing. The RNA was retrotranscribed and sequenced using the high-throughput Illumina platform. Bioinformatics analyses of 93,526,915 reads identified 135 conserved miRNA, belonging to 22 miRNA families in the olive. In addition, 38 putative novel miRNAs were discovered in the datasets. Expression of olive tree miRNAs varied greatly among the six libraries, indicating the contribution of diverse miRNA in balancing between reproductive and vegetative phases. Predicted targets of miRNA were categorized into 108 process ontology groups with significance abundance. Among those, potential alternate bearing-associated processes were found, such as development, hormone-mediated signaling and organ morphogenesis. The KEGG analyses revealed that the miRNA-targeted genes are involved in seven main pathways, belonging to carbohydrate metabolism and hormone signal-transduction pathways. Conclusion A comprehensive study on olive miRNA related to alternate bearing was performed. Regulation of miRNA under different developmental phases and tissues indicated that control of nutrition and hormone, together with flowering processes had a noteworthy impact on the olive tree alternate bearing. Our results also provide significant data

34A, miRNA-944, miRNA-101 and miRNA-218 in cervical cancer

African Journals Online (AJOL)

RNAs (21 - 24 nucleotides in length) that are critical for many important processes such as development, ... RNA extraction and reverse transcription. Total RNA was extracted from each of the experimental groups using ... used as an endogenous control to normalize the expression of miRNA-143, miRNA-34A, miRNA-.

Targeting MicroRNA Function in Respiratory Diseases: Mini-review

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Steven eMaltby

2016-02-01

Full Text Available MicroRNAs (miRNAs are small non-coding RNA molecules that modulate expression of the majority of genes by inhibiting protein translation. Growing literature has identified functional roles for miRNAs across a broad range of biological processes. As such, miRNAs are recognised as potential disease biomarkers and novel targets for therapies. While several miRNA-targeted therapies are currently in clinical trials (e.g. for the treatment of hepatitis C virus infection and cancer, no therapies have targeted miRNAs in respiratory diseases in the clinic. In this mini-review, we review the current knowledge on miRNA expression and function in respiratory diseases, intervention strategies to target miRNA function and considerations specific to respiratory diseases. Altered miRNA expression profiles have been reported in a number of respiratory diseases, including asthma, chronic obstructive pulmonary disease, cystic fibrosis and idiopathic pulmonary fibrosis. These include alterations in isolated lung tissue, as well as sputum, bronchoalveolar lavage fluids and peripheral blood or serum. The observed alterations in easily accessible body fluids (e.g. serum have been proposed as new biomarkers that may inform disease diagnosis and patient management. In a subset of studies, miRNA-targeted interventions also improved disease outcomes, indicating functional roles for altered miRNA expression in disease pathogenesis. In fact, direct administration of miRNA-targeting molecules to the lung has yielded promising results in a number of animal models. The ability to directly administer compounds to the lung holds considerable promise and may limit potential off-target effects and side effects caused by the systemic administration required to treat other diseases.

miRTrail - a comprehensive webserver for analyzing gene and miRNA patterns to enhance the understanding of regulatory mechanisms in diseases

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Laczny Cedric

2012-02-01

Full Text Available Abstract Background Expression profiling provides new insights into regulatory and metabolic processes and in particular into pathogenic mechanisms associated with diseases. Besides genes, non-coding transcripts as microRNAs (miRNAs gained increasing relevance in the last decade. To understand the regulatory processes of miRNAs on genes, integrative computer-aided approaches are essential, especially in the light of complex human diseases as cancer. Results Here, we present miRTrail, an integrative tool that allows for performing comprehensive analyses of interactions of genes and miRNAs based on expression profiles. The integrated analysis of mRNA and miRNA data should generate more robust and reliable results on deregulated pathogenic processes and may also offer novel insights into the regulatory interactions between miRNAs and genes. Our web-server excels in carrying out gene sets analysis, analysis of miRNA sets as well as the combination of both in a systems biology approach. To this end, miRTrail integrates information on 20.000 genes, almost 1.000 miRNAs, and roughly 280.000 putative interactions, for Homo sapiens and accordingly for Mus musculus and Danio rerio. The well-established, classical Chi-squared test is one of the central techniques of our tool for the joint consideration of miRNAs and their targets. For interactively visualizing obtained results, it relies on the network analyzers and viewers BiNA or Cytoscape-web, also enabling direct access to relevant literature. We demonstrated the potential of miRTrail by applying our tool to mRNA and miRNA data of malignant melanoma. MiRTrail identified several deregulated miRNAs that target deregulated mRNAs including miRNAs hsa-miR-23b and hsa-miR-223, which target the highest numbers of deregulated mRNAs and regulate the pathway "basal cell carcinoma". In addition, both miRNAs target genes like PTCH1 and RASA1 that are involved in many oncogenic processes. Conclusions The application

From moderately severe to severe hypertriglyceridemia induced acute pancreatitis: circulating miRNAs play role as potential biomarkers.

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Fangmei An

Full Text Available The incidence of hypertriglyceridemia induced acute pancreatitis (HTAP continues to rise in China. It has systemic complications and high mortality, making the early assessment of the severity of this disease even more important. Circulating microRNAs (miRNAs could be novel, non-invasive biomarkers for disease progression judgment. This study aimed to identify the potential role of serum miRNAs as novel biomarkers of HTAP progression. HTAP patients were divided into two groups: moderately severe (HTMSAP and severe (HTSAP, healthy people were used as control group. The serum miRNA expression profiles of these three groups were determined by microarray and verified by qRT-PCR. The functions and pathways of the targeted genes of deregulated miRNAs were predicted, using bioinformatics analysis; miRNA-mRNA network was generated. Moreover, the correlation between miR-181a-5p and pancreatitis metabolism related substances were studied and the serum concentration of inflammatory cytokines and miRNAs at different time points during the MSAP and SAP were investigated, respectively. Finally, the receiver operating characteristic (ROC curve of miRNAs was studied. Significant changes in the serum concentration of the following miRNAs of HTAP patients (P<0.05 were discovered: miR24-3p, 361-5p, 1246, and 222-3p (constantly upregulated, and 181a-5p (constantly downregulated (P<0.05. Bioinformatics analysis predicted that 13 GOs and 36 pathways regulated by overlap miRNAs were involved in glucose, fat, calcium (Ca++, and insulin metabolism (P<0.001. miRNA-mRNA network revealed that the overlap miRNAs targeted genes participating in pancreas metabolism and miR-181a-5p, the only downregulated miRNA, had good negative correlation with triglyceride (TG, total cholesterol (TC, and fast blood glucose (FBG, but a positive correlation with Ca++. When compared with inflammatory cytokines, the changes of all five overlap miRNAs were more stable. It was found that when

Sox9-regulated miRNA-574-3p inhibits chondrogenic differentiation of mesenchymal stem cells.

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David Guérit

Full Text Available The aim of this study was to identify new microRNAs (miRNAs that are modulated during the differentiation of mesenchymal stem cells (MSCs toward chondrocytes. Using large scale miRNA arrays, we compared the expression of miRNAs in MSCs (day 0 and at early time points (day 0.5 and 3 after chondrogenesis induction. Transfection of premiRNA or antagomiRNA was performed on MSCs before chondrogenesis induction and expression of miRNAs and chondrocyte markers was evaluated at different time points during differentiation by RT-qPCR. Among miRNAs that were modulated during chondrogenesis, we identified miR-574-3p as an early up-regulated miRNA. We found that miR-574-3p up-regulation is mediated via direct binding of Sox9 to its promoter region and demonstrated by reporter assay that retinoid X receptor (RXRα is one gene specifically targeted by the miRNA. In vitro transfection of MSCs with premiR-574-3p resulted in the inhibition of chondrogenesis demonstrating its role during the commitment of MSCs towards chondrocytes. In vivo, however, both up- and down-regulation of miR-574-3p expression inhibited differentiation toward cartilage and bone in a model of heterotopic ossification. In conclusion, we demonstrated that Sox9-dependent up-regulation of miR-574-3p results in RXRα down-regulation. Manipulating miR-574-3p levels both in vitro and in vivo inhibited chondrogenesis suggesting that miR-574-3p might be required for chondrocyte lineage maintenance but also that of MSC multipotency.

A comprehensive survey of 3′ animal miRNA modification events and a possible role for 3′ adenylation in modulating miRNA targeting effectiveness

OpenAIRE

Burroughs, A. Maxwell; Ando, Yoshinari; de Hoon, Michiel J.L.; Tomaru, Yasuhiro; Nishibu, Takahiro; Ukekawa, Ryo; Funakoshi, Taku; Kurokawa, Tsutomu; Suzuki, Harukazu; Hayashizaki, Yoshihide; Daub, Carsten O.

2010-01-01

Animal microRNA sequences are subject to 3′ nucleotide addition. Through detailed analysis of deep-sequenced short RNA data sets, we show adenylation and uridylation of miRNA is globally present and conserved across Drosophila and vertebrates. To better understand 3′ adenylation function, we deep-sequenced RNA after knockdown of nucleotidyltransferase enzymes. The PAPD4 nucleotidyltransferase adenylates a wide range of miRNA loci, but adenylation does not appear to affect miRNA stability on a...

Reference miRNAs for miRNAome analysis of urothelial carcinomas.

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Nadine Ratert

Full Text Available BACKGROUND/OBJECTIVE: Reverse transcription quantitative real-time PCR (RT-qPCR is widely used in microRNA (miRNA expression studies on cancer. To compensate for the analytical variability produced by the multiple steps of the method, relative quantification of the measured miRNAs is required, which is based on normalization to endogenous reference genes. No study has been performed so far on reference miRNAs for normalization of miRNA expression in urothelial carcinoma. The aim of this study was to identify suitable reference miRNAs for miRNA expression studies by RT-qPCR in urothelial carcinoma. METHODS: Candidate reference miRNAs were selected from 24 urothelial carcinoma and normal bladder tissue samples by miRNA microarrays. The usefulness of these candidate reference miRNAs together with the commonly for normalization purposes used small nuclear RNAs RNU6B, RNU48, and Z30 were thereafter validated by RT-qPCR in 58 tissue samples and analyzed by the algorithms geNorm, NormFinder, and BestKeeper. PRINCIPAL FINDINGS: Based on the miRNA microarray data, a total of 16 miRNAs were identified as putative reference genes. After validation by RT-qPCR, miR-101, miR-125a-5p, miR-148b, miR-151-5p, miR-181a, miR-181b, miR-29c, miR-324-3p, miR-424, miR-874, RNU6B, RNU48, and Z30 were used for geNorm, NormFinder, and BestKeeper analyses that gave different combinations of recommended reference genes for normalization. CONCLUSIONS: The present study provided the first systematic analysis for identifying suitable reference miRNAs for miRNA expression studies of urothelial carcinoma by RT-qPCR. Different combinations of reference genes resulted in reliable expression data for both strongly and less strongly altered miRNAs. Notably, RNU6B, which is the most frequently used reference gene for miRNA studies, gave inaccurate normalization. The combination of four (miR-101, miR-125a-5p, miR-148b, and miR-151-5p or three (miR-148b, miR-181b, and miR-874

Highly selective and sensitive detection of miRNA based on toehold-mediated strand displacement reaction and DNA tetrahedron substrate.

Science.gov (United States)

Li, Wei; Jiang, Wei; Ding, Yongshun; Wang, Lei

2015-09-15

MicroRNAs (miRNAs) play important roles in a variety of biological processes and have been regarded as tumor biomarkers in cancer diagnosis and prognosis. In this work, a single-molecule counting method for miRNA analysis was proposed based on toehold-mediated strand displacement reaction (SDR) and DNA tetrahedron substrate. Firstly, a specially designed DNA tetrahedron was assembled with a hairpin at one of the vertex, which has an overhanging toehold domain. Then, the DNA tetrahedron was immobilized on the epoxy-functional glass slide by epoxy-amine reaction, forming a DNA tetrahedron substrate. Next, the target miRNA perhybridized with the toehold domain and initiated a strand displacement reaction along with the unfolding of the hairpin, realizing the selective recognization of miRNA. Finally, a biotin labeled detection DNA was hybridized with the new emerging single strand and the streptavidin coated QDs were used as fluorescent probes. Fluorescent images were acquired via epi-fluorescence microscopy, the numbers of fluorescence dots were counted one by one for quantification. The detection limit is 5 fM, which displayed an excellent sensitivity. Moreover, the proposed method which can accurately be identified the target miRNA among its family members, demonstrated an admirable selectivity. Furthermore, miRNA analysis in total RNA samples from human lung tissues was performed, suggesting the feasibility of this method for quantitative detection of miRNA in biomedical research and early clinical diagnostics. Copyright © 2015 Elsevier B.V. All rights reserved.

Integrated approaches to miRNAs target definition: time-series analysis in an osteosarcoma differentiative model.

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Grilli, A; Sciandra, M; Terracciano, M; Picci, P; Scotlandi, K

2015-06-30

microRNAs (miRs) are small non-coding RNAs involved in the fine regulation of several cellular processes by inhibiting their target genes at post-transcriptional level. Osteosarcoma (OS) is a tumor thought to be related to a molecular blockade of the normal process of osteoblast differentiation. The current paper explores temporal transcriptional modifications comparing an osteosarcoma cell line, Saos-2, and clones stably transfected with CD99, a molecule which was found to drive OS cells to terminally differentiate. Parental cell line and CD99 transfectants were cultured up to 14 days in differentiating medium. In this setting, OS cells were profiled by gene and miRNA expression arrays. Integration of gene and miRNA profiling was performed by both sequence complementarity and expression correlation. Further enrichment and network analyses were carried out to focus on the modulated pathways and on the interactions between transcriptome and miRNome. To track the temporal transcriptional modification, a PCA analysis with differentiated human MSC was performed. We identified a strong (about 80 %) gene down-modulation where reversion towards the osteoblast-like phenotype matches significant enrichment in TGFbeta signaling players like AKT1 and SMADs. In parallel, we observed the modulation of several cancer-related microRNAs like miR-34a, miR-26b or miR-378. To decipher their impact on the modified transcriptional program in CD99 cells, we correlated gene and microRNA time-series data miR-34a, in particular, was found to regulate a distinct subnetwork of genes with respect to the rest of the other differentially expressed miRs and it appeared to be the main mediator of several TGFbeta signaling genes at initial and middle phases of differentiation. Integration studies further highlighted the involvement of TGFbeta pathway in the differentiation of OS cells towards osteoblasts and its regulation by microRNAs. These data underline that the expression of miR-34a and down

Identifying miRNA and gene modules of colon cancer associated with pathological stage by weighted gene co-expression network analysis

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Zhou X

2018-05-01

characterize the results of WGCNA.Results: Two gene modules (Gmagenta and Ggreen and one miRNA module were associated with the pathological stage. Six hub genes (COL1A2, THBS2, BGN, COL1A1, TAGLN and DACT3 were related to prognosis and validated to be associated with the pathological stage. Five hub miRNAs were identified to be related to prognosis (hsa-miR-125b-5p, hsa-miR-145-5p, hsa-let-7c-5p, hsa-miR-218-5p and hsa-miR-125b-2-3p. A total of 18 hub genes and seven hub miRNAs were predominantly expressed in tumor stroma. Proteoglycans in cancer, focal adhesion, extracellular matrix (ECM–receptor interaction and so on were common pathways of the three modules. Hsa-let-7c-5p was located at the core of miRNA–gene network.Conclusion: These findings help to advance the understanding of tumor stroma in the progression of CAC and provide prognostic biomarkers as well as therapeutic targets. Keywords: colon adenocarcinoma, weighted gene co-expression network analysis, differentially expressed genes, differentially expressed miRNA, tumor stroma

Identification of microRNAs in Caragana intermedia by high-throughput sequencing and expression analysis of 12 microRNAs and their targets under salt stress.

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Zhu, Jianfeng; Li, Wanfeng; Yang, Wenhua; Qi, Liwang; Han, Suying

2013-09-01

142 miRNAs were identified and 38 miRNA targets were predicted, 4 of which were validated, in C. intermedia . The expression of 12 miRNAs in salt-stressed leaves was assessed by qRT-PCR. MicroRNAs (miRNAs) are endogenous small RNAs that play important roles in various biological and metabolic processes in plants. Caragana intermedia is an important ecological and economic tree species prominent in the desert environment of west and northwest China. To date, no investigation into C. intermedia miRNAs has been reported. In this study, high-throughput sequencing of small RNAs and analysis of transcriptome data were performed to identify both conserved and novel miRNAs, and also their target mRNA genes in C. intermedia. Based on sequence similarity and hairpin structure prediction, 132 putative conserved miRNAs (12 of which were confirmed to form hairpin precursors) belonging to 31 known miRNA families were identified. Ten novel miRNAs (including the miRNA* sequences of three novel miRNAs) were also discovered. Furthermore, 36 potential target genes of 17 known miRNA families and 2 potential target genes of 1 novel miRNA were predicted; 4 of these were validated by 5' RACE. The expression of 12 miRNAs was validated in different tissues, and these and five target mRNAs were assessed by qRT-PCR after salt treatment. The expression levels of seven miRNAs (cin-miR157a, cin-miR159a, cin-miR165a, cin-miR167b, cin-miR172b, cin-miR390a and cin-miR396a) were upregulated, while cin-miR398a expression was downregulated after salt treatment. The targets of cin-miR157a, cin-miR165a, cin-miR172b and cin-miR396a were downregulated and showed an approximately negative correlation with their corresponding miRNAs under salt treatment. These results would help further understanding of miRNA regulation in response to abiotic stress in C. intermedia.

Infinity: An In-Silico Tool for Genome-Wide Prediction of Specific DNA Matrices in miRNA Genomic Loci.

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Falcone, Emmanuela; Grandoni, Luca; Garibaldi, Francesca; Manni, Isabella; Filligoi, Giancarlo; Piaggio, Giulia; Gurtner, Aymone

2016-01-01

miRNAs are potent regulators of gene expression and modulate multiple cellular processes in physiology and pathology. Deregulation of miRNAs expression has been found in various cancer types, thus, miRNAs may be potential targets for cancer therapy. However, the mechanisms through which miRNAs are regulated in cancer remain unclear. Therefore, the identification of transcriptional factor-miRNA crosstalk is one of the most update aspects of the study of miRNAs regulation. In the present study we describe the development of a fast and user-friendly software, named infinity, able to find the presence of DNA matrices, such as binding sequences for transcriptional factors, on ~65kb (kilobase) of 939 human miRNA genomic sequences, simultaneously. Of note, the power of this software has been validated in vivo by performing chromatin immunoprecipitation assays on a subset of new in silico identified target sequences (CCAAT) for the transcription factor NF-Y on colon cancer deregulated miRNA loci. Moreover, for the first time, we have demonstrated that NF-Y, through its CCAAT binding activity, regulates the expression of miRNA-181a, -181b, -21, -17, -130b, -301b in colon cancer cells. The infinity software that we have developed is a powerful tool to underscore new TF/miRNA regulatory networks. Infinity was implemented in pure Java using Eclipse framework, and runs on Linux and MS Windows machine, with MySQL database. The software is freely available on the web at https://github.com/bio-devel/infinity. The website is implemented in JavaScript, PHP and HTML with all major browsers supported.

Infinity: An In-Silico Tool for Genome-Wide Prediction of Specific DNA Matrices in miRNA Genomic Loci.

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Emmanuela Falcone

Full Text Available miRNAs are potent regulators of gene expression and modulate multiple cellular processes in physiology and pathology. Deregulation of miRNAs expression has been found in various cancer types, thus, miRNAs may be potential targets for cancer therapy. However, the mechanisms through which miRNAs are regulated in cancer remain unclear. Therefore, the identification of transcriptional factor-miRNA crosstalk is one of the most update aspects of the study of miRNAs regulation.In the present study we describe the development of a fast and user-friendly software, named infinity, able to find the presence of DNA matrices, such as binding sequences for transcriptional factors, on ~65kb (kilobase of 939 human miRNA genomic sequences, simultaneously. Of note, the power of this software has been validated in vivo by performing chromatin immunoprecipitation assays on a subset of new in silico identified target sequences (CCAAT for the transcription factor NF-Y on colon cancer deregulated miRNA loci. Moreover, for the first time, we have demonstrated that NF-Y, through its CCAAT binding activity, regulates the expression of miRNA-181a, -181b, -21, -17, -130b, -301b in colon cancer cells.The infinity software that we have developed is a powerful tool to underscore new TF/miRNA regulatory networks.Infinity was implemented in pure Java using Eclipse framework, and runs on Linux and MS Windows machine, with MySQL database. The software is freely available on the web at https://github.com/bio-devel/infinity. The website is implemented in JavaScript, PHP and HTML with all major browsers supported.

Novel Insights into miRNA in Lung and Heart Inflammatory Diseases

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Amit Kishore

2014-01-01

Full Text Available MicroRNAs (miRNAs are noncoding regulatory sequences that govern posttranscriptional inhibition of genes through binding mainly at regulatory regions. The regulatory mechanism of miRNAs are influenced by complex crosstalk among single nucleotide polymorphisms (SNPs within miRNA seed region and epigenetic modifications. Circulating miRNAs exhibit potential characteristics as stable biomarker. Functionally, miRNAs are involved in basic regulatory mechanisms of cells including inflammation. Thus, miRNA dysregulation, resulting in aberrant expression of a gene, is suggested to play an important role in disease susceptibility. This review focuses on the role of miRNA as diagnostic marker in pathogenesis of lung inflammatory diseases and in cardiac remodelling events during inflammation. From recent reports, In this context, the information about the models in which miRNAs expression were investigated including types of biological samples, as well as on the methods for miRNA validation and prediction/definition of their gene targets are emphasized in the review. Besides disease pathogenesis, promising role of miRNAs in early disease diagnosis and prognostication is also discussed. However, some miRNAs are also indicated with protective role. Thus, identifications and usage of such potential miRNAs as well as disruption of disease susceptible miRNAs using antagonists, antagomirs, are imperative and may provide a novel therapeutic approach towards combating the disease progression.

MirZ: an integrated microRNA expression atlas and target prediction resource.

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Hausser, Jean; Berninger, Philipp; Rodak, Christoph; Jantscher, Yvonne; Wirth, Stefan; Zavolan, Mihaela

2009-07-01

MicroRNAs (miRNAs) are short RNAs that act as guides for the degradation and translational repression of protein-coding mRNAs. A large body of work showed that miRNAs are involved in the regulation of a broad range of biological functions, from development to cardiac and immune system function, to metabolism, to cancer. For most of the over 500 miRNAs that are encoded in the human genome the functions still remain to be uncovered. Identifying miRNAs whose expression changes between cell types or between normal and pathological conditions is an important step towards characterizing their function as is the prediction of mRNAs that could be targeted by these miRNAs. To provide the community the possibility of exploring interactively miRNA expression patterns and the candidate targets of miRNAs in an integrated environment, we developed the MirZ web server, which is accessible at www.mirz.unibas.ch. The server provides experimental and computational biologists with statistical analysis and data mining tools operating on up-to-date databases of sequencing-based miRNA expression profiles and of predicted miRNA target sites in species ranging from Caenorhabditis elegans to Homo sapiens.

miRNAs in Tuberculosis: New Avenues for Diagnosis and Host-Directed Therapy

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Naveed Sabir

2018-03-01

Full Text Available Tuberculosis (TB is one of the most fatal infectious diseases and a leading cause of mortality, with 95% of these deaths occurring in developing countries. The causative agent, Mycobacterium tuberculosis (Mtb, has a well-established ability to circumvent the host’s immune system for its intracellular survival. microRNAs (miRNAs are small, non-coding RNAs having an important function at the post-transcriptional level and are involved in shaping immunity by regulating the repertoire of genes expressed in immune cells. It has been established in recent studies that the innate immune response against TB is significantly regulated by miRNAs. Moreover, differential expression of miRNA in Mtb infection can reflect the disease progression and may help distinguish between active and latent TB infection (LTBI. These findings encouraged the application of miRNAs as potential biomarkers. Similarly, active participation of miRNAs in modulation of autophagy and apoptosis responses against Mtb opens an exciting avenue for the exploitation of miRNAs as host directed therapy (HDT against TB. Nanoparticles mediated delivery of miRNAs to treat various diseases has been reported and this technology has a great potential to be used in TB. In reality, this exploitation of miRNAs as biomarkers and in HDT is still in its infancy stage, and more studies using animal models mimicking human TB are advocated to assess the role of miRNAs as biomarkers and therapeutic targets. In this review, we attempt to summarize the recent advancements in the role of miRNAs in TB as immune modulator, miRNAs’ capability to distinguish between active and latent TB and, finally, usage of miRNAs as therapeutic targets against TB.

Big endothelin changes the cellular miRNA environment in TMOb osteoblasts and increases mineralization.

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Johnson, Michael G; Kristianto, Jasmin; Yuan, Baozhi; Konicke, Kathryn; Blank, Robert

2014-08-01

Endothelin (ET1) promotes the growth of osteoblastic breast and prostate cancer metastases. Conversion of big ET1 to mature ET1, catalyzed primarily by endothelin converting enzyme 1 (ECE1), is necessary for ET1's biological activity. We previously identified the Ece1, locus as a positional candidate gene for a pleiotropic quantitative trait locus affecting femoral size, shape, mineralization, and biomechanical performance. We exposed TMOb osteoblasts continuously to 25 ng/ml big ET1. Cells were grown for 6 days in growth medium and then switched to mineralization medium for an additional 15 days with or without big ET1, by which time the TMOb cells form mineralized nodules. We quantified mineralization by alizarin red staining and analyzed levels of miRNAs known to affect osteogenesis. Micro RNA 126-3p was identified by search as a potential regulator of sclerostin (SOST) translation. TMOb cells exposed to big ET1 showed greater mineralization than control cells. Big ET1 repressed miRNAs targeting transcripts of osteogenic proteins. Big ET1 increased expression of miRNAs that target transcripts of proteins that inhibit osteogenesis. Big ET1 increased expression of 126-3p 121-fold versus control. To begin to assess the effect of big ET1 on SOST production we analyzed both SOST transcription and protein production with and without the presence of big ET1 demonstrating that transcription and translation were uncoupled. Our data show that big ET1 signaling promotes mineralization. Moreover, the results suggest that big ET1's osteogenic effects are potentially mediated through changes in miRNA expression, a previously unrecognized big ET1 osteogenic mechanism.

Exploring miRNA based approaches in cancer diagnostics and therapeutics.

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Mishra, Shivangi; Yadav, Tanuja; Rani, Vibha

2016-02-01

MicroRNAs (miRNAs), a highly conserved class of tissue specific, small non-protein coding RNAs maintain cell homeostasis by negative gene regulation. Proper controlling of miRNA expression is required for a balanced physiological environment, as these small molecules influence almost every genetic pathway from cell cycle checkpoint, cell proliferation to apoptosis, with a wide range of target genes. Deregulation in miRNAs expression correlates with various cancers by acting as tumor suppressors and oncogenes. Although promising therapies exist to control tumor development and progression, there is a lack of efficient diagnostic and therapeutic approaches for delineating various types of cancer. The molecularly different tumors can be differentiated by specific miRNA profiling as their phenotypic signatures, which can hence be exploited to surmount the diagnostic and therapeutic challenges. Present review discusses the involvement of miRNAs in oncogenesis with the analysis of patented research available on miRNAs. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

MicroRNA transfection and AGO-bound CLIP-seq data sets reveal distinct determinants of miRNA action

DEFF Research Database (Denmark)

Wen, Jiayu; Parker, Brian J; Jacobsen, Anders

2011-01-01

the predictive effect of target flanking features. We observe distinct target determinants between expression-based and CLIP-based data. Target flanking features such as flanking region conservation are an important AGO-binding determinant-we hypothesize that CLIP experiments have a preference for strongly bound......Microarray expression analyses following miRNA transfection/inhibition and, more recently, Argonaute cross-linked immunoprecipitation (CLIP)-seq assays have been used to detect miRNA target sites. CLIP and expression approaches measure differing stages of miRNA functioning-initial binding of the mi...... miRNP-target interactions involving adjacent RNA-binding proteins that increase the strength of cross-linking. In contrast, seed-related features are major determinants in expression-based studies, but less so for CLIP-seq studies, and increased miRNA concentrations typical of transfection studies...

Identification of microRNAs and their targets in Finger millet by high throughput sequencing.

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Usha, S; Jyothi, M N; Sharadamma, N; Dixit, Rekha; Devaraj, V R; Nagesh Babu, R

2015-12-15

MicroRNAs are short non-coding RNAs which play an important role in regulating gene expression by mRNA cleavage or by translational repression. The majority of identified miRNAs were evolutionarily conserved; however, others expressed in a species-specific manner. Finger millet is an important cereal crop; nonetheless, no practical information is available on microRNAs to date. In this study, we have identified 95 conserved microRNAs belonging to 39 families and 3 novel microRNAs by high throughput sequencing. For the identified conserved and novel miRNAs a total of 507 targets were predicted. 11 miRNAs were validated and tissue specificity was determined by stem loop RT-qPCR, Northern blot. GO analyses revealed targets of miRNA were involved in wide range of regulatory functions. This study implies large number of known and novel miRNAs found in Finger millet which may play important role in growth and development. Copyright © 2015 Elsevier B.V. All rights reserved.

Analysis of Deregulated microRNAs and Their Target Genes in Gastric Cancer.

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Simonas Juzėnas

Full Text Available MicroRNAs (miRNAs are widely studied non-coding RNAs that modulate gene expression. MiRNAs are deregulated in different tumors including gastric cancer (GC and have potential diagnostic and prognostic implications. The aim of our study was to determine miRNA profile in GC tissues, followed by evaluation of deregulated miRNAs in plasma of GC patients. Using available databases and bioinformatics methods we also aimed to evaluate potential target genes of confirmed differentially expressed miRNA and validate these findings in GC tissues.The study included 51 GC patients and 51 controls. Initially, we screened miRNA expression profile in 13 tissue samples of GC and 12 normal gastric tissues with TaqMan low density array (TLDA. In the second stage, differentially expressed miRNAs were validated in a replication cohort using qRT-PCR in tissue and plasma samples. Subsequently, we analyzed potential target genes of deregulated miRNAs using bioinformatics approach, determined their expression in GC tissues and performed correlation analysis with targeting miRNAs.Profiling with TLDA revealed 15 deregulated miRNAs in GC tissues compared to normal gastric mucosa. Replication analysis confirmed that miR-148a-3p, miR-204-5p, miR-223-3p and miR-375 were consistently deregulated in GC tissues. Analysis of GC patients' plasma samples showed significant down-regulation of miR-148a-3p, miR-375 and up-regulation of miR-223-3p compared to healthy subjects. Further, using bioinformatic tools we identified targets of replicated miRNAs and performed disease-associated gene enrichment analysis. Ultimately, we evaluated potential target gene BCL2 and DNMT3B expression by qRT-PCR in GC tissue, which correlated with targeting miRNA expression.Our study revealed miRNA profile in GC tissues and showed that miR-148a-3p, miR-223-3p and miR-375 are deregulated in GC plasma samples, but these circulating miRNAs showed relatively weak diagnostic performance as sole biomarkers

A construct with fluorescent indicators for conditional expression of miRNA

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Xia Xugang

2008-10-01

Full Text Available Abstract Background Transgenic RNAi holds promise as a simple, low-cost, and fast method for reverse genetics in mammals. It may be particularly useful for producing animal models for hypomorphic gene function. Inducible RNAi that permits spatially and temporally controllable gene silencing in vivo will enhance the power of transgenic RNAi approach. Furthermore, because microRNA (miRNA targeting specific genes can be expressed simultaneously with protein coding genes, incorporation of fluorescent marker proteins can simplify the screening and analysis of transgenic RNAi animals. Results We sought to optimally express a miRNA simultaneously with a fluorescent marker. We compared two construct designs. One expressed a red fluorescent protein (RFP and a miRNA placed in its 3' untranslated region (UTR. The other expressed the same RFP and miRNA, but the precursor miRNA (pre-miRNA coding sequence was placed in an intron that was inserted into the 3'-UTR. We found that the two constructs expressed comparable levels of miRNA. However, the intron-containing construct expressed a significantly higher level of RFP than the intron-less construct. Further experiments indicate that the 3'-UTR intron enhances RFP expression by its intrinsic gene-expression-enhancing activity and by eliminating the inhibitory effect of the pre-miRNA on the expression of RFP. Based on these findings, we incorporated the intron-embedded pre-miRNA design into a conditional expression construct that employed the Cre-loxP system. This construct initially expressed EGFP gene, which was flanked by loxP sites. After exposure to Cre recombinase, the transgene stopped EGFP expression and began expression of RFP and a miRNA, which silenced the expression of specific cellular genes. Conclusion We have designed and tested a conditional miRNA-expression construct and showed that this construct expresses both the marker genes strongly and can silence the target gene efficiently upon Cre

Profiling micro rnas and their targets in radish (raphanus sativus l.)

International Nuclear Information System (INIS)

Barozai, M.Y.; Din, M.

2015-01-01

MicroRNAs (miRNAs) are tiny, non-protein coding and negative regulatory RNAs approximately 21 nucleotides in length. The comparative genomic methodology due to their conserved nature is a reasonable approach for the novel miRNAs discovery. In this research, total 25 novel miRNAs from 18 families (ras-miR-156, 160, 162, 163, 164, 167, 168, 319, 399, 408, 413, 414, 841, 1310, 2936, 5030 and 5661) are identified in an important vegetable radish (Raphanus sativus L.). The 25 miRNA precursor sequences showed secondary structures with the mature miRNAs in the stem region. Total 42 putative targets are also identified for the novel 25 radish miRNAs. These findings suggest that more thorough understanding of the function of such miRNAs will help to unravel the mysteries role in plant biology. (author)

Alterations of serum levels of BDNF-related miRNAs in patients with depression.

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You-Jie Li

Full Text Available Depression is a serious and potentially life-threatening mental disorder with unknown etiology. Emerging evidence shows that brain-derived neurotrophic factor (BDNF and microRNAs (miRNAs play critical roles in the etiology of depression. Here this study was aimed to identify and characterize the roles of BDNF and its putative regulatory miRNAs in depression. First, we identified that miR-182 may be a putative miRNA that regulates BDNF levels by bioinformatic studies, and characterized the effects of miR-182 on the BDNF levels using cell-based studies, side by side with miR-132 (a known miRNA that regulates BDNF expression. We showed that treatment of miR-132 and miR-182 respectively decreased the BDNF protein levels in a human neuronal cell model, supporting the regulatory roles of miR-132 and miR-182 on the BDNF expression. Furthermore, we explored the roles of miR-132 and miR-182 on the BDNF levels in depression using human subjects by assessing their serum levels. Compared with the healthy controls, patients with depression showed lower serum BDNF levels (via the enzyme-linked immunosorbent assays and higher serum miR-132 and miR-182 levels (via the real-time PCR. Finally, the Pearson's (or Spearman's correlation coefficient was calculated to study whether there was a relationship among the Self-Rating Depression Scale score, the serum BDNF levels, and serum BDNF-related miRNA levels. Our results revealed that there was a significant negative correlation between the SDS scores and the serum BDNF levels, and a positive correlation between the SDS scores and miR-132 levels. In addition, we found a reverse relationship between the serum BDNF levels and the miR-132/miR-182 levels in depression. Collectively, we provided evidence supporting that miR-182 is a putative BDNF-regulatory miRNA, and suggested that the serum BDNF and its related miRNAs may be utilized as important biomarkers in the diagnosis or as therapeutic targets of depression.

Functional miRNAs in breast cancer drug resistance

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Hu WZ

2018-03-01

Full Text Available Weizi Hu,1–3,* Chunli Tan,1–3,* Yunjie He,4 Guangqin Zhang,2 Yong Xu,3,5 Jinhai Tang1 1Department of General Surgery, The First Affiliated Hospital of Nanjing Medical University, 2School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, 3Nanjing Medical University Affiliated Cancer Hospital, 4The First Clinical School of Nanjing Medical University, 5Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment, Nanjing Medical University, Nanjing, People’s Republic of China *These authors contributed equally to this work Abstract: Owing to improved early surveillance and advanced therapy strategies, the current death rate due to breast cancer has decreased; nevertheless, drug resistance and relapse remain obstacles on the path to successful systematic treatment. Multiple mechanisms responsible for drug resistance have been elucidated, and miRNAs seem to play a major part in almost every aspect of cancer progression, including tumorigenesis, metastasis, and drug resistance. In recent years, exosomes have emerged as novel modes of intercellular signaling vehicles, initiating cell–cell communication through their fusion with target cell membranes, delivering functional molecules including miRNAs and proteins. This review particularly focuses on enumerating functional miRNAs involved in breast cancer drug resistance as well as their targets and related mechanisms. Subsequently, we discuss the prospects and challenges of miRNA function in drug resistance and highlight valuable approaches for the investigation of the role of exosomal miRNAs in breast cancer progression and drug resistance. Keywords: microRNA, exosome, breast cancer, drug resistance

Combined analysis of mRNA and miRNA identifies dehydration and salinity responsive key molecular players in citrus roots.

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Xie, Rangjin; Zhang, Jin; Ma, Yanyan; Pan, Xiaoting; Dong, Cuicui; Pang, Shaoping; He, Shaolan; Deng, Lie; Yi, Shilai; Zheng, Yongqiang; Lv, Qiang

2017-02-06

Citrus is one of the most economically important fruit crops around world. Drought and salinity stresses adversely affected its productivity and fruit quality. However, the genetic regulatory networks and signaling pathways involved in drought and salinity remain to be elucidated. With RNA-seq and sRNA-seq, an integrative analysis of miRNA and mRNA expression profiling and their regulatory networks were conducted using citrus roots subjected to dehydration and salt treatment. Differentially expressed (DE) mRNA and miRNA profiles were obtained according to fold change analysis and the relationships between miRNAs and target mRNAs were found to be coherent and incoherent in the regulatory networks. GO enrichment analysis revealed that some crucial biological processes related to signal transduction (e.g. 'MAPK cascade'), hormone-mediated signaling pathways (e.g. abscisic acid- activated signaling pathway'), reactive oxygen species (ROS) metabolic process (e.g. 'hydrogen peroxide catabolic process') and transcription factors (e.g., 'MYB, ZFP and bZIP') were involved in dehydration and/or salt treatment. The molecular players in response to dehydration and salt treatment were partially overlapping. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis further confirmed the results from RNA-seq and sRNA-seq analysis. This study provides new insights into the molecular mechanisms how citrus roots respond to dehydration and salt treatment.

TGF-β/Smad2/3 signaling directly regulates several miRNAs in mouse ES cells and early embryos.

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Nicholas Redshaw

Full Text Available The Transforming Growth Factor-β (TGF-β signaling pathway is one of the major pathways essential for normal embryonic development and tissue homeostasis, with anti-tumor but also pro-metastatic properties in cancer. This pathway directly regulates several target genes that mediate its downstream functions, however very few microRNAs (miRNAs have been identified as targets. miRNAs are modulators of gene expression with essential roles in development and a clear association with diseases including cancer. Little is known about the transcriptional regulation of the primary transcripts (pri-miRNA, pri-miR from which several mature miRNAs are often derived. Here we present the identification of miRNAs regulated by TGF-β signaling in mouse embryonic stem (ES cells and early embryos. We used an inducible ES cell system to maintain high levels of the TGF-β activated/phosphorylated Smad2/3 effectors, which are the transcription factors of the pathway, and a specific inhibitor that blocks their activation. By performing short RNA deep-sequencing after 12 hours Smad2/3 activation and after 16 hours inhibition, we generated a database of responsive miRNAs. Promoter/enhancer analysis of a subset of these miRNAs revealed that the transcription of pri-miR-181c/d and the pri-miR-341∼3072 cluster were found to depend on activated Smad2/3. Several of these miRNAs are expressed in early mouse embryos, when the pathway is known to play an essential role. Treatment of embryos with TGF-β inhibitor caused a reduction of their levels confirming that they are targets of this pathway in vivo. Furthermore, we showed that pri-miR-341∼3072 transcription also depends on FoxH1, a known Smad2/3 transcription partner during early development. Together, our data show that miRNAs are regulated directly by the TGF-β/Smad2/3 pathway in ES cells and early embryos. As somatic abnormalities in functions known to be regulated by the TGF-β/Smad2/3 pathway underlie tumor

Functional integration of complex miRNA networks in central and peripheral lesion and axonal regeneration.

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Ghibaudi, M; Boido, M; Vercelli, A

2017-11-01

New players are emerging in the game of peripheral and central nervous system injury since their physiopathological mechanisms remain partially elusive. These mechanisms are characterized by several molecules whose activation and/or modification following a trauma is often controlled at transcriptional level. In this scenario, microRNAs (miRNAs/miRs) have been identified as main actors in coordinating important molecular pathways in nerve or spinal cord injury (SCI). miRNAs are small non-coding RNAs whose functionality at network level is now emerging as a new level of complexity. Indeed they can act as an organized network to provide a precise control of several biological processes. Here we describe the functional synergy of some miRNAs in case of SCI and peripheral damage. In particular we show how several small RNAs can cooperate in influencing simultaneously the molecular pathways orchestrating axon regeneration, inflammation, apoptosis and remyelination. We report about the networks for which miRNA-target bindings have been experimentally demonstrated or inferred based on target prediction data: in both cases, the connection between one miRNA and its downstream pathway is derived from a validated observation or is predicted from the literature. Hence, we discuss the importance of miRNAs in some pathological processes focusing on their functional structure as participating in a cooperative and/or convergence network. Copyright © 2017 Elsevier Ltd. All rights reserved.

Identification of Novel and Conserved miRNAs from Extreme Halophyte, Oryza coarctata, a Wild Relative of Rice.

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Mondal, Tapan Kumar; Ganie, Showkat Ahmad; Debnath, Ananda Bhusan

2015-01-01

Oryza coarctata, a halophyte and wild relative of rice, is grown normally in saline water. MicroRNAs (miRNAs) are non-coding RNAs that play pivotal roles in every domain of life including stress response. There are very few reports on the discovery of salt-responsive miRNAs from halophytes. In this study, two small RNA libraries, one each from the control and salt-treated (450 mM NaCl for 24 h) leaves of O. coarctata were sequenced, which yielded 338 known and 95 novel miRNAs. Additionally, we used publicly available transcriptomics data of O. coarctata which led to the discovery of additional 48 conserved miRNAs along with their pre-miRNA sequences through in silico analysis. In total, 36 known and 7 novel miRNAs were up-regulated whereas, 12 known and 7 novel miRNAs were down-regulated under salinity stress. Further, 233 and 154 target genes were predicted for 48 known and 14 novel differentially regulated miRNAs respectively. These targets with the help of gene ontology analysis were found to be involved in several important biological processes that could be involved in salinity tolerance. Relative expression trends of majority of the miRNAs as detected by real time-PCR as well as predicted by Illumina sequencing were found to be coherent. Additionally, expression of most of the target genes was negatively correlated with their corresponding miRNAs. Thus, the present study provides an account of miRNA-target networking that is involved in salinity adaption of O. coarctata.

Widespread dysregulation of MiRNAs by MYCN amplification and chromosomal imbalances in neuroblastoma: association of miRNA expression with survival.

LENUS (Irish Health Repository)

Bray, Isabella

2009-01-01

MiRNAs regulate gene expression at a post-transcriptional level and their dysregulation can play major roles in the pathogenesis of many different forms of cancer, including neuroblastoma, an often fatal paediatric cancer originating from precursor cells of the sympathetic nervous system. We have analyzed a set of neuroblastoma (n = 145) that is broadly representative of the genetic subtypes of this disease for miRNA expression (430 loci by stem-loop RT qPCR) and for DNA copy number alterations (array CGH) to assess miRNA involvement in disease pathogenesis. The tumors were stratified and then randomly split into a training set (n = 96) and a validation set (n = 49) for data analysis. Thirty-seven miRNAs were significantly over- or under-expressed in MYCN amplified tumors relative to MYCN single copy tumors, indicating a potential role for the MYCN transcription factor in either the direct or indirect dysregulation of these loci. In addition, we also determined that there was a highly significant correlation between miRNA expression levels and DNA copy number, indicating a role for large-scale genomic imbalances in the dysregulation of miRNA expression. In order to directly assess whether miRNA expression was predictive of clinical outcome, we used the Random Forest classifier to identify miRNAs that were most significantly associated with poor overall patient survival and developed a 15 miRNA signature that was predictive of overall survival with 72.7% sensitivity and 86.5% specificity in the validation set of tumors. We conclude that there is widespread dysregulation of miRNA expression in neuroblastoma tumors caused by both over-expression of the MYCN transcription factor and by large-scale chromosomal imbalances. MiRNA expression patterns are also predicative of clinical outcome, highlighting the potential for miRNA mediated diagnostics and therapeutics.

MSDD: a manually curated database of experimentally supported associations among miRNAs, SNPs and human diseases.

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Yue, Ming; Zhou, Dianshuang; Zhi, Hui; Wang, Peng; Zhang, Yan; Gao, Yue; Guo, Maoni; Li, Xin; Wang, Yanxia; Zhang, Yunpeng; Ning, Shangwei; Li, Xia

2018-01-04

The MiRNA SNP Disease Database (MSDD, http://www.bio-bigdata.com/msdd/) is a manually curated database that provides comprehensive experimentally supported associations among microRNAs (miRNAs), single nucleotide polymorphisms (SNPs) and human diseases. SNPs in miRNA-related functional regions such as mature miRNAs, promoter regions, pri-miRNAs, pre-miRNAs and target gene 3'-UTRs, collectively called 'miRSNPs', represent a novel category of functional molecules. miRSNPs can lead to miRNA and its target gene dysregulation, and resulting in susceptibility to or onset of human diseases. A curated collection and summary of miRSNP-associated diseases is essential for a thorough understanding of the mechanisms and functions of miRSNPs. Here, we describe MSDD, which currently documents 525 associations among 182 human miRNAs, 197 SNPs, 153 genes and 164 human diseases through a review of more than 2000 published papers. Each association incorporates information on the miRNAs, SNPs, miRNA target genes and disease names, SNP locations and alleles, the miRNA dysfunctional pattern, experimental techniques, a brief functional description, the original reference and additional annotation. MSDD provides a user-friendly interface to conveniently browse, retrieve, download and submit novel data. MSDD will significantly improve our understanding of miRNA dysfunction in disease, and thus, MSDD has the potential to serve as a timely and valuable resource. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

OmniSearch: a semantic search system based on the Ontology for MIcroRNA Target (OMIT) for microRNA-target gene interaction data.

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Huang, Jingshan; Gutierrez, Fernando; Strachan, Harrison J; Dou, Dejing; Huang, Weili; Smith, Barry; Blake, Judith A; Eilbeck, Karen; Natale, Darren A; Lin, Yu; Wu, Bin; Silva, Nisansa de; Wang, Xiaowei; Liu, Zixing; Borchert, Glen M; Tan, Ming; Ruttenberg, Alan

2016-01-01

As a special class of non-coding RNAs (ncRNAs), microRNAs (miRNAs) perform important roles in numerous biological and pathological processes. The realization of miRNA functions depends largely on how miRNAs regulate specific target genes. It is therefore critical to identify, analyze, and cross-reference miRNA-target interactions to better explore and delineate miRNA functions. Semantic technologies can help in this regard. We previously developed a miRNA domain-specific application ontology, Ontology for MIcroRNA Target (OMIT), whose goal was to serve as a foundation for semantic annotation, data integration, and semantic search in the miRNA field. In this paper we describe our continuing effort to develop the OMIT, and demonstrate its use within a semantic search system, OmniSearch, designed to facilitate knowledge capture of miRNA-target interaction data. Important changes in the current version OMIT are summarized as: (1) following a modularized ontology design (with 2559 terms imported from the NCRO ontology); (2) encoding all 1884 human miRNAs (vs. 300 in previous versions); and (3) setting up a GitHub project site along with an issue tracker for more effective community collaboration on the ontology development. The OMIT ontology is free and open to all users, accessible at: http://purl.obolibrary.org/obo/omit.owl. The OmniSearch system is also free and open to all users, accessible at: http://omnisearch.soc.southalabama.edu/index.php/Software.

Computational identification of conserved microRNAs and their targets from expression sequence tags of blueberry (Vaccinium corybosum).

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Li, Xuyan; Hou, Yanming; Zhang, Li; Zhang, Wenhao; Quan, Chen; Cui, Yuhai; Bian, Shaomin

2014-01-01

MicroRNAs (miRNAs) are a class of endogenous, approximately 21nt in length, non-coding RNA, which mediate the expression of target genes primarily at post-transcriptional levels. miRNAs play critical roles in almost all plant cellular and metabolic processes. Although numerous miRNAs have been identified in the plant kingdom, the miRNAs in blueberry, which is an economically important small fruit crop, still remain totally unknown. In this study, we reported a computational identification of miRNAs and their targets in blueberry. By conducting an EST-based comparative genomics approach, 9 potential vco-miRNAs were discovered from 22,402 blueberry ESTs according to a series of filtering criteria, designated as vco-miR156-5p, vco-miR156-3p, vco-miR1436, vco-miR1522, vco-miR4495, vco-miR5120, vco-miR5658, vco-miR5783, and vco-miR5986. Based on sequence complementarity between miRNA and its target transcript, 34 target ESTs from blueberry and 70 targets from other species were identified for the vco-miRNAs. The targets were found to be involved in transcription, RNA splicing and binding, DNA duplication, signal transduction, transport and trafficking, stress response, as well as synthesis and metabolic process. These findings will greatly contribute to future research in regard to functions and regulatory mechanisms of blueberry miRNAs.

Expression of miRNAs confers enhanced tolerance to drought and salt stress in Finger millet (Eleusine coracona

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Nageshbabu R.

2013-08-01

Full Text Available Plants respond to the environmental cues in various ways, recent knowledge of RNA interference in conferring stress tolerance had become a new hope of developing tolerant varieties. Here we attempt to unfold the molecular mechanism of stress tolerance through miRNA profiling and expression analysis in Finger millet (Eleusine coracona under salt and drought stress conditions. The expression analysis of 12 stress specific conserved miRNAs was studied using semi-quantitative real time PCR and Northern blot assay. Our studies revealed that, although most of the miRNAs responded to the stresses, the expression of particular miRNA differed with the nature of stress and the tissue. The expression analysis was correlated with the existing data of their target genes. Abiotic stress up-regulated miRNAs are expected to target negative regulators of stress responses or positive regulators of processes that are inhibited by stresses. On the other hand, stress down-regulated miRNAs may repress the expression of positive regulators and/or stress up-regulated genes. Thus the current study of miRNAs and their targets under abiotic stress conditions displays miRNAs may be good candidates to attribute the stress tolerance in plants by transgenic technology.

High-Throughput Sequencing Identifies MicroRNAs from Posterior Intestine of Loach (Misgurnus anguillicaudatus) and Their Response to Intestinal Air-Breathing Inhibition.

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Huang, Songqian; Cao, Xiaojuan; Tian, Xianchang; Wang, Weimin

2016-01-01

MicroRNAs (miRNAs) exert important roles in animal growth, immunity, and development, and regulate gene expression at the post-transcriptional level. Knowledges about the diversities of miRNAs and their roles in accessory air-breathing organs (ABOs) of fish remain unknown. In this work, we used high-throughput sequencing to identify known and novel miRNAs from the posterior intestine, an important ABO, in loach (Misgurnus anguillicaudatus) under normal and intestinal air-breathing inhibited conditions. A total of 204 known and 84 novel miRNAs were identified, while 47 miRNAs were differentially expressed between the two small RNA libraries (i.e. between the normal and intestinal air-breathing inhibited group). Potential miRNA target genes were predicted by combining our transcriptome data of the posterior intestine of the loach under the same conditions, and then annotated using COG, GO, KEGG, Swissprot and Nr databases. The regulatory networks of miRNAs and their target genes were analyzed. The abundances of nine known miRNAs were validated by qRT-PCR. The relative expression profiles of six known miRNAs and their eight corresponding target genes, and two novel potential miRNAs were also detected. Histological characteristics of the posterior intestines in both normal and air-breathing inhibited group were further analyzed. This study contributes to our understanding on the functions and molecular regulatory mechanisms of miRNAs in accessory air-breathing organs of fish.

High-Throughput Sequencing Identifies MicroRNAs from Posterior Intestine of Loach (Misgurnus anguillicaudatus and Their Response to Intestinal Air-Breathing Inhibition.

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Songqian Huang

Full Text Available MicroRNAs (miRNAs exert important roles in animal growth, immunity, and development, and regulate gene expression at the post-transcriptional level. Knowledges about the diversities of miRNAs and their roles in accessory air-breathing organs (ABOs of fish remain unknown. In this work, we used high-throughput sequencing to identify known and novel miRNAs from the posterior intestine, an important ABO, in loach (Misgurnus anguillicaudatus under normal and intestinal air-breathing inhibited conditions. A total of 204 known and 84 novel miRNAs were identified, while 47 miRNAs were differentially expressed between the two small RNA libraries (i.e. between the normal and intestinal air-breathing inhibited group. Potential miRNA target genes were predicted by combining our transcriptome data of the posterior intestine of the loach under the same conditions, and then annotated using COG, GO, KEGG, Swissprot and Nr databases. The regulatory networks of miRNAs and their target genes were analyzed. The abundances of nine known miRNAs were validated by qRT-PCR. The relative expression profiles of six known miRNAs and their eight corresponding target genes, and two novel potential miRNAs were also detected. Histological characteristics of the posterior intestines in both normal and air-breathing inhibited group were further analyzed. This study contributes to our understanding on the functions and molecular regulatory mechanisms of miRNAs in accessory air-breathing organs of fish.

miRSponge: a manually curated database for experimentally supported miRNA sponges and ceRNAs.

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Wang, Peng; Zhi, Hui; Zhang, Yunpeng; Liu, Yue; Zhang, Jizhou; Gao, Yue; Guo, Maoni; Ning, Shangwei; Li, Xia

2015-01-01

In this study, we describe miRSponge, a manually curated database, which aims at providing an experimentally supported resource for microRNA (miRNA) sponges. Recent evidence suggests that miRNAs are themselves regulated by competing endogenous RNAs (ceRNAs) or 'miRNA sponges' that contain miRNA binding sites. These competitive molecules can sequester miRNAs to prevent them interacting with their natural targets to play critical roles in various biological and pathological processes. It has become increasingly important to develop a high quality database to record and store ceRNA data to support future studies. To this end, we have established the experimentally supported miRSponge database that contains data on 599 miRNA-sponge interactions and 463 ceRNA relationships from 11 species following manual curating from nearly 1200 published articles. Database classes include endogenously generated molecules including coding genes, pseudogenes, long non-coding RNAs and circular RNAs, along with exogenously introduced molecules including viral RNAs and artificial engineered sponges. Approximately 70% of the interactions were identified experimentally in disease states. miRSponge provides a user-friendly interface for convenient browsing, retrieval and downloading of dataset. A submission page is also included to allow researchers to submit newly validated miRNA sponge data. Database URL: http://www.bio-bigdata.net/miRSponge. © The Author(s) 2015. Published by Oxford University Press.

miRNA-135a promotes breast cancer cell migration and invasion by targeting HOXA10

International Nuclear Information System (INIS)

Chen, Yating; Zhang, Hongwei; Ma, Duan; Zhang, Jin; Wang, Huijun; Zhao, Jiayi; Xu, Cheng; Du, Yingying; Luo, Xin; Zheng, Fengyun; Liu, Rui

2012-01-01

miRNAs are a group of small RNA molecules regulating target genes by inducing mRNA degradation or translational repression. Aberrant expression of miRNAs correlates with various cancers. Although miR-135a has been implicated in several other cancers, its role in breast cancer is unknown. HOXA10 however, is associated with multiple cancer types and was recently shown to induce p53 expression in breast cancer cells and reduce their invasive ability. Because HOXA10 is a confirmed miR-135a target in more than one tissue, we examined miR-135a levels in relation to breast cancer phenotypes to determine if miR-135a plays role in this cancer type. Expression levels of miR-135a in tissues and cells were determined by poly (A)-RT PCR. The effect of miR-135a on proliferation was evaluated by CCK8 assay, cell migration and invasion were evaluated by transwell migration and invasion assays, and target protein expression was determined by western blotting. GFP and luciferase reporter plasmids were constructed to confirm the action of miR-135a on downstream target genes including HOXA10. Results are reported as means ± S.D. and differences were tested for significance using 2-sided Student's t-test. Here we report that miR-135a was highly expressed in metastatic breast tumors. We found that the expression of miR-135a was required for the migration and invasion of breast cancer cells, but not their proliferation. HOXA10, which encodes a transcription factor required for embryonic development and is a metastasis suppressor in breast cancer, was shown to be a direct target of miR-135a in breast cancer cells. Our analysis showed that miR-135a suppressed the expression of HOXA10 both at the mRNA and protein level, and its ability to promote cellular migration and invasion was partially reversed by overexpression of HOXA10. In summary, our results indicate that miR-135a is an onco-miRNA that can promote breast cancer cell migration and invasion. HOXA10 is a target gene for mi

Progress risk assessment of oral premalignant lesions with saliva miRNA analysis

International Nuclear Information System (INIS)

Yang, Ya; Li, Yue-xiu; Yang, Xi; Jiang, Long; Zhou, Zuo-jun; Zhu, Ya-qin

2013-01-01

Oral cancer develops through multi-stages: from normal to mild (low grade) dysplasia (LGD), moderate dysplasia, and severe (high grade) dysplasia (HGD), to carcinoma in situ (CIS) and finally invasive oral squamous cell carcinomas (OSCC). Clinical and histological assessments are not reliable in predicting which precursor lesions will progress. The aim of this study was to assess the potential of a noninvasive approach to assess progress risk of oral precancerous lesions. We first used microRNA microarray to profile progressing LGD oral premaligant lesions (OPLs) from non-progressing LGD OPLs in order to explore the possible microRNAs deregulated in low grade OPLs which later progressed to HGD or OSCC. We then used RT-qPCR to detect miRNA targets from the microarray results in saliva samples of these patients. We identified a specific miRNA signature that is aberrantly expressed in progressing oral LGD leukoplakias. Similar expression patterns were detected in saliva samples from these patients. These results show promise for using saliva miRNA signature for monitoring of cancer precursor lesions and early detection of disease progression

MTUS1 tumor suppressor and its miRNA regulators in fibroadenoma and breast cancer.

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Kara, Murat; Kaplan, Mehmet; Bozgeyik, Ibrahim; Ozcan, Onder; Celik, Ozgur Ilhan; Bozgeyik, Esra; Yumrutas, Onder

2016-08-10

Breast cancer is major public health problem predominantly effects female population. Current therapeutic approaches to deal with breast cancer are still lack of effectiveness. Thus, identifying/developing novel strategies to fight against breast cancer is very important. The frequent deletions at 8p21.3-22 chromosomal location nearby D8S254 marker enabled the discovery of a novel tumor suppressor gene, MTUS1. Subsequently, MTUS1 was demonstrated to be less expressed in a variety cancer types including breast cancer. Also, it is obvious that gene expression is widely regulated by miRNAs. Here, we aimed to report differential expression of MTUS1 and its regulatory miRNAs in breast cancer and fibroadenoma tissues. Dynamic analysis of MTUS1 expression levels and its miRNAs regulators were attained by Fluidigm 96×96 Dynamic Array Expression chips and reactions were performed in Fluidigm BioMark™ HD System qPCR. Consequently, MTUS1 mRNA levels were significantly diminished in breast cancer tissues and elevated in fibroadenoma tissues. Also, among MTUS1 targeting miRNAs, miR-183-5p was identified to be overexpressed in breast cancer and down-regulated in fibroadenoma tissues. Also, expression levels of MTUS1 and miR-183-5p were well correlated with clinical parameters. In particular, MTUS1 expression was found to be diminished and miR-183-5p expression was elevated with the advancing stage. In conclusion, as a potential therapeutic target, miR-183-5p can be a chief regulator of MTUS1 and MTUS1-miR-183-5p axis may have significant influence in the pathology of breast cancer. Copyright © 2016 Elsevier B.V. All rights reserved.

Micro-RNA-128 (miRNA-128) down-regulation in glioblastoma targets ARP5 (ANGPTL6), Bmi-1 and E2F-3a, key regulators of brain cell proliferation.

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Cui, J G; Zhao, Y; Sethi, P; Li, Y Y; Mahta, A; Culicchia, F; Lukiw, W J

2010-07-01

High density micro-RNA (miRNA) arrays, fluorescent-reporter miRNA assay and Northern miRNA dot-blot analysis show that a brain-enriched miRNA-128 is significantly down-regulated in glioblastoma multiforme (GBM) and in GBM cell lines when compared to age-matched controls. The down-regulation of miRNA-128 was found to inversely correlate with WHO tumor grade. Three bioinformatics-verified miRNA-128 targets, angiopoietin-related growth factor protein 5 (ARP5; ANGPTL6), a transcription suppressor that promotes stem cell renewal and inhibits the expression of known tumor suppressor genes involved in senescence and differentiation, Bmi-1, and a transcription factor critical for the control of cell-cycle progression, E2F-3a, were found to be up-regulated. Addition of exogenous miRNA-128 to CRL-1690 and CRL-2610 GBM cell lines (a) restored 'homeostatic' ARP5 (ANGPTL6), Bmi-1 and E2F-3a expression, and (b) significantly decreased the proliferation of CRL-1690 and CRL-2610 cell lines. Our data suggests that down-regulation of miRNA-128 may contribute to glioma and GBM, in part, by coordinately up-regulating ARP5 (ANGPTL6), Bmi-1 and E2F-3a, resulting in the proliferation of undifferentiated GBM cells.

Mi-DISCOVERER: A bioinformatics tool for the detection of mi-RNA in human genome.

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Arshad, Saadia; Mumtaz, Asia; Ahmad, Freed; Liaquat, Sadia; Nadeem, Shahid; Mehboob, Shahid; Afzal, Muhammad

2010-11-27

MicroRNAs (miRNAs) are 22 nucleotides non-coding RNAs that play pivotal regulatory roles in diverse organisms including the humans and are difficult to be identified due to lack of either sequence features or robust algorithms to efficiently identify. Therefore, we made a tool that is Mi-Discoverer for the detection of miRNAs in human genome. The tools used for the development of software are Microsoft Office Access 2003, the JDK version 1.6.0, BioJava version 1.0, and the NetBeans IDE version 6.0. All already made miRNAs softwares were web based; so the advantage of our project was to make a desktop facility to the user for sequence alignment search with already identified miRNAs of human genome present in the database. The user can also insert and update the newly discovered human miRNA in the database. Mi-Discoverer, a bioinformatics tool successfully identifies human miRNAs based on multiple sequence alignment searches. It's a non redundant database containing a large collection of publicly available human miRNAs.

In Silico Analysis of Small RNAs Suggest Roles for Novel and Conserved miRNAs in the Formation of Epigenetic Memory in Somatic Embryos of Norway Spruce.

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Yakovlev, Igor A; Fossdal, Carl G

2017-01-01

Epigenetic memory in Norway spruce affects the timing of bud burst and bud set, vitally important adaptive traits for this long-lived forest species. Epigenetic memory is established in response to the temperature conditions during embryogenesis. Somatic embryogenesis at different epitype inducing (EpI) temperatures closely mimics the natural processes of epigenetic memory formation in seeds, giving rise to epigenetically different clonal plants in a reproducible and predictable manner, with respect to altered bud phenology. MicroRNAs (miRNAs) and other small non-coding RNAs (sRNAs) play an essential role in the regulation of plant gene expression and may affect this epigenetic mechanism. We used NGS sequencing and computational in silico methods to identify and profile conserved and novel miRNAs among small RNAs in embryogenic tissues of Norway spruce at three EpI temperatures (18, 23 and 28°C). We detected three predominant classes of sRNAs related to a length of 24 nt, followed by a 21-22 nt class and a third 31 nt class of sRNAs. More than 2100 different miRNAs within the prevailing length 21-22 nt were identified. Profiling these putative miRNAs allowed identification of 1053 highly expressed miRNAs, including 523 conserved and 530 novels. 654 of these miRNAs were found to be differentially expressed (DEM) depending on EpI temperature. For most DEMs, we defined their putative mRNA targets. The targets represented mostly by transcripts of multiple-repeats proteins, like TIR, NBS-LRR, PPR and TPR repeat, Clathrin/VPS proteins, Myb-like, AP2, etc. Notably, 124 DE miRNAs targeted 203 differentially expressed epigenetic regulators. Developing Norway spruce embryos possess a more complex sRNA structure than that reported for somatic tissues. A variety of the predicted miRNAs showed distinct EpI temperature dependent expression patterns. These putative EpI miRNAs target spruce genes with a wide range of functions, including genes known to be involved in epigenetic

In Silico Analysis of Small RNAs Suggest Roles for Novel and Conserved miRNAs in the Formation of Epigenetic Memory in Somatic Embryos of Norway Spruce

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Igor A. Yakovlev

2017-09-01

Full Text Available Epigenetic memory in Norway spruce affects the timing of bud burst and bud set, vitally important adaptive traits for this long-lived forest species. Epigenetic memory is established in response to the temperature conditions during embryogenesis. Somatic embryogenesis at different epitype inducing (EpI temperatures closely mimics the natural processes of epigenetic memory formation in seeds, giving rise to epigenetically different clonal plants in a reproducible and predictable manner, with respect to altered bud phenology. MicroRNAs (miRNAs and other small non-coding RNAs (sRNAs play an essential role in the regulation of plant gene expression and may affect this epigenetic mechanism. We used NGS sequencing and computational in silico methods to identify and profile conserved and novel miRNAs among small RNAs in embryogenic tissues of Norway spruce at three EpI temperatures (18, 23 and 28°C. We detected three predominant classes of sRNAs related to a length of 24 nt, followed by a 21–22 nt class and a third 31 nt class of sRNAs. More than 2100 different miRNAs within the prevailing length 21–22 nt were identified. Profiling these putative miRNAs allowed identification of 1053 highly expressed miRNAs, including 523 conserved and 530 novels. 654 of these miRNAs were found to be differentially expressed (DEM depending on EpI temperature. For most DEMs, we defined their putative mRNA targets. The targets represented mostly by transcripts of multiple-repeats proteins, like TIR, NBS-LRR, PPR and TPR repeat, Clathrin/VPS proteins, Myb-like, AP2, etc. Notably, 124 DE miRNAs targeted 203 differentially expressed epigenetic regulators. Developing Norway spruce embryos possess a more complex sRNA structure than that reported for somatic tissues. A variety of the predicted miRNAs showed distinct EpI temperature dependent expression patterns. These putative EpI miRNAs target spruce genes with a wide range of functions, including genes known to be

Covalent Strategies for Targeting Messenger and Non-Coding RNAs: An Updated Review on siRNA, miRNA and antimiR Conjugates

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Santiago Grijalvo

2018-02-01

Full Text Available Oligonucleotide-based therapy has become an alternative to classical approaches in the search of novel therapeutics involving gene-related diseases. Several mechanisms have been described in which demonstrate the pivotal role of oligonucleotide for modulating gene expression. Antisense oligonucleotides (ASOs and more recently siRNAs and miRNAs have made important contributions either in reducing aberrant protein levels by sequence-specific targeting messenger RNAs (mRNAs or restoring the anomalous levels of non-coding RNAs (ncRNAs that are involved in a good number of diseases including cancer. In addition to formulation approaches which have contributed to accelerate the presence of ASOs, siRNAs and miRNAs in clinical trials; the covalent linkage between non-viral vectors and nucleic acids has also added value and opened new perspectives to the development of promising nucleic acid-based therapeutics. This review article is mainly focused on the strategies carried out for covalently modifying siRNA and miRNA molecules. Examples involving cell-penetrating peptides (CPPs, carbohydrates, polymers, lipids and aptamers are discussed for the synthesis of siRNA conjugates whereas in the case of miRNA-based drugs, this review article makes special emphasis in using antagomiRs, locked nucleic acids (LNAs, peptide nucleic acids (PNAs as well as nanoparticles. The biomedical applications of siRNA and miRNA conjugates are also discussed.

Preferential microRNA targeting revealed by in vivo competitive binding and differential Argonaute immunoprecipitation.

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Werfel, Stanislas; Leierseder, Simon; Ruprecht, Benjamin; Kuster, Bernhard; Engelhardt, Stefan

2017-09-29

MicroRNAs (miRNAs) have been described to simultaneously inhibit hundreds of targets, albeit to a modest extent. It was recently proposed that there could exist more specific, exceptionally strong binding to a subgroup of targets. However, it is unknown, whether this is the case and how such targets can be identified. Using Argonaute2-ribonucleoprotein immunoprecipitation and in vivo competitive binding assays, we demonstrate for miRNAs-21, -199-3p and let-7 exceptional regulation of a subset of targets, which are characterized by preferential miRNA binding. We confirm this finding by analysis of independent quantitative proteome and transcriptome datasets obtained after miRNA silencing. Our data suggest that mammalian miRNA activity is guided by preferential binding of a small set of 3'-untranslated regions, thereby shaping a steep gradient of regulation between potential targets. Our approach can be applied for transcriptome-wide identification of such targets independently of the presence of seed complementary sequences or other predictors. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Proteomic screening of human targets of viral microRNAs reveals functions associated with immune evasion and angiogenesis.

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Amelia M Gallaher

Full Text Available Kaposi's sarcoma (KS is caused by infection with Kaposi's sarcoma-associated herpesvirus (KSHV. The virus expresses unique microRNAs (miRNAs, but the targets and functions of these miRNAs are not completely understood. In order to identify human targets of viral miRNAs, we measured protein expression changes caused by multiple KSHV miRNAs using pulsed stable labeling with amino acids in cell culture (pSILAC in primary endothelial cells. This led to the identification of multiple human genes that are repressed at the protein level, but not at the miRNA level. Further analysis also identified that KSHV miRNAs can modulate activity or expression of upstream regulatory factors, resulting in suppressed activation of a protein involved in leukocyte recruitment (ICAM1 following lysophosphatidic acid treatment, as well as up-regulation of a pro-angiogenic protein (HIF1α, and up-regulation of a protein involved in stimulating angiogenesis (HMOX1. This study aids in our understanding of miRNA mechanisms of repression and miRNA contributions to viral pathogenesis.

MicroRNA-145 targets YES and STAT1 in colon cancer cells

DEFF Research Database (Denmark)

Gregersen, Lea H; Jacobsen, Anders B; Frankel, Lisa

2010-01-01

miRNA overexpression. Gene Ontology analysis showed an overrepresentation of genes involved in cell death, cellular growth and proliferation, cell cycle, gene expression and cancer. A number of the identified miRNA targets have previously been implicated in cancer, including YES, FSCN1, ADAM17, BIRC2......, VANGL1 as well as the transcription factor STAT1. Both YES and STAT1 were verified as direct miR-145 targets. CONCLUSIONS/SIGNIFICANCE: The study identifies and validates new cancer-relevant direct targets of miR-145 in colon cancer cells and hereby adds important mechanistic understanding of the tumor......BACKGROUND: MicroRNAs (miRNAs) have emerged as important gene regulators and are recognized as key players in tumorigenesis. miR-145 is reported to be down-regulated in several cancers, but knowledge of its targets in colon cancer remains limited. METHODOLOGY/PRINCIPAL FINDINGS: To investigate...

Transcriptome-Wide Analysis of Botrytis elliptica Responsive microRNAs and Their Targets in Lilium Regale Wilson by High-Throughput Sequencing and Degradome Analysis

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Xue Gao

2017-05-01

Full Text Available MicroRNAs, as master regulators of gene expression, have been widely identified and play crucial roles in plant-pathogen interactions. A fatal pathogen, Botrytis elliptica, causes the serious folia disease of lily, which reduces production because of the high susceptibility of most cultivated species. However, the miRNAs related to Botrytis infection of lily, and the miRNA-mediated gene regulatory networks providing resistance to B. elliptica in lily remain largely unexplored. To systematically dissect B. elliptica-responsive miRNAs and their target genes, three small RNA libraries were constructed from the leaves of Lilium regale, a promising Chinese wild Lilium species, which had been subjected to mock B. elliptica treatment or B. elliptica infection for 6 and 24 h. By high-throughput sequencing, 71 known miRNAs belonging to 47 conserved families and 24 novel miRNA were identified, of which 18 miRNAs were downreguleted and 13 were upregulated in response to B. elliptica. Moreover, based on the lily mRNA transcriptome, 22 targets for 9 known and 1 novel miRNAs were identified by the degradome sequencing approach. Most target genes for elliptica-responsive miRNAs were involved in metabolic processes, few encoding different transcription factors, including ELONGATION FACTOR 1 ALPHA (EF1a and TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR 2 (TCP2. Furthermore, the expression patterns of a set of elliptica-responsive miRNAs and their targets were validated by quantitative real-time PCR. This study represents the first transcriptome-based analysis of miRNAs responsive to B. elliptica and their targets in lily. The results reveal the possible regulatory roles of miRNAs and their targets in B. elliptica interaction, which will extend our understanding of the mechanisms of this disease in lily.

Expression profiles of miRNA-122 and its target CAT1 in minipigs (Sus scrofa) fed a high-cholesterol diet

DEFF Research Database (Denmark)

Cirera Salicio, Susanna; Birck, Malene Muusfeldt; Busk, Peter Kamp

2010-01-01

The Göttingen minipig is an excellent model for studying effects of dietary high-fat intake on obesity. In this study, we analyzed the expression level of microRNA-122 (miRNA-122) and its target mRNA, CAT1, in intact young male minipigs fed either high-cholesterol or standard diet for 11 wk. Mi...... with a decrease in the expression of miRNA-122, confirming the implication of this microRNA in obesity. Gene expression levels of CAT1 did not differ between groups.......RNA-122 and CAT1 are known to be important regulators of lipid metabolism. The weight of the young minipigs was monitored once a week during the feeding period; measurements of total cholesterol, triglycerides, high-density lipoproteins, and low-density lipoproteins were recorded at 4 time points (8, 14...

Hepatitis A virus-encoded miRNAs attenuate the accumulation of viral genomic RNAs in infected cells.

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Shi, Jiandong; Sun, Jing; Wu, Meini; Hu, Ningzhu; Hu, Yunzhang

2016-06-01

The establishment of persistent infection with hepatitis A virus (HAV) is the common result of most HAV/cell culture systems. Previous observations show that the synthesis of viral RNAs is reduced during infection. However, the underlying mechanism is poorly understood. We characterized three HAV-encoded miRNAs in our previous study. In this study, we aim to investigate the impact of these miRNAs on the accumulation of viral RNAs. The results indicated that the synthesis of viral genomic RNAs was dramatically reduced (more than 75 % reduction, P viral miRNA mimics. Conversely, they were significantly increased (more than 3.3-fold addition, P viral miRNA inhibitors. The luciferase reporter assay of miRNA targets showed that viral miRNAs were fully complementary to specific sites of the viral plus or minus strand RNA and strongly inhibited their expressions. Further data showed that the relative abundance of viral genomic RNA fragments that contain miRNA targets was also dramatically reduced (more than 80 % reduction, P viral miRNAs were overexpressed with miRNA mimics. In contrast, they were significantly increased (approximately 2-fold addition, P viral miRNAs were inhibited with miRNA inhibitors. In conclusion, these data suggest a possible mechanism for the reduction of viral RNA synthesis during HAV infection. Thus, we propose that it is likely that RNA virus-derived miRNA could serve as a self-mediated feedback regulator during infection.

Transcriptome and Degradome of microRNAs and Their Targets in Response to Drought Stress in the Plants of a Diploid and Its Autotetraploid Paulownia australis.

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Suyan Niu

Full Text Available MicroRNAs (miRNAs are small, non-coding RNAs that play vital roles in plant growth, development, and stress response. Increasing numbers of studies aimed at discovering miRNAs and analyzing their functions in plants are being reported. In this study, we investigated the effect of drought stress on the expression of miRNAs and their targets in plants of a diploid and derived autotetraploid Paulownia australis. Four small RNA (sRNA libraries and four degradome libraries were constructed from diploid and autotetraploid P. australis plants treated with either 75% or 25% relative soil water content. A total of 33 conserved and 104 novel miRNAs (processing precision value > 0.1 were identified, and 125 target genes were identified for 36 of the miRNAs by using the degradome sequencing. Among the identified miRNAs, 54 and 68 were differentially expressed in diploid and autotetraploid plants under drought stress (25% relative soil water content, respectively. The expressions of miRNAs and target genes were also validated by quantitative real-time PCR. The results showed that the relative expression trends of the randomly selected miRNAs were similar to the trends predicted by Illumina sequencing. And the correlations between miRNAs and their target genes were also analyzed. Furthermore, the functional analysis showed that most of these miRNAs and target genes were associated with plant development and environmental stress response. This study provided molecular evidence for the possible involvement of certain miRNAs in the drought response and/or tolerance in P. australis, and certain level of differential expression between diploid and autotetraploid plants.

A systems biology approach identified different regulatory networks targeted by KSHV miR-K12-11 in B cells and endothelial cells.

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Yang, Yajie; Boss, Isaac W; McIntyre, Lauren M; Renne, Rolf

2014-08-08

Kaposi's sarcoma associated herpes virus (KSHV) is associated with tumors of endothelial and lymphoid origin. During latent infection, KSHV expresses miR-K12-11, an ortholog of the human tumor gene hsa-miR-155. Both gene products are microRNAs (miRNAs), which are important post-transcriptional regulators that contribute to tissue specific gene expression. Advances in target identification technologies and molecular interaction databases have allowed a systems biology approach to unravel the gene regulatory networks (GRNs) triggered by miR-K12-11 in endothelial and lymphoid cells. Understanding the tissue specific function of miR-K12-11 will help to elucidate underlying mechanisms of KSHV pathogenesis. Ectopic expression of miR-K12-11 differentially affected gene expression in BJAB cells of lymphoid origin and TIVE cells of endothelial origin. Direct miRNA targeting accounted for a small fraction of the observed transcriptome changes: only 29 genes were identified as putative direct targets of miR-K12-11 in both cell types. However, a number of commonly affected biological pathways, such as carbohydrate metabolism and interferon response related signaling, were revealed by gene ontology analysis. Integration of transcriptome profiling, bioinformatic algorithms, and databases of protein-protein interactome from the ENCODE project identified different nodes of GRNs utilized by miR-K12-11 in a tissue-specific fashion. These effector genes, including cancer associated transcription factors and signaling proteins, amplified the regulatory potential of a single miRNA, from a small set of putative direct targets to a larger set of genes. This is the first comparative analysis of miRNA-K12-11's effects in endothelial and B cells, from tissues infected with KSHV in vivo. MiR-K12-11 was able to broadly modulate gene expression in both cell types. Using a systems biology approach, we inferred that miR-K12-11 establishes its GRN by both repressing master TFs and influencing

miRNA profiles in cerebrospinal fluid from patients with central hypersomnias

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Holm, Anja; Bang-Berthelsen, Claus Heiner; Knudsen, Stine

2014-01-01

addressed whether miRNA levels are altered in the cerebrospinal fluid (CSF) of patients with central hypersomnias. We conducted high-throughput analyses of miRNAs in CSF from patients using quantitative real-time polymerase chain reaction panels. We identified 13, 9, and 11 miRNAs with a more than two...

Evaluation of a new high-dimensional miRNA profiling platform

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Lamblin Anne-Francoise

2009-08-01

Full Text Available Abstract Background MicroRNAs (miRNAs are a class of approximately 22 nucleotide long, widely expressed RNA molecules that play important regulatory roles in eukaryotes. To investigate miRNA function, it is essential that methods to quantify their expression levels be available. Methods We evaluated a new miRNA profiling platform that utilizes Illumina's existing robust DASL chemistry as the basis for the assay. Using total RNA from five colon cancer patients and four cell lines, we evaluated the reproducibility of miRNA expression levels across replicates and with varying amounts of input RNA. The beta test version was comprised of 735 miRNA targets of Illumina's miRNA profiling application. Results Reproducibility between sample replicates within a plate was good (Spearman's correlation 0.91 to 0.98 as was the plate-to-plate reproducibility replicates run on different days (Spearman's correlation 0.84 to 0.98. To determine whether quality data could be obtained from a broad range of input RNA, data obtained from amounts ranging from 25 ng to 800 ng were compared to those obtained at 200 ng. No effect across the range of RNA input was observed. Conclusion These results indicate that very small amounts of starting material are sufficient to allow sensitive miRNA profiling using the Illumina miRNA high-dimensional platform. Nonlinear biases were observed between replicates, indicating the need for abundance-dependent normalization. Overall, the performance characteristics of the Illumina miRNA profiling system were excellent.

Growth promotion-related miRNAs in Oncidium orchid roots colonized by the endophytic fungus Piriformospora indica.

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Wei Ye

Full Text Available Piriformospora indica, an endophytic fungus of Sebacinales, colonizes the roots of a wide range of host plants and establishes various benefits for the plants. In this work, we describe miRNAs which are upregulated in Oncidium orchid roots after colonization by the fungus. Growth promotion and vigorous root development were observed in Oncidium hybrid orchid, while seedlings were colonized by P. indica. We performed a genome-wide expression profiling of small RNAs in Oncidium orchid roots either colonized or not-colonized by P. indica. After sequencing, 24,570,250 and 24744,141 clean reads were obtained from two libraries. 13,736 from 17,036,953 unique sequences showed homology to either 86 miRNA families described in 41 plant species, or to 46 potential novel miRNAs, or to 51 corresponding miRNA precursors. The predicted target genes of these miRNAs are mainly involved in auxin signal perception and transduction, transcription, development and plant defense. The expression analysis of miRNAs and target genes demonstrated the regulatory functions they may participate in. This study revealed that growth stimulation of the Oncidium orchid after colonization by P. indica includes an intricate network of miRNAs and their targets. The symbiotic function of P. indica on Oncidium orchid resembles previous findings on Chinese cabbage. This is the first study on growth regulation and development of Oncidium orchid by miRNAs induced by the symbiotic fungus P. indica.

Evolutionary Transitions of MicroRNA-Target Pairs

KAUST Repository

Nozawa, Masafumi; Fujimi, Mai; Iwamoto, Chie; Onizuka, Kanako; Fukuda, Nana; Ikeo, Kazuho; Gojobori, Takashi

2016-01-01

How newly generated microRNA (miRNA) genes are integrated into gene regulatory networks during evolution is fundamental in understanding the molecular and evolutionary bases of robustness and plasticity in gene regulation. A recent model proposed that after the birth of a miRNA, the miRNA is generally integrated into the network by decreasing the number of target genes during evolution. However, this decreasing model remains to be carefully examined by considering in vivo conditions. In this study, we therefore compared the number of target genes among miRNAs with different ages, combining experiments with bioinformatics predictions. First, we focused on three Drosophila miRNAs with different ages. As a result, we found that an older miRNA has a greater number of target genes than a younger miRNA, suggesting the increasing number of targets for each miRNA during evolution (increasing model). To further confirm our results, we also predicted all target genes for all miRNAs in D. melanogaster, considering co-expression of miRNAs and mRNAs in vivo. The results obtained also do not support the decreasing model but are reasonably consistent with the increasing model of miRNA-target pairs. Furthermore, our large-scale analyses of currently available experimental data of miRNA-target pairs also showed a weak but the same trend in humans. These results indicate that the current decreasing model of miRNA-target pairs should be reconsidered and the increasing model may be more appropriate to explain the evolutionary transitions of miRNA-target pairs in many organisms.

Evolutionary Transitions of MicroRNA-Target Pairs

KAUST Repository

Nozawa, Masafumi

2016-04-27

How newly generated microRNA (miRNA) genes are integrated into gene regulatory networks during evolution is fundamental in understanding the molecular and evolutionary bases of robustness and plasticity in gene regulation. A recent model proposed that after the birth of a miRNA, the miRNA is generally integrated into the network by decreasing the number of target genes during evolution. However, this decreasing model remains to be carefully examined by considering in vivo conditions. In this study, we therefore compared the number of target genes among miRNAs with different ages, combining experiments with bioinformatics predictions. First, we focused on three Drosophila miRNAs with different ages. As a result, we found that an older miRNA has a greater number of target genes than a younger miRNA, suggesting the increasing number of targets for each miRNA during evolution (increasing model). To further confirm our results, we also predicted all target genes for all miRNAs in D. melanogaster, considering co-expression of miRNAs and mRNAs in vivo. The results obtained also do not support the decreasing model but are reasonably consistent with the increasing model of miRNA-target pairs. Furthermore, our large-scale analyses of currently available experimental data of miRNA-target pairs also showed a weak but the same trend in humans. These results indicate that the current decreasing model of miRNA-target pairs should be reconsidered and the increasing model may be more appropriate to explain the evolutionary transitions of miRNA-target pairs in many organisms.

miRNA Signatures of Insulin Resistance in Obesity.

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Jones, Angela; Danielson, Kirsty M; Benton, Miles C; Ziegler, Olivia; Shah, Ravi; Stubbs, Richard S; Das, Saumya; Macartney-Coxson, Donia

2017-10-01

Extracellular microRNAs (miRNAs) represent functional biomarkers for obesity and related disorders; this study investigated plasma miRNAs in insulin resistance phenotypes in obesity. One hundred seventy-five miRNAs were analyzed in females with obesity (insulin sensitivity, n = 11; insulin resistance, n = 19; type 2 diabetes, n = 15) and without obesity (n = 12). Correlations between miRNA level and clinical parameters and levels of 15 miRNAs in a murine obesity model were investigated. One hundred six miRNAs were significantly (adjusted P ≤ 0.05) different between controls and at least one obesity phenotype, including miRNAs with the following attributes: previously reported roles in obesity and altered circulating levels (e.g., miR-122, miR-192); known roles in obesity but no reported changes in circulating levels (e.g., miR-378a); and no current reported role in, or association with, obesity (e.g., miR-28-5p, miR-374b, miR-32). The miRNAs in the latter group were found to be associated with extracellular vesicles. Forty-eight miRNAs showed significant correlations with clinical parameters; stepwise regression retained let-7b, miR-144-5p, miR-34a, and miR-532-5p in a model predictive of insulin resistance (R 2  = 0.57, P = 7.5 × 10 -8 ). Both miR-378a and miR-122 were perturbed in metabolically relevant tissues in a murine model of obesity. This study expands on the role of extracellular miRNAs in insulin-resistant phenotypes of obesity and identifies candidate miRNAs not previously associated with obesity. © 2017 The Obesity Society.

Deep sequencing of small RNAs identifies canonical and non-canonical miRNA and endogenous siRNAs in mammalian somatic tissues.

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Castellano, Leandro; Stebbing, Justin

2013-03-01

MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression. They are characterized by specific maturation processes defined by canonical and non-canonical biogenic pathways. Analysis of ∼0.5 billion sequences from mouse data sets derived from different tissues, developmental stages and cell types, partly characterized by either ablation or mutation of the main proteins belonging to miRNA processor complexes, reveals 66 high-confidence new genomic loci coding for miRNAs that could be processed in a canonical or non-canonical manner. A proportion of the newly discovered miRNAs comprises mirtrons, for which we define a new sub-class. Notably, some of these newly discovered miRNAs are generated from untranslated and open reading frames of coding genes, and we experimentally validate these. We also show that many annotated miRNAs do not present miRNA-like features, as they are neither processed by known processing complexes nor loaded on AGO2; this indicates that the current miRNA miRBase database list should be refined and re-defined. Accordingly, a group of them map on ribosomal RNA molecules, whereas others cannot undergo genuine miRNA biogenesis. Notably, a group of annotated miRNAs are Dgcr8 independent and DICER dependent endogenous small interfering RNAs that derive from a unique hairpin formed from a short interspersed nuclear element.

Strategies to identify microRNA targets: New advances

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MicroRNAs (miRNAs) are small regulatory RNA molecules functioning to modulate gene expression at the post-transcriptional level, and playing an important role in many developmental and physiological processes. Ten thousand miRNAs have been discovered in various organisms. Although considerable progr...

Transcriptome Analysis of mRNA and miRNA in Somatic Embryos of Larix leptolepis Subjected to Hydrogen Treatment

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Yali Liu

2016-11-01

Full Text Available Hydrogen is a therapeutic antioxidant that has been used extensively in clinical trials. It also acts as a bioactive molecule that can alleviate abiotic stress in plants. However, the biological effects of hydrogen in somatic embryos and the underlying molecular basis remain largely unknown. In this study, the morphological and physiological influence of exogenous H2 treatment during somatic embryogenesis was characterized in Larix leptolepis Gordon. The results showed that exposure to hydrogen increased the proportions of active pro-embryogenic cells and normal somatic embryos. We sequenced mRNA and microRNA (miRNA libraries to identify global transcriptome changes at different time points during H2 treatment of larch pro-embryogenic masses (PEMs. A total of 45,393 mRNAs and 315 miRNAs were obtained. Among them, 4253 genes and 96 miRNAs were differentially expressed in the hydrogen-treated libraries compared with the control. Further, a large number of the differentially expressed mRNAs and miRNAs were related to reactive oxygen species (ROS homeostasis and cell cycle regulation. We also identified 4399 potential target genes for 285 of the miRNAs. The differential expression data and the mRNA-miRNA interaction network described here provide new insights into the molecular mechanisms that determine the performance of PEMs exposed to H2 during somatic embryogenesis.

A compilation of Web-based research tools for miRNA analysis.

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Shukla, Vaibhav; Varghese, Vinay Koshy; Kabekkodu, Shama Prasada; Mallya, Sandeep; Satyamoorthy, Kapaettu

2017-09-01

Since the discovery of microRNAs (miRNAs), a class of noncoding RNAs that regulate the gene expression posttranscriptionally in sequence-specific manner, there has been a release of number of tools useful for both basic and advanced applications. This is because of the significance of miRNAs in many pathophysiological conditions including cancer. Numerous bioinformatics tools that have been developed for miRNA analysis have their utility for detection, expression, function, target prediction and many other related features. This review provides a comprehensive assessment of web-based tools for the miRNA analysis that does not require prior knowledge of any computing languages. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Circulating Serum miRNAs as Diagnostic Markers for Colorectal Cancer.

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Abdel-Rahman N Zekri

Full Text Available The study was designed to assess the possibility of using circulating miRNAs (serum miRNAs as diagnostic biomarkers in colorectal cancer (CRC and to identify their possibility as candidates for targeted therapy.The study involved two sample sets: 1- a training set which included 90 patients with colorectal related disease (30 with CRC, 18 with inflammatory bowel disease (IBD, 18 with colonic polyps (CP and 24 with different colonic symptoms but without any colonoscopic abnormality who were enrolled as control group and 2- a validation set which included 100 CRC patients. Serum miRNAs were extracted from all subjects to assess the expression profiles for the following miRNAs (miR-17, miR-18a, miR-19a, miR-19b, miR-20a, miR-21, miR-146a, miR-223, miR-24, miR-454, miR-183, miR-135a, miR- 135b and miR- 92a using the custom miScript miRNA PCR-based sybergreen array. The area under the receiver operating characteristic curve (AUC was used to evaluate the diagnostic performance of the studied miRNAs for colorectal cancer diagnosis.Data analysis of miRNA from the training set showed that; compared to control group, only miR-19b was significantly up-regulated in patients with IBD group (fold change = 5.24, p = 0.016, whereas in patients with colonic polyps, miR-18a was significantly up-regulated (fold change = 3.49, p-value = 0.018. On the other hand, miR-17, miR-19a, miR-20a and miR-223 were significantly up-regulated (fold change = 2.35, 3.07, 2.38 and 10.35; respectively and p-value = 0.02, 0.015, 0.017 and 0.016; respectively in CRC patients. However, the validation set showed that only miR-223 was significantly up-regulated in CRC patients (fold change = 4.06, p-value = 0.04.Aberrant miRNA expressions are highly involved in the cascade of colorectal carcinogenesis. We have found that (miR-17, miR-19a, miR-20a and miR-223 could be used as diagnostic biomarkers for CRC. On the other hand, miR-19b and miR-18a could be used as diagnostic biomarkers for

ARMOUR – A Rice miRNA: mRNA Interaction Resource

OpenAIRE

Neeti Sanan-Mishra; Anita Tripathi; Kavita Goswami; Rohit N. Shukla; Madavan Vasudevan; Hitesh Goswami

2018-01-01

ARMOUR was developed as ARice miRNA:mRNA interaction resource. This informative and interactive database includes the experimentally validated expression profiles of miRNAs under different developmental and abiotic stress conditions across seven Indian rice cultivars. This comprehensive database covers 689 known and 1664 predicted novel miRNAs and their expression profiles in more than 38 different tissues or conditions along with their predicted/known target transcripts. The understanding of...

Phage-mediated counting by the naked eye of miRNA molecules at attomolar concentrations in a Petri dish

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Zhou, Xin; Cao, Peng; Zhu, Ye; Lu, Wuguang; Gu, Ning; Mao, Chuanbin

2015-10-01

The ability to count biomolecules such as cancer-biomarker miRNAs with the naked eye is seemingly impossible in molecular diagnostics. Here, we show an ultrasensitive naked-eye-counting strategy for quantifying miRNAs by employing T7 phage--a bacteria-specific virus nanoparticle--as a surrogate. The phage is genetically engineered to become fluorescent and capable of binding a miRNA-capturing gold nanoparticle (GNP) in a one-to-one manner. Target miRNAs crosslink the resultant phage-GNP couple and miRNA-capturing magnetic microparticles, forming a sandwich complex containing equimolar phage and miRNA. The phage is then released from the complex and developed into one macroscopic fluorescent plaque in a Petri dish by plating it in a host bacterial medium. Counting the plaques by the naked eye enables the quantification of miRNAs with detection limits of ~3 and ~5 aM for single-target and two-target miRNAs, respectively. This approach offers ultrasensitive and convenient quantification of disease biomarkers by the naked eye.

Distinctive serum miRNA profile in mouse models of striated muscular pathologies.

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Nicolas Vignier

Full Text Available Biomarkers are critically important for disease diagnosis and monitoring. In particular, close monitoring of disease evolution is eminently required for the evaluation of therapeutic treatments. Classical monitoring methods in muscular dystrophies are largely based on histological and molecular analyses of muscle biopsies. Such biopsies are invasive and therefore difficult to obtain. The serum protein creatine kinase is a useful biomarker, which is however not specific for a given pathology and correlates poorly with the severity or course of the muscular pathology. The aim of the present study was the systematic evaluation of serum microRNAs (miRNAs as biomarkers in striated muscle pathologies. Mouse models for five striated muscle pathologies were investigated: Duchenne muscular dystrophy (DMD, limb-girdle muscular dystrophy type 2D (LGMD2D, limb-girdle muscular dystrophy type 2C (LGMD2C, Emery-Dreifuss muscular dystrophy (EDMD and hypertrophic cardiomyopathy (HCM. Two-step RT-qPCR methodology was elaborated, using two different RT-qPCR miRNA quantification technologies. We identified miRNA modulation in the serum of all the five mouse models. The most highly dysregulated serum miRNAs were found to be commonly upregulated in DMD, LGMD2D and LGMD2C mouse models, which all exhibit massive destruction of striated muscle tissues. Some of these miRNAs were down rather than upregulated in the EDMD mice, a model without massive myofiber destruction. The dysregulated miRNAs identified in the HCM model were different, with the exception of one dysregulated miRNA common to all pathologies. Importantly, a specific and distinctive circulating miRNA profile was identified for each studied pathological mouse model. The differential expression of a few dysregulated miRNAs in the DMD mice was further evaluated in DMD patients, providing new candidates of circulating miRNA biomarkers for DMD.

Efficient Identification of miRNAs for Classification of Tumor Origin

DEFF Research Database (Denmark)

Søkilde, Rolf; Vincent, Martin; Møller, Anne K

2014-01-01

Carcinomas of unknown primary origin constitute 3% to 5% of all newly diagnosed metastatic cancers, with the primary source difficult to classify with current histological methods. Effective cancer treatment depends on early and accurate identification of the tumor; patients with metastases...... of unknown origin have poor prognosis and short survival. Because miRNA expression is highly tissue specific, the miRNA profile of a metastasis may be used to identify its origin. We therefore evaluated the potential of miRNA profiling to identify the primary tumor of known metastases. Two hundred eight...... formalin-fixed, paraffin-embedded samples, representing 15 different histologies, were profiled on a locked nucleic acid-enhanced microarray platform, which allows for highly sensitive and specific detection of miRNA. On the basis of these data, we developed and cross-validated a novel classification...

Differentially expressed miRNAs after GnRH treatment and their potential roles in FSH regulation in porcine anterior pituitary cell.

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Rui-Song Ye

Full Text Available Hypothalamic gonadotropin-releasing hormone (GnRH is a major regulator of follicle-stimulating hormone (FSH secretion in gonadotrope cell in the anterior pituitary gland. microRNAs (miRNAs are small RNA molecules that control gene expression by imperfect binding to the 3'-untranslated region (3'-UTR of mRNA at the post-transcriptional level. It has been proven that miRNAs play an important role in hormone response and/or regulation. However, little is known about miRNAs in the regulation of FSH secretion. In this study, primary anterior pituitary cells were treated with 100 nM GnRH. The supernatant of pituitary cell was collected for FSH determination by enzyme-linked immunosorbent assay (ELISA at 3 hours and 6 hours post GnRH treatment respectively. Results revealed that GnRH significantly promoted FSH secretion at 3 h and 6 h post-treatment by 1.40-fold and 1.80-fold, respectively. FSHβ mRNA at 6 h post GnRH treatment significantly increased by 1.60-fold. At 6 hours, cells were collected for miRNA expression profile analysis using MiRCURY LNA Array and quantitative PCR (qPCR. Consequently, 21 up-regulated and 10 down-regulated miRNAs were identified, and qPCR verification of 10 randomly selected miRNAs showed a strong correlation with microarray results. Chromosome location analysis indicated that 8 miRNAs were mapped to chromosome 12 and 4 miRNAs to chromosome X. Target and pathway analysis showed that some miRNAs may be associated with GnRH regulation pathways. In addition, In-depth analysis indicated that 10 up-regulated and 3 down-regulated miRNAs probably target FSHβ mRNA 3'-UTR directly, including miR-361-3p, a highly conserved X-linked miRNA. Most importantly, functional experimental results showed that miR-361-3p was involved in FSH secretion regulation, and up-regulated miR-361-3p expression inhibited FSH secretion, while down-regulated miR-361-3p expression promoted FSH secretion in pig pituitary cell model. These differentially

Microprocessor Activity Controls Differential miRNA Biogenesis In Vivo

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Thomas Conrad

2014-10-01

Full Text Available In miRNA biogenesis, pri-miRNA transcripts are converted into pre-miRNA hairpins. The in vivo properties of this process remain enigmatic. Here, we determine in vivo transcriptome-wide pri-miRNA processing using next-generation sequencing of chromatin-associated pri-miRNAs. We identify a distinctive Microprocessor signature in the transcriptome profile from which efficiency of the endogenous processing event can be accurately quantified. This analysis reveals differential susceptibility to Microprocessor cleavage as a key regulatory step in miRNA biogenesis. Processing is highly variable among pri-miRNAs and a better predictor of miRNA abundance than primary transcription itself. Processing is also largely stable across three cell lines, suggesting a major contribution of sequence determinants. On the basis of differential processing efficiencies, we define functionality for short sequence features adjacent to the pre-miRNA hairpin. In conclusion, we identify Microprocessor as the main hub for diversified miRNA output and suggest a role for uncoupling miRNA biogenesis from host gene expression.

miRNAs in brain development

International Nuclear Information System (INIS)

Petri, Rebecca; Malmevik, Josephine; Fasching, Liana; Åkerblom, Malin; Jakobsson, Johan

2014-01-01

MicroRNAs (miRNAs) are small, non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. In the brain, a large number of miRNAs are expressed and there is a growing body of evidence demonstrating that miRNAs are essential for brain development and neuronal function. Conditional knockout studies of the core components in the miRNA biogenesis pathway, such as Dicer and DGCR8, have demonstrated a crucial role for miRNAs during the development of the central nervous system. Furthermore, mice deleted for specific miRNAs and miRNA-clusters demonstrate diverse functional roles for different miRNAs during the development of different brain structures. miRNAs have been proposed to regulate cellular functions such as differentiation, proliferation and fate-determination of neural progenitors. In this review we summarise the findings from recent studies that highlight the importance of miRNAs in brain development with a focus on the mouse model. We also discuss the technical limitations of current miRNA studies that still limit our understanding of this family of non-coding RNAs and propose the use of novel and refined technologies that are needed in order to fully determine the impact of specific miRNAs in brain development. - Highlights: • miRNAs are essential for brain development and neuronal function. • KO of Dicer is embryonically lethal. • Conditional Dicer KO results in defective proliferation or increased apoptosis. • KO of individual miRNAs or miRNA families is necessary to determine function

Genome-wide identification of microRNA targets in the neglected disease pathogens of the genus Echinococcus.

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Macchiaroli, Natalia; Maldonado, Lucas L; Zarowiecki, Magdalena; Cucher, Marcela; Gismondi, María Inés; Kamenetzky, Laura; Rosenzvit, Mara Cecilia

2017-06-01

MicroRNAs (miRNAs), a class of small non-coding RNAs, are key regulators of gene expression at post-transcriptional level and play essential roles in biological processes such as development. MiRNAs silence target mRNAs by binding to complementary sequences in the 3'untranslated regions (3'UTRs). The parasitic helminths of the genus Echinococcus are the causative agents of echinococcosis, a zoonotic neglected disease. In previous work, we performed a comprehensive identification and characterization of Echinococcus miRNAs. However, current knowledge about their targets is limited. Since target prediction algorithms rely on complementarity between 3'UTRs and miRNA sequences, a major limitation is the lack of accurate sequence information of 3'UTR for most species including parasitic helminths. We performed RNA-seq and developed a pipeline that integrates the transcriptomic data with available genomic data of this parasite in order to identify 3'UTRs of Echinococcus canadensis. The high confidence set of 3'UTRs obtained allowed the prediction of miRNA targets in Echinococcus through a bioinformatic approach. We performed for the first time a comparative analysis of miRNA targets in Echinococcus and Taenia. We found that many evolutionarily conserved target sites in Echinococcus and Taenia may be functional and under selective pressure. Signaling pathways such as MAPK and Wnt were among the most represented pathways indicating miRNA roles in parasite growth and development. Genome-wide identification and characterization of miRNA target genes in Echinococcus provide valuable information to guide experimental studies in order to understand miRNA functions in the parasites biology. miRNAs involved in essential functions, especially those being absent in the host or showing sequence divergence with respect to host orthologs, might be considered as novel therapeutic targets for echinococcosis control. Copyright © 2017 Elsevier B.V. All rights reserved.

Epigenetic regulation of normal human mammary cell type-specific miRNAs

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Vrba, Lukas [Univ. of Arizona, Tucson, AZ (United States). Arizona Cancer Center; Inst. of Plant Molecular Biology, Ceske Budejovice (Czech Republic). Biology Centre ASCR; Garbe, James C. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Life Sciences Center; Stampfer, Martha R. [Univ. of Arizona, Tucson, AZ (United States). Arizona Cancer Center; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Life Sciences Center; Futscher, Bernard W. [Univ. of Arizona, Tucson, AZ (United States). Arizona Cancer Center and Dept. of Pharmacology & Toxicology

2011-08-26

Epigenetic mechanisms are important regulators of cell type–specific genes, including miRNAs. In order to identify cell type-specific miRNAs regulated by epigenetic mechanisms, we undertook a global analysis of miRNA expression and epigenetic states in three isogenic pairs of human mammary epithelial cells (HMEC) and human mammary fibroblasts (HMF), which represent two differentiated cell types typically present within a given organ, each with a distinct phenotype and a distinct epigenotype. While miRNA expression and epigenetic states showed strong interindividual concordance within a given cell type, almost 10% of the expressed miRNA showed a cell type–specific pattern of expression that was linked to the epigenetic state of their promoter. The tissue-specific miRNA genes were epigenetically repressed in nonexpressing cells by DNA methylation (38%) and H3K27me3 (58%), with only a small set of miRNAs (21%) showing a dual epigenetic repression where both DNA methylation and H3K27me3 were present at their promoters, such as MIR10A and MIR10B. Individual miRNA clusters of closely related miRNA gene families can each display cell type–specific repression by the same or complementary epigenetic mechanisms, such as the MIR200 family, and MIR205, where fibroblasts repress MIR200C/141 by DNA methylation, MIR200A/200B/429 by H3K27me3, and MIR205 by both DNA methylation and H3K27me3. Since deregulation of many of the epigenetically regulated miRNAs that we identified have been linked to disease processes such as cancer, it is predicted that compromise of the epigenetic control mechanisms is important for this process. Overall, these results highlight the importance of epigenetic regulation in the control of normal cell type–specific miRNA expression.

Wolbachia-induced aae-miR-12 miRNA negatively regulates the expression of MCT1 and MCM6 genes in Wolbachia-infected mosquito cell line.

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Solomon Osei-Amo

Full Text Available BACKGROUND: Best recognized for its role in manipulating host reproduction, the parasitic gram-negative Wolbachia pipientis is known to colonize a wide range of invertebrates. The endosymbiotic bacterium has recently been shown to cause a life-shortening effect as well as inhibiting replication of arboviruses in Aedes aegypti; although the molecular mechanisms behind these effects are largely unknown. MicroRNAs (miRNAs have been determined to have a wide range of roles in regulating gene expression in eukaryotes. A recent study showed that several A. aegypti mosquito miRNAs are differentially expressed when infected with Wolbachia. METHODOLOGY/PRINCIPAL FINDINGS: Based on the prior knowledge that one of these miRNAs, aae-miR-12, is differentially expressed in mosquitoes infected with Wolbachia, we aimed to determine any significance of this mediation. We also set out to characterize the target genes of this miRNA in the A. aegpyti genome. Bioinformatic approaches predicted a list of potential target genes and subsequent functional analyses confirmed that two of these, DNA replication licensing (MCM6 and monocarboxylate transporter (MCT1, are under the regulative control of aae-miR-12. We also demonstrated that aae-miR-12 is critical in the persistence of Wolbachia in the host cell. CONCLUSIONS/SIGNIFICANCE: Our study has identified two target genes of aae-miR-12, a differentially expressed mosquito miRNA in Wolbachia-infected cells, and determined that the miRNA affects Wolbachia density in the host cells.

Microvesicles derived from adult human bone marrow and tissue specific mesenchymal stem cells shuttle selected pattern of miRNAs.

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Federica Collino

Full Text Available BACKGROUND: Cell-derived microvesicles (MVs have been described as a new mechanism of cell-to-cell communication. MVs after internalization within target cells may deliver genetic information. Human bone marrow derived mesenchymal stem cells (MSCs and liver resident stem cells (HLSCs were shown to release MVs shuttling functional mRNAs. The aim of the present study was to evaluate whether MVs derived from MSCs and HLSCs contained selected micro-RNAs (miRNAs. METHODOLOGY/PRINCIPAL FINDINGS: MVs were isolated from MSCs and HLSCs. The presence in MVs of selected ribonucleoproteins involved in the traffic and stabilization of RNA was evaluated. We observed that MVs contained TIA, TIAR and HuR multifunctional proteins expressed in nuclei and stress granules, Stau1 and 2 implicated in the transport and stability of mRNA and Ago2 involved in miRNA transport and processing. RNA extracted from MVs and cells of origin was profiled for 365 known human mature miRNAs by real time PCR. Hierarchical clustering and similarity analysis of miRNAs showed 41 co-expressed miRNAs in MVs and cells. Some miRNAs were accumulated within MVs and absent in the cells after MV release; others were retained within the cells and not secreted in MVs. Gene ontology analysis of predicted and validated targets showed that the high expressed miRNAs in cells and MVs could be involved in multi-organ development, cell survival and differentiation. Few selected miRNAs shuttled by MVs were also associated with the immune system regulation. The highly expressed miRNAs in MVs were transferred to target cells after MV incorporation. CONCLUSIONS: This study demonstrated that MVs contained ribonucleoproteins involved in the intracellular traffic of RNA and selected pattern of miRNAs, suggesting a dynamic regulation of RNA compartmentalization in MVs. The observation that MV-highly expressed miRNAs were transferred to target cells, rises the possibility that the biological effect of stem

DIANA-microT web server: elucidating microRNA functions through target prediction.

Science.gov (United States)

Maragkakis, M; Reczko, M; Simossis, V A; Alexiou, P; Papadopoulos, G L; Dalamagas, T; Giannopoulos, G; Goumas, G; Koukis, E; Kourtis, K; Vergoulis, T; Koziris, N; Sellis, T; Tsanakas, P; Hatzigeorgiou, A G

2009-07-01

Computational microRNA (miRNA) target prediction is one of the key means for deciphering the role of miRNAs in development and disease. Here, we present the DIANA-microT web server as the user interface to the DIANA-microT 3.0 miRNA target prediction algorithm. The web server provides extensive information for predicted miRNA:target gene interactions with a user-friendly interface, providing extensive connectivity to online biological resources. Target gene and miRNA functions may be elucidated through automated bibliographic searches and functional information is accessible through Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The web server offers links to nomenclature, sequence and protein databases, and users are facilitated by being able to search for targeted genes using different nomenclatures or functional features, such as the genes possible involvement in biological pathways. The target prediction algorithm supports parameters calculated individually for each miRNA:target gene interaction and provides a signal-to-noise ratio and a precision score that helps in the evaluation of the significance of the predicted results. Using a set of miRNA targets recently identified through the pSILAC method, the performance of several computational target prediction programs was assessed. DIANA-microT 3.0 achieved there with 66% the highest ratio of correctly predicted targets over all predicted targets. The DIANA-microT web server is freely available at www.microrna.gr/microT.

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

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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.

miRNAs in human subcutaneous adipose tissue: Effects of weight loss induced by hypocaloric diet and exercise.

Science.gov (United States)

Kristensen, Malene M; Davidsen, Peter K; Vigelsø, Andreas; Hansen, Christina N; Jensen, Lars J; Jessen, Niels; Bruun, Jens M; Dela, Flemming; Helge, Jørn W

2017-03-01

Obesity is central in the development of insulin resistance. However, the underlying mechanisms still need elucidation. Dysregulated microRNAs (miRNAs; post-transcriptional regulators) in adipose tissue may present an important link. The miRNA expression in subcutaneous adipose tissue from 19 individuals with severe obesity (10 women and 9 men) before and after a 15-week weight loss intervention was studied using genome-wide microarray analysis. The microarray results were validated with RT-qPCR, and pathway enrichment analysis of in silico predicted targets was performed to elucidate the biological consequences of the miRNA dysregulation. Lastly, the messenger RNA (mRNA) and/or protein expression of multiple predicted targets as well as several proteins involved in lipolysis were investigated. The intervention led to upregulation of miR-29a-3p and miR-29a-5p and downregulation of miR-20b-5p. The mRNA and protein expression of predicted targets was not significantly affected by the intervention. However, negative correlations between miR-20b-5p and the protein levels of its predicted target, acyl-CoA synthetase long-chain family member 1, were observed. Several other miRNA-target relationships correlated negatively, indicating possible miRNA regulation, including miR-29a-3p and lipoprotein lipase mRNA levels. Proteins involved in lipolysis were not affected by the intervention. Weight loss influenced several miRNAs, some of which were negatively correlated with predicted targets. These dysregulated miRNAs may affect adipocytokine signaling and forkhead box protein O signaling. © 2017 The Obesity Society.

miRNA profiling of naive, effector and memory CD8 T cells.

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Haoquan Wu

Full Text Available microRNAs have recently emerged as master regulators of gene expression during development and cell differentiation. Although profound changes in gene expression also occur during antigen-induced T cell differentiation, the role of miRNAs in the process is not known. We compared the miRNA expression profiles between antigen-specific naïve, effector and memory CD8+ T cells using 3 different methods--small RNA cloning, miRNA microarray analysis and real-time PCR. Although many miRNAs were expressed in all the T cell subsets, the frequency of 7 miRNAs (miR-16, miR-21, miR-142-3p, miR-142-5p, miR-150, miR-15b and let-7f alone accounted for approximately 60% of all miRNAs, and their expression was several fold higher than the other expressed miRNAs. Global downregulation of miRNAs (including 6/7 dominantly expressed miRNAs was observed in effector T cells compared to naïve cells and the miRNA expression levels tended to come back up in memory T cells. However, a few miRNAs, notably miR-21 were higher in effector and memory T cells compared to naïve T cells. These results suggest that concomitant with profound changes in gene expression, miRNA profile also changes dynamically during T cell differentiation. Sequence analysis of the cloned mature miRNAs revealed an extensive degree of end polymorphism. While 3'end polymorphisms dominated, heterogeneity at both ends, resembling drosha/dicer processing shift was also seen in miR-142, suggesting a possible novel mechanism to generate new miRNA and/or to diversify miRNA target selection. Overall, our results suggest that dynamic changes in the expression of miRNAs may be important for the regulation of gene expression during antigen-induced T cell differentiation. Our study also suggests possible novel mechanisms for miRNA biogenesis and function.

Comparative MiRNA Expressional Profiles and Molecular Networks in Human Small Bowel Tissues of Necrotizing Enterocolitis and Spontaneous Intestinal Perforation.

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Pak Cheung Ng

Full Text Available Necrotizing enterocolitis (NEC and spontaneous intestinal perforation (SIP are acute intestinal conditions which could result in mortality and severe morbidity in preterm infants. Our objective was to identify dysregulated micro-RNAs (miRNAs in small bowel tissues of NEC and SIP, and their possible roles in disease pathophysiology.We performed differential miRNA arrays on tissues of NEC (n = 4, SIP (n = 4 and surgical-control (Surg-CTL; n = 4, and validated target miRNAs by qPCR (n = 10 each group. The association of target miRNAs with 52 dysregulated mRNAs was investigated by bioinformatics on functional and base-pair sequence algorithms, and correlation in same tissue samples.We presented the first miRNA profiles of NEC, SIP and Surg-CTL intestinal tissues in preterm infants. Of 28 validated miRNAs, 21 were significantly different between NEC or SIP and Surg-CTL. Limited overlapping in the aberrant expression of miRNAs between NEC and SIP indicated their distinct molecular mechanisms. A proposed network of dysregulated miRNA/mRNA pairs in NEC suggested interaction at bacterial receptor TLR4 (miR-31, miR-451, miR-203, miR-4793-3p, mediated via key transcription factors NFKB2 (miR-203, AP-1/FOSL1 (miR-194-3p, FOXA1 (miR-21-3p, miR-431 and miR-1290 and HIF1A (miR-31, and extended downstream to pathways of angiogenesis, arginine metabolism, cell adhesion and chemotaxis, extracellular matrix remodeling, hypoxia/oxidative stress, inflammation and muscle contraction. In contrast, upregulation of miR-451 and miR-223 in SIP suggested modulation of G-protein-mediated muscle contraction.The robust response of miRNA dysregulation in NEC and SIP, and concerted involvement of specific miRNAs in the molecular networks indicated their crucial roles in mucosa integrity and disease pathophysiology.

Positive radionuclide imaging of miRNA expression using RILES and the human sodium iodide symporter as reporter gene is feasible and supports a protective role of miRNA-23a in response to muscular atrophy.

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Viorel Simion

Full Text Available MicroRNAs (miRNAs are key players in many biological processes and are considered as an emerging class of pharmacology drugs for diagnosis and therapy. However to fully exploit the therapeutic potential of miRNAs, it is becoming crucial to monitor their expression pattern using medical imaging modalities. Recently, we developed a method called RILES, for RNAi-Inducible Luciferase Expression System that relies on an engineered regulatable expression system to switch-ON the expression of the luciferase gene when a miRNA of interest is expressed in cells. Here we investigated whether replacing the luciferase reporter gene with the human sodium iodide symporter (hNIS reporter gene will be also suited to monitor the expression of miRNAs in a clinical setting context. We provide evidence that radionuclide imaging of miRNA expression using hNIS is feasible although it is not as robust as when the luciferase reporter gene is used. However, under appropriate conditions, we monitored the expression of several miRNAs in cells, in the liver and in the tibialis anterior muscle of mice undergoing muscular atrophy. We demonstrated that radiotracer accumulation in transfected cells correlated with the induction of hNIS and with the expression of miRNAs detected by real time PCR. We established the kinetic of miRNA-23a expression in mice and demonstrated that this miRNA follows a biphasic expression pattern characterized by a loss of expression at a late time point of muscular atrophy. At autopsy, we found an opposite expression pattern between miRNA-23a and one of the main transcriptional target of this miRNA, APAF-1, and as downstream target, Caspase 9. Our results report the first positive monitoring of endogenously expressed miRNAs in a nuclear medicine imaging context and support the development of additional work to establish the potential therapeutic value of miRNA-23 to prevent the damaging effects of muscular atrophy.

Positive radionuclide imaging of miRNA expression using RILES and the human sodium iodide symporter as reporter gene is feasible and supports a protective role of miRNA-23a in response to muscular atrophy.

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Simion, Viorel; Sobilo, Julien; Clemoncon, Rudy; Natkunarajah, Sharuja; Ezzine, Safia; Abdallah, Florence; Lerondel, Stephanie; Pichon, Chantal; Baril, Patrick

2017-01-01

MicroRNAs (miRNAs) are key players in many biological processes and are considered as an emerging class of pharmacology drugs for diagnosis and therapy. However to fully exploit the therapeutic potential of miRNAs, it is becoming crucial to monitor their expression pattern using medical imaging modalities. Recently, we developed a method called RILES, for RNAi-Inducible Luciferase Expression System that relies on an engineered regulatable expression system to switch-ON the expression of the luciferase gene when a miRNA of interest is expressed in cells. Here we investigated whether replacing the luciferase reporter gene with the human sodium iodide symporter (hNIS) reporter gene will be also suited to monitor the expression of miRNAs in a clinical setting context. We provide evidence that radionuclide imaging of miRNA expression using hNIS is feasible although it is not as robust as when the luciferase reporter gene is used. However, under appropriate conditions, we monitored the expression of several miRNAs in cells, in the liver and in the tibialis anterior muscle of mice undergoing muscular atrophy. We demonstrated that radiotracer accumulation in transfected cells correlated with the induction of hNIS and with the expression of miRNAs detected by real time PCR. We established the kinetic of miRNA-23a expression in mice and demonstrated that this miRNA follows a biphasic expression pattern characterized by a loss of expression at a late time point of muscular atrophy. At autopsy, we found an opposite expression pattern between miRNA-23a and one of the main transcriptional target of this miRNA, APAF-1, and as downstream target, Caspase 9. Our results report the first positive monitoring of endogenously expressed miRNAs in a nuclear medicine imaging context and support the development of additional work to establish the potential therapeutic value of miRNA-23 to prevent the damaging effects of muscular atrophy.

A path-based measurement for human miRNA functional similarities using miRNA-disease associations

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Ding, Pingjian; Luo, Jiawei; Xiao, Qiu; Chen, Xiangtao

2016-09-01

Compared with the sequence and expression similarity, miRNA functional similarity is so important for biology researches and many applications such as miRNA clustering, miRNA function prediction, miRNA synergism identification and disease miRNA prioritization. However, the existing methods always utilized the predicted miRNA target which has high false positive and false negative to calculate the miRNA functional similarity. Meanwhile, it is difficult to achieve high reliability of miRNA functional similarity with miRNA-disease associations. Therefore, it is increasingly needed to improve the measurement of miRNA functional similarity. In this study, we develop a novel path-based calculation method of miRNA functional similarity based on miRNA-disease associations, called MFSP. Compared with other methods, our method obtains higher average functional similarity of intra-family and intra-cluster selected groups. Meanwhile, the lower average functional similarity of inter-family and inter-cluster miRNA pair is obtained. In addition, the smaller p-value is achieved, while applying Wilcoxon rank-sum test and Kruskal-Wallis test to different miRNA groups. The relationship between miRNA functional similarity and other information sources is exhibited. Furthermore, the constructed miRNA functional network based on MFSP is a scale-free and small-world network. Moreover, the higher AUC for miRNA-disease prediction indicates the ability of MFSP uncovering miRNA functional similarity.

The Role of miRNA in Papillary Thyroid Cancer in the Context of miRNA Let-7 Family

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Ewelina Perdas

2016-06-01

Full Text Available Papillary thyroid carcinoma (PTC is the most common endocrine malignancy. RET/PTC rearrangement is the most common genetic modification identified in this category of cancer, increasing proliferation and dedifferentiation by the activation of the RET/PTC-RAS-BRAF-MAPK-ERK signaling pathway. Recently, let-7 miRNA was found to reduce RAS levels, acting as a tumor suppressor gene. Circulating miRNA profiles of the let-7 family may be used as novel noninvasive diagnostic, prognostic, treatment and surveillance markers for PTC.

Identification of conserved microRNAs and their targets in chickpea (Cicer arietinum L.).

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Hu, Jihong; Sun, Lulu; Ding, Yi

2013-04-01

The microRNAs (miRNAs) are a new class of non-protein coding small RNAs that regulate gene expression at the post-transcriptional level in plants. Although thousands of miRNAs have been identified in many plant species, little studies have been reported about chickpea microRNAs. In this study, 28 potential miRNA candidates belonging to 20 families were identified from 16 ESTs and 12 GSSs in chickpea using a comparative genome-based computational analysis. A total of 664 miRNA targets were predicted and some of them encoded transcription factors as well as genes that function in stress response, signal transduction, methylation and a variety of other metabolic processes. These findings lay the foundation for further understanding of miRNA function in the development of chickpea.

Breast cancer stem cells, EMT and therapeutic targets

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Kotiyal, Srishti; Bhattacharya, Susinjan, E-mail: s.bhattacharya@jiit.ac.in

2014-10-10

Highlights: • Therapeutic targeting or inhibition of the key molecules of signaling pathways can control growth of breast cancer stem cells (BCSCs). • Development of BCSCs also involves miRNA interactions. • Therapeutic achievement can be done by targeting identified targets in the BCSC pathways. - Abstract: A small heterogeneous population of breast cancer cells acts as seeds to induce new tumor growth. These seeds or breast cancer stem cells (BCSCs) exhibit great phenotypical plasticity which allows them to undergo “epithelial to mesenchymal transition” (EMT) at the site of primary tumor and a future reverse transition. Apart from metastasis they are also responsible for maintaining the tumor and conferring it with drug and radiation resistance and a tendency for post-treatment relapse. Many of the signaling pathways involved in induction of EMT are involved in CSC generation and regulation. Here we are briefly reviewing the mechanism of TGF-β, Wnt, Notch, TNF-α, NF-κB, RTK signalling pathways which are involved in EMT as well as BCSCs maintenance. Therapeutic targeting or inhibition of the key/accessory players of these pathways could control growth of BCSCs and hence malignant cancer. Additionally several miRNAs are dysregulated in cancer stem cells indicating their roles as oncogenes or tumor suppressors. This review also lists the miRNA interactions identified in BCSCs and discusses on some newly identified targets in the BCSC regulatory pathways like SHIP2, nicastrin, Pin 1, IGF-1R, pro-inflammatory cytokines and syndecan which can be targeted for therapeutic achievements.

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

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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.

SmD1 Modulates the miRNA Pathway Independently of Its Pre-mRNA Splicing Function.

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Xiao-Peng Xiong

2015-08-01

Full Text Available microRNAs (miRNAs are a class of endogenous regulatory RNAs that play a key role in myriad biological processes. Upon transcription, primary miRNA transcripts are sequentially processed by Drosha and Dicer ribonucleases into ~22-24 nt miRNAs. Subsequently, miRNAs are incorporated into the RNA-induced silencing complexes (RISCs that contain Argonaute (AGO family proteins and guide RISC to target RNAs via complementary base pairing, leading to post-transcriptional gene silencing by a combination of translation inhibition and mRNA destabilization. Select pre-mRNA splicing factors have been implicated in small RNA-mediated gene silencing pathways in fission yeast, worms, flies and mammals, but the underlying molecular mechanisms are not well understood. Here, we show that SmD1, a core component of the Drosophila small nuclear ribonucleoprotein particle (snRNP implicated in splicing, is required for miRNA biogenesis and function. SmD1 interacts with both the microprocessor component Pasha and pri-miRNAs, and is indispensable for optimal miRNA biogenesis. Depletion of SmD1 impairs the assembly and function of the miRISC without significantly affecting the expression of major canonical miRNA pathway components. Moreover, SmD1 physically and functionally associates with components of the miRISC, including AGO1 and GW182. Notably, miRNA defects resulting from SmD1 silencing can be uncoupled from defects in pre-mRNA splicing, and the miRNA and splicing machineries are physically and functionally distinct entities. Finally, photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP analysis identifies numerous SmD1-binding events across the transcriptome and reveals direct SmD1-miRNA interactions. Our study suggests that SmD1 plays a direct role in miRNA-mediated gene silencing independently of its pre-mRNA splicing activity and indicates that the dual roles of splicing factors in post-transcriptional gene regulation may be

Identification of circulating miRNA involved in meat yield of Korean cattle.

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Lee, Surim; Park, Seung-Ju; Cheong, Jae-Kyoung; Ko, Jong-Youl; Bong, Jinjong; Baik, Myunggi

2017-07-01

Cattle plays an important role in providing essential nutrients through meat production. Thus, we focused on epigenetic factors associated with meat yield. To investigate circulating miRNAs that are involved with meat yield and connect biofluids and longissimus dorsi (LD) muscle in Korean cattle, we performed analyses of the carcass characteristics, miRNA array, qPCR, and bioinformatics. Carcass characteristics relative to the yield grade (YG) showed that the yield index and rib eye area were the highest, whereas the backfat thickness was the lowest for YG A (equal to high YG) cattle among the three YGs. miRNA array sorted the circulating miRNAs that connect biofluids and LD muscle. miRNA qPCR showed that miR-15a (r = 0.84), miR-26b (r = 0.91), and miR-29c (r = 0.92) had positive relationships with biofluids and LD muscle. In YG A cattle, miR-26b was considered to be a circulating miRNA connecting biofluids and LD muscle because the target genes of miR-26b were more involved with myogenesis. Then, miR-26b-targeted genes, DIAPH3 and YOD1, were downregulated in YG A cattle. Our results suggest that miR-15a, miR-26b, and miR-29c are upregulated in biofluids and LD muscle, whereas DIAPH3 and YOD1 are downregulated in the LD muscle of finishing cattle steers. © 2017 International Federation for Cell Biology.

microRNA expression profiling in fetal single ventricle malformation identified by deep sequencing.

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Yu, Zhang-Bin; Han, Shu-Ping; Bai, Yun-Fei; Zhu, Chun; Pan, Ya; Guo, Xi-Rong

2012-01-01

microRNAs (miRNAs) have emerged as key regulators in many biological processes, particularly cardiac growth and development, although the specific miRNA expression profile associated with this process remains to be elucidated. This study aimed to characterize the cellular microRNA profile involved in the development of congenital heart malformation, through the investigation of single ventricle (SV) defects. Comprehensive miRNA profiling in human fetal SV cardiac tissue was performed by deep sequencing. Differential expression of 48 miRNAs was revealed by sequencing by oligonucleotide ligation and detection (SOLiD) analysis. Of these, 38 were down-regulated and 10 were up-regulated in differentiated SV cardiac tissue, compared to control cardiac tissue. This was confirmed by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. Predicted target genes of the 48 differentially expressed miRNAs were analyzed by gene ontology and categorized according to cellular process, regulation of biological process and metabolic process. Pathway-Express analysis identified the WNT and mTOR signaling pathways as the most significant processes putatively affected by the differential expression of these miRNAs. The candidate genes involved in cardiac development were identified as potential targets for these differentially expressed microRNAs and the collaborative network of microRNAs and cardiac development related-mRNAs was constructed. These data provide the basis for future investigation of the mechanism of the occurrence and development of fetal SV malformations.

Identification and Target Prediction of MicroRNAs in Ulmus pumila L. Seedling Roots under Salt Stress by High-Throughput Sequencing

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Jianfeng Zhu

2016-12-01

Full Text Available MicroRNAs (miRNAs are a class of endogenous small RNAs with important roles in plant growth, development, and environmental stress responses. Ulmus pumila L., a deciduous broadleaved tree species of northern temperate regions, is widely distributed in central and northern Asia and has important economic and ecological value. With the spread and aggravation of soil salinization, salt stress has become a major abiotic stress affecting the normal growth and development of U. pumila. However, the influence of salt stress on U. pumila miRNA expression has not been investigated. To identify miRNAs and predict their target mRNA genes under salt stress, three small RNA libraries were generated and sequenced from roots of U. pumila seedlings treated with various concentrations of NaCl corresponding to no salt stress, light short-term salt stress, and medium-heavy long-term salt stress. Integrative analysis identified 254 conserved miRNAs representing 29 families and 49 novel miRNAs; 232 potential targets of the miRNAs were also predicted. Expression profiling of miRNAs between libraries was performed, and the expression of six miRNAs was validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR. Our findings provide an overview of potential miRNAs and corresponding targets involved in regulating U. pumila salt defense responses. These results lay the foundation for further research into molecular mechanisms involved in salt stress resistance in U. pumila and other Ulmaceae species.

Sensing miRNA: Signal Amplification by Cognate RISC for Intracellular Detection of miRNA in Live Cells.

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Kavishwar, Amol; Medarova, Zdravka

2016-01-01

The ability to detect miRNA expression in live cells would leave these cells available for further manipulation or culture. Here, we describe the design of a miRNA sensor oligonucleotide whose sequence mimics the target mRNA. The sensor has a fluorescent label on one end of the oligo and a quencher on the other. When inside the cell, the sensor is recognized by its cognate miRNA-RISC and gets cleaved, setting the fluorophore free from its quencher. This results in fluorescence "turn on." Since cleavage by the RISC complex is an enzymatic process, the described approach has a very high level of sensitivity (nM). The rate of nonspecific cleavage of the sensor is very slow permitting the collection of meaningful signal over a long period of time.

Polymorphisms in miRNA genes and their involvement in autoimmune diseases susceptibility.

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Latini, Andrea; Ciccacci, Cinzia; Novelli, Giuseppe; Borgiani, Paola

2017-08-01

MicroRNAs (miRNAs) are small non-coding RNA molecules that negatively regulate the expression of multiple protein-encoding genes at the post-transcriptional level. MicroRNAs are involved in different pathways, such as cellular proliferation and differentiation, signal transduction and inflammation, and play crucial roles in the development of several diseases, such as cancer, diabetes, and cardiovascular diseases. They have recently been recognized to play a role also in the pathogenesis of autoimmune diseases. Although the majority of studies are focused on miRNA expression profiles investigation, a growing number of studies have been investigating the role of polymorphisms in miRNA genes in the autoimmune diseases development. Indeed, polymorphisms affecting the miRNA genes can modify the set of targets they regulate or the maturation efficiency. This review is aimed to give an overview about the available studies that have investigated the association of miRNA gene polymorphisms with the susceptibility to various autoimmune diseases and to their clinical phenotypes.

Single-Step Incubation Determination of miRNAs in Cancer Cells Using an Amperometric Biosensor Based on Competitive Hybridization onto Magnetic Beads

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Eva Vargas

2018-03-01

Full Text Available This work reports an amperometric biosensor for the determination of miRNA-21, a relevant oncogene. The methodology involves a competitive DNA-target miRNA hybridization assay performed on the surface of magnetic microbeads (MBs and amperometric transduction at screen-printed carbon electrodes (SPCEs. The target miRNA competes with a synthetic fluorescein isothiocyanate (FITC-modified miRNA with an identical sequence for hybridization with a biotinylated and complementary DNA probe (b-Cp immobilized on the surface of streptavidin-modified MBs (b-Cp-MBs. Upon labeling, the FITC-modified miRNA attached to the MBs with horseradish peroxidase (HRP-conjugated anti-FITC Fab fragments and magnetic capturing of the MBs onto the working electrode surface of SPCEs. The cathodic current measured at −0.20 V (versus the Ag pseudo-reference electrode was demonstrated to be inversely proportional to the concentration of the target miRNA. This convenient biosensing method provided a linear range between 0.7 and 10.0 nM and a limit of detection (LOD of 0.2 nM (5 fmol in 25 μL of sample for the synthetic target miRNA without any amplification step. An acceptable selectivity towards single-base mismatched oligonucleotides, a high storage stability of the b-Cp-MBs, and usefulness for the accurate determination of miRNA-21 in raw total RNA (RNAt extracted from breast cancer cells (MCF-7 were demonstrated.

Monitoring the Spatiotemporal Activities of miRNAs in Small Animal Models Using Molecular Imaging Modalities

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Patrick Baril

2015-03-01

Full Text Available MicroRNAs (miRNAs are a class of small non-coding RNAs that regulate gene expression by binding mRNA targets via sequence complementary inducing translational repression and/or mRNA degradation. A current challenge in the field of miRNA biology is to understand the functionality of miRNAs under physiopathological conditions. Recent evidence indicates that miRNA expression is more complex than simple regulation at the transcriptional level. MiRNAs undergo complex post-transcriptional regulations such miRNA processing, editing, accumulation and re-cycling within P-bodies. They are dynamically regulated and have a well-orchestrated spatiotemporal localization pattern. Real-time and spatio-temporal analyses of miRNA expression are difficult to evaluate and often underestimated. Therefore, important information connecting miRNA expression and function can be lost. Conventional miRNA profiling methods such as Northern blot, real-time PCR, microarray, in situ hybridization and deep sequencing continue to contribute to our knowledge of miRNA biology. However, these methods can seldom shed light on the spatiotemporal organization and function of miRNAs in real-time. Non-invasive molecular imaging methods have the potential to address these issues and are thus attracting increasing attention. This paper reviews the state-of-the-art of methods used to detect miRNAs and discusses their contribution in the emerging field of miRNA biology and therapy.

Monitoring the spatiotemporal activities of miRNAs in small animal models using molecular imaging modalities.

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Baril, Patrick; Ezzine, Safia; Pichon, Chantal

2015-03-04

MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression by binding mRNA targets via sequence complementary inducing translational repression and/or mRNA degradation. A current challenge in the field of miRNA biology is to understand the functionality of miRNAs under physiopathological conditions. Recent evidence indicates that miRNA expression is more complex than simple regulation at the transcriptional level. MiRNAs undergo complex post-transcriptional regulations such miRNA processing, editing, accumulation and re-cycling within P-bodies. They are dynamically regulated and have a well-orchestrated spatiotemporal localization pattern. Real-time and spatio-temporal analyses of miRNA expression are difficult to evaluate and often underestimated. Therefore, important information connecting miRNA expression and function can be lost. Conventional miRNA profiling methods such as Northern blot, real-time PCR, microarray, in situ hybridization and deep sequencing continue to contribute to our knowledge of miRNA biology. However, these methods can seldom shed light on the spatiotemporal organization and function of miRNAs in real-time. Non-invasive molecular imaging methods have the potential to address these issues and are thus attracting increasing attention. This paper reviews the state-of-the-art of methods used to detect miRNAs and discusses their contribution in the emerging field of miRNA biology and therapy.

Differential expression of miRNAs and their relation to active tuberculosis.

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Xu, Zhihong; Zhou, Aiping; Ni, Jinjing; Zhang, Qiufen; Wang, Ying; Lu, Jie; Wu, Wenjuan; Karakousis, Petros C; Lu, Shuihua; Yao, Yufeng

2015-07-01

The aim of this work was to screen miRNA signatures dysregulated in tuberculosis to improve our understanding of the biological role of miRNAs involved in the disease. Datasets deposited in publically available databases from microarray studies on infectious diseases and malignancies were retrieved, screened, and subjected to further analysis. Effect sizes were combined using the inverse-variance model and between-study heterogeneity was evaluated by the random effects model. 35 miRNAs were differentially expressed (12 up-regulated, 23 down-regulated; p tuberculosis and other infectious diseases. 15 miRNAs were found to be significantly differentially regulated (7 up-regulated, 8 down-regulated; p tuberculosis and malignancies. Most of the miRNA signatures identified in this study were found to be involved in immune responses and metabolism. Expression of these miRNA signatures in serum samples from TB subjects (n = 11) as well as healthy controls (n = 10) was examined by TaqMan miRNA array. Taken together, the results revealed differential expression of miRNAs in TB, but available datasets are limited and these miRNA signatures should be validated in future studies. Copyright © 2015 Elsevier Ltd. All rights reserved.

miR-155, identified as anti-metastatic by global miRNA profiling of a metastasis model, inhibits cancer cell extravasation and colonization in vivo and causes significant signaling alterations

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Gravgaard, Karina Hedelund; Terp, Mikkel G; Lund, Rikke R

2015-01-01

To gain insight into miRNA regulation in metastasis formation, we used a metastasis cell line model that allows investigation of extravasation and colonization of circulating cancer cells to lungs in mice. Using global miRNA profiling, 28 miRNAs were found to exhibit significantly altered...... proliferation or apoptosis in established lung tumors. To identify proteins regulated by miR-155 and thus delineate its function in our cell model, we compared the proteome of xenograft tumors derived from miR-155-overexpressing CL16 cells and CL16 control cells using mass spectrometry-based proteomics. >4......,000 proteins were identified, of which 92 were consistently differentially expressed. Network analysis revealed that the altered proteins were associated with cellular functions such as movement, growth and survival as well as cell-to-cell signaling and interaction. Downregulation of the three metastasis...

Dissecting miRNA gene repression on single cell level with an advanced fluorescent reporter system

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Lemus-Diaz, Nicolas; Böker, Kai O.; Rodriguez-Polo, Ignacio; Mitter, Michael; Preis, Jasmin; Arlt, Maximilian; Gruber, Jens

2017-01-01

Despite major advances on miRNA profiling and target predictions, functional readouts for endogenous miRNAs are limited and frequently lead to contradicting conclusions. Numerous approaches including functional high-throughput and miRISC complex evaluations suggest that the functional miRNAome differs from the predictions based on quantitative sRNA profiling. To resolve the apparent contradiction of expression versus function, we generated and applied a fluorescence reporter gene assay enabling single cell analysis. This approach integrates and adapts a mathematical model for miRNA-driven gene repression. This model predicts three distinct miRNA-groups with unique repression activities (low, mid and high) governed not just by expression levels but also by miRNA/target-binding capability. Here, we demonstrate the feasibility of the system by applying controlled concentrations of synthetic siRNAs and in parallel, altering target-binding capability on corresponding reporter-constructs. Furthermore, we compared miRNA-profiles with the modeled predictions of 29 individual candidates. We demonstrate that expression levels only partially reflect the miRNA function, fitting to the model-projected groups of different activities. Furthermore, we demonstrate that subcellular localization of miRNAs impacts functionality. Our results imply that miRNA profiling alone cannot define their repression activity. The gene regulatory function is a dynamic and complex process beyond a minimalistic conception of “highly expressed equals high repression”. PMID:28338079

Multistep Model of Cervical Cancer: Participation of miRNAs and Coding Genes

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Angelica Judith Granados López

2014-09-01

Full Text Available Aberrant miRNA expression is well recognized as an important step in the development of cancer. Close to 70 microRNAs (miRNAs have been implicated in cervical cancer up to now, nevertheless it is unknown if aberrant miRNA expression causes the onset of cervical cancer. One of the best ways to address this issue is through a multistep model of carcinogenesis. In the progression of cervical cancer there are three well-established steps to reach cancer that we used in the model proposed here. The first step of the model comprises the gene changes that occur in normal cells to be transformed into immortal cells (CIN 1, the second comprises immortal cell changes to tumorigenic cells (CIN 2, the third step includes cell changes to increase tumorigenic capacity (CIN 3, and the final step covers tumorigenic changes to carcinogenic cells. Altered miRNAs and their target genes are located in each one of the four steps of the multistep model of carcinogenesis. miRNA expression has shown discrepancies in different works; therefore, in this model we include miRNAs recording similar results in at least two studies. The present model is a useful insight into studying potential prognostic, diagnostic, and therapeutic miRNAs.

Splenic marginal zone lymphoma: comprehensive analysis of gene expression and miRNA profiling.

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Arribas, Alberto J; Gómez-Abad, Cristina; Sánchez-Beato, Margarita; Martinez, Nerea; Dilisio, Lorena; Casado, Felipe; Cruz, Miguel A; Algara, Patrocinio; Piris, Miguel A; Mollejo, Manuela

2013-07-01

Splenic marginal zone lymphoma is a small B-cell neoplasm whose molecular pathogenesis is still essentially unknown and whose differentiation from other small B-cell lymphomas is hampered by the lack of specific markers. We have analyzed the gene expression and miRNA profiles of 31 splenic marginal zone lymphoma cases. For comparison, 7 spleens with reactive lymphoid hyperplasia, 10 spleens infiltrated by chronic lymphocytic leukemia, 12 spleens with follicular lymphoma, 6 spleens infiltrated by mantle cell lymphoma and 15 lymph nodes infiltrated by nodal marginal zone lymphoma were included. The results were validated by qRT-PCR in an independent series including 77 paraffin-embedded splenic marginal zone lymphomas. The splenic marginal zone lymphoma miRNA signature had deregulated expression of 51 miRNAs. The most highly overexpressed miRNAs were miR-155, miR-21, miR-34a, miR-193b and miR-100, while the most repressed miRNAs were miR-377, miR-27b, miR-145, miR-376a and miR-424. MiRNAs located in 14q32-31 were underexpressed in splenic marginal zone lymphoma compared with reactive lymphoid tissues and other B-cell lymphomas. Finally, the gene expression data were integrated with the miRNA profile to identify functional relationships between genes and deregulated miRNAs. Our study reveals miRNAs that are deregulated in splenic marginal zone lymphoma and identifies new candidate diagnostic molecules for splenic marginal zone lymphoma.

Sparse Modeling Reveals miRNA Signatures for Diagnostics of Inflammatory Bowel Disease.

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Matthias Hübenthal

Full Text Available The diagnosis of inflammatory bowel disease (IBD still remains a clinical challenge and the most accurate diagnostic procedure is a combination of clinical tests including invasive endoscopy. In this study we evaluated whether systematic miRNA expression profiling, in conjunction with machine learning techniques, is suitable as a non-invasive test for the major IBD phenotypes (Crohn's disease (CD and ulcerative colitis (UC. Based on microarray technology, expression levels of 863 miRNAs were determined for whole blood samples from 40 CD and 36 UC patients and compared to data from 38 healthy controls (HC. To further discriminate between disease-specific and general inflammation we included miRNA expression data from other inflammatory diseases (inflammation controls (IC: 24 chronic obstructive pulmonary disease (COPD, 23 multiple sclerosis, 38 pancreatitis and 45 sarcoidosis cases as well as 70 healthy controls from previous studies. Classification problems considering 2, 3 or 4 groups were solved using different types of penalized support vector machines (SVMs. The resulting models were assessed regarding sparsity and performance and a subset was selected for further investigation. Measured by the area under the ROC curve (AUC the corresponding median holdout-validated accuracy was estimated as ranging from 0.75 to 1.00 (including IC and 0.89 to 0.98 (excluding IC, respectively. In combination, the corresponding models provide tools for the distinction of CD and UC as well as CD, UC and HC with expected classification error rates of 3.1 and 3.3%, respectively. These results were obtained by incorporating not more than 16 distinct miRNAs. Validated target genes of these miRNAs have been previously described as being related to IBD. For others we observed significant enrichment for IBD susceptibility loci identified in earlier GWAS. These results suggest that the proposed miRNA signature is of relevance for the etiology of IBD. Its diagnostic

Analyzing the interactions of mRNAs, miRNAs, lncRNAs and circRNAs to predict competing endogenous RNA networks in glioblastoma.

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Yuan, Yang; Jiaoming, Li; Xiang, Wang; Yanhui, Liu; Shu, Jiang; Maling, Gou; Qing, Mao

2018-05-01

Cross-talk between competitive endogenous RNAs (ceRNAs) may play a critical role in revealing potential mechanisms of tumor development and physiology. Glioblastoma is the most common type of malignant primary brain tumor, and the mechanisms of tumor genesis and development in glioblastoma are unclear. Here, to investigate the role of non-coding RNAs and the ceRNA network in glioblastoma, we performed paired-end RNA sequencing and microarray analyses to obtain the expression profiles of mRNAs, lncRNAs, circRNAs and miRNAs. We identified that the expression of 501 lncRNAs, 1999 mRNAs, 2038 circRNAs and 143 miRNAs were often altered between glioblastoma and matched normal brain tissue. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed on these differentially expressed mRNAs and miRNA-mediated target genes of lncRNAs and circRNAs. Furthermore, we used a multi-step computational framework and several bioinformatics methods to construct a ceRNA network combining mRNAs, miRNAs, lncRNAs and circRNA, based on co-expression analysis between the differentially expressed RNAs. We identified that plenty of lncRNAs, CircRNAs and their downstream target genes in the ceRNA network are related to glutamatergic synapse, suggesting that glutamate metabolism is involved in glioma biological functions. Our results will accelerate the understanding of tumorigenesis, cancer progression and even therapeutic targeting in glioblastoma.

Global MicroRNA Profiling in Human Bone Marrow Skeletal—Stromal or Mesenchymal–Stem Cells Identified Candidates for Bone Regeneration

DEFF Research Database (Denmark)

Chang, Chi Chih; Venø, Morten T.; Chen, Li

2018-01-01

Bone remodeling and regeneration are highly regulated multistep processes involving posttranscriptional regulation by microRNAs (miRNAs). Here, we performed a global profiling of differentially expressed miRNAs in bone-marrow-derived skeletal cells (BMSCs; also known as stromal or mesenchymal stem......RNAs for enhancing bone tissue regeneration. Scaffolds functionalized with miRNA nano-carriers enhanced osteoblastogenesis in 3D culture and retained this ability at least 2 weeks after storage. Additionally, anti-miR-222 enhanced in vivo ectopic bone formation through targeting the cell-cycle inhibitor CDKN1B...... cells) during in vitro osteoblast differentiation. We functionally validated the regulatory effects of several miRNAs on osteoblast differentiation and identified 15 miRNAs, most significantly miR-222 and miR-423, as regulators of osteoblastogenesis. In addition, we tested the possible targeting of mi...

Identification of a set of endogenous reference genes for miRNA expression studies in Parkinson's disease blood samples.

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Serafin, Alice; Foco, Luisa; Blankenburg, Hagen; Picard, Anne; Zanigni, Stefano; Zanon, Alessandra; Pramstaller, Peter P; Hicks, Andrew A; Schwienbacher, Christine

2014-10-10

Research on microRNAs (miRNAs) is becoming an increasingly attractive field, as these small RNA molecules are involved in several physiological functions and diseases. To date, only few studies have assessed the expression of blood miRNAs related to Parkinson's disease (PD) using microarray and quantitative real-time PCR (qRT-PCR). Measuring miRNA expression involves normalization of qRT-PCR data using endogenous reference genes for calibration, but their choice remains a delicate problem with serious impact on the resulting expression levels. The aim of the present study was to evaluate the suitability of a set of commonly used small RNAs as normalizers and to identify which of these miRNAs might be considered reliable reference genes in qRT-PCR expression analyses on PD blood samples. Commonly used reference genes snoRNA RNU24, snRNA RNU6B, snoRNA Z30 and miR-103a-3p were selected from the literature. We then analyzed the effect of using these genes as reference, alone or in any possible combination, on the measured expression levels of the target genes miR-30b-5p and miR-29a-3p, which have been previously reported to be deregulated in PD blood samples. We identified RNU24 and Z30 as a reliable and stable pair of reference genes in PD blood samples.

DIANA-microT web server v5.0: service integration into miRNA functional analysis workflows.

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Paraskevopoulou, Maria D; Georgakilas, Georgios; Kostoulas, Nikos; Vlachos, Ioannis S; Vergoulis, Thanasis; Reczko, Martin; Filippidis, Christos; Dalamagas, Theodore; Hatzigeorgiou, A G

2013-07-01

MicroRNAs (miRNAs) are small endogenous RNA molecules that regulate gene expression through mRNA degradation and/or translation repression, affecting many biological processes. DIANA-microT web server (http://www.microrna.gr/webServer) is dedicated to miRNA target prediction/functional analysis, and it is being widely used from the scientific community, since its initial launch in 2009. DIANA-microT v5.0, the new version of the microT server, has been significantly enhanced with an improved target prediction algorithm, DIANA-microT-CDS. It has been updated to incorporate miRBase version 18 and Ensembl version 69. The in silico-predicted miRNA-gene interactions in Homo sapiens, Mus musculus, Drosophila melanogaster and Caenorhabditis elegans exceed 11 million in total. The web server was completely redesigned, to host a series of sophisticated workflows, which can be used directly from the on-line web interface, enabling users without the necessary bioinformatics infrastructure to perform advanced multi-step functional miRNA analyses. For instance, one available pipeline performs miRNA target prediction using different thresholds and meta-analysis statistics, followed by pathway enrichment analysis. DIANA-microT web server v5.0 also supports a complete integration with the Taverna Workflow Management System (WMS), using the in-house developed DIANA-Taverna Plug-in. This plug-in provides ready-to-use modules for miRNA target prediction and functional analysis, which can be used to form advanced high-throughput analysis pipelines.

Identification and characterization of miRNAs transcriptome in the South African abalone, Haliotis midae.

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Picone, Barbara; Rhode, Clint; Roodt-Wilding, Rouvay

2017-02-01

Aquatic animal diseases are one of the most important limitations to the growth of aquaculture. miRNAs represent an important class of small ncRNAs able to modulate host immune and stress responses. In Mollusca, a large phylum of invertebrates, miRNAs have been identified in several species. The current preliminary study identified known miRNAs from the South African abalone, Haliotis midae. The economic and ecological importance of abalone makes this species a suitable model for studying and understanding stress response in marine gastropods. Furthermore, the identification of miRNA, represents an alternative and powerful tool to combat infectious disease. Copyright © 2016 Elsevier B.V. All rights reserved.

Circulating microRNA (miRNA Expression Profiling in Plasma of Patients with Gestational Diabetes Mellitus Reveals Upregulation of miRNA miR-330-3p

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Guido Sebastiani

2017-12-01

Full Text Available Gestational diabetes mellitus (GDM is characterized by insulin resistance accompanied by low/absent beta-cell compensatory adaptation to the increased insulin demand. Although the molecular mechanisms and factors acting on beta-cell compensatory response during pregnancy have been partially elucidated and reported, those inducing an impaired beta-cell compensation and function, thus evolving in GDM, have yet to be fully addressed. MicroRNAs (miRNAs are a class of small endogenous non-coding RNAs, which negatively modulate gene expression through their sequence-specific binding to 3′UTR of mRNA target. They have been described as potent modulators of cell survival and proliferation and, furthermore, as orchestrating molecules of beta-cell compensatory response and function in diabetes. Moreover, it has been reported that miRNAs can be actively secreted by cells and found in many biological fluids (e.g., serum/plasma, thus representing both optimal candidate disease biomarkers and mediators of tissues crosstalk(s. Here, we analyzed the expression profiles of circulating miRNAs in plasma samples obtained from n = 21 GDM patients and from n = 10 non-diabetic control pregnant women (24–33 weeks of gestation using TaqMan array microfluidics cards followed by RT-real-time PCR single assay validation. The results highlighted the upregulation of miR-330-3p in plasma of GDM vs non-diabetics. Furthermore, the analysis of miR-330-3p expression levels revealed a bimodally distributed GDM patients group characterized by high or low circulating miR-330 expression and identified as GDM-miR-330high and GDM-miR-330low. Interestingly, GDM-miR-330high subgroup retained lower levels of insulinemia, inversely correlated to miR-330-3p expression levels, and a significant higher rate of primary cesarean sections. Finally, miR-330-3p target genes analysis revealed major modulators of beta-cell proliferation and of insulin secretion, such as the

Revised annotation of Plutella xylostella microRNAs and their genome-wide target identification.

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Etebari, K; Asgari, S

2016-12-01

The diamondback moth, Plutella xylostella, is the most devastating pest of brassica crops worldwide. Although 128 mature microRNAs (miRNAs) have been annotated from this species in miRBase, there is a need to extend and correct the current P. xylostella miRNA repertoire as a result of its recently improved genome assembly and more available small RNA sequence data. We used our new ultra-deep sequence data and bioinformatics to re-annotate the P. xylostella genome for high confidence miRNAs with the correct 5p and 3p arm features. Furthermore, all the P. xylostella annotated genes were also screened to identify potential miRNA binding sites using three target-predicting algorithms. In total, 203 mature miRNAs were annotated, including 33 novel miRNAs. We identified 7691 highly confident binding sites for 160 pxy-miRNAs. The data provided here will facilitate future studies involving functional analyses of P. xylostella miRNAs as a platform to introduce novel approaches for sustainable management of this destructive pest. © 2016 The Royal Entomological Society.

A multiplexed miRNA and transgene expression platform for simultaneous repression and expression of protein coding sequences.

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Seyhan, Attila A

2016-01-01

Knockdown of single or multiple gene targets by RNA interference (RNAi) is necessary to overcome escape mutants or isoform redundancy. It is also necessary to use multiple RNAi reagents to knockdown multiple targets. It is also desirable to express a transgene or positive regulatory elements and inhibit a target gene in a coordinated fashion. This study reports a flexible multiplexed RNAi and transgene platform using endogenous intronic primary microRNAs (pri-miRNAs) as a scaffold located in the green fluorescent protein (GFP) as a model for any functional transgene. The multiplexed intronic miRNA - GFP transgene platform was designed to co-express multiple small RNAs within the polycistronic cluster from a Pol II promoter at more moderate levels to reduce potential vector toxicity. The native intronic miRNAs are co-transcribed with a precursor GFP mRNA as a single transcript and presumably cleaved out of the precursor-(pre) mRNA by the RNA splicing machinery, spliceosome. The spliced intron with miRNA hairpins will be further processed into mature miRNAs or small interfering RNAs (siRNAs) capable of triggering RNAi effects, while the ligated exons become a mature messenger RNA for the translation of the functional GFP protein. Data show that this approach led to robust RNAi-mediated silencing of multiple Renilla Luciferase (R-Luc)-tagged target genes and coordinated expression of functional GFP from a single transcript in transiently transfected HeLa cells. The results demonstrated that this design facilitates the coordinated expression of all mature miRNAs either as individual miRNAs or as multiple miRNAs and the associated protein. The data suggest that, it is possible to simultaneously deliver multiple negative (miRNA or shRNA) and positive (transgene) regulatory elements. Because many cellular processes require simultaneous repression and activation of downstream pathways, this approach offers a platform technology to achieve that dual manipulation efficiently

miRNA-135b Contributes to Triple Negative Breast Cancer Molecular Heterogeneity: Different Expression Profile in Basal-like Versus non-Basal-like Phenotypes.

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Uva, Paolo; Cossu-Rocca, Paolo; Loi, Federica; Pira, Giovanna; Murgia, Luciano; Orrù, Sandra; Floris, Matteo; Muroni, Maria Rosaria; Sanges, Francesca; Carru, Ciriaco; Angius, Andrea; De Miglio, Maria Rosaria

2018-01-01

The clinical and genetic heterogeneity of Triple Negative Breast Cancer (TNBC) and the lack of unambiguous molecular targets contribute to the inadequacy of current therapeutic options for these variants. MicroRNAs (miRNA) are a class of small highly conserved regulatory endogenous non-coding RNA, which can alter the expression of genes encoding proteins and may play a role in the dysregulation of cellular pathways. Our goal was to improve the knowledge of the molecular pathogenesis of TNBC subgroups analyzing the miRNA expression profile, and to identify new prognostic and predictive biomarkers. We conducted a human miRNome analysis by TaqMan Low Density Array comparing different TNBC subtypes, defined by immunohistochemical basal markers EGFR and CK5/6. RT-qPCR confirmed differential expression of microRNAs. To inspect the function of the selected targets we perform Gene Ontology and KEGG enrichment analysis. We identified a single miRNA signature given by miR-135b expression level, which was strictly related to TNBC with basal-like phenotype. miR-135b target analysis revealed a role in the TGF-beta, WNT and ERBB pathways. A significant positive correlation was identified between neoplastic proliferative index and miR-135b expression. These findings confirm the oncogenic roles of miR-135b in the pathogenesis of TNBC expressing basal markers. A potential negative prognostic role of miR-135b overexpression might be related to the positive correlation with high proliferative index. Our study implies potential clinical applications: miR-135b could be a potential therapeutic target in basal-like TNBCs.

The First Report of miRNAs from a Thysanopteran Insect, Thrips palmi Karny Using High-Throughput Sequencing.

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K B Rebijith

Full Text Available Thrips palmi Karny (Thysanoptera: Thripidae is the sole vector of Watermelon bud necrosis tospovirus, where the crop loss has been estimated to be around USD 50 million annually. Chemical insecticides are of limited use in the management of T. palmi due to the thigmokinetic behaviour and development of high levels of resistance to insecticides. There is an urgent need to find out an effective futuristic management strategy, where the small RNAs especially microRNAs hold great promise as a key player in the growth and development. miRNAs are a class of short non-coding RNAs involved in regulation of gene expression either by mRNA cleavage or by translational repression. We identified and characterized a total of 77 miRNAs from T. palmi using high-throughput deep sequencing. Functional classifications of the targets for these miRNAs revealed that majority of them are involved in the regulation of transcription and translation, nucleotide binding and signal transduction. We have also validated few of these miRNAs employing stem-loop RT-PCR, qRT-PCR and Northern blot. The present study not only provides an in-depth understanding of the biological and physiological roles of miRNAs in governing gene expression but may also lead as an invaluable tool for the management of thysanopteran insects in the future.

Comparison of miRNA and gene expression profiles between metastatic and primary prostate cancer.

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Guo, Kaimin; Liang, Zuowen; Li, Fubiao; Wang, Hongliang

2017-11-01

The present study aimed to identify the regulatory mechanisms associated with the metastasis of prostate cancer (PC). The microRNA (miRNA/miR) microarray dataset GSE21036 and gene transcript dataset GSE21034 were downloaded from the Gene Expression Omnibus database. Following pre-processing, differentially expressed miRNAs (DEMs) and differentially expressed genes (DEGs) between samples from patients with primary prostate cancer (PPC) and metastatic prostate cancer (MPC) with |log 2 fold change (FC)| >1 and a false discovery rate terms (36 terms), followed by miR-494 (24 terms), miR-30d (18 terms), miR-181a (15 terms), hsa-miR-196a (8 terms), miR-708 (7 terms) and miR-486-5p (2 terms). Therefore, these miRNAs may serve roles in the metastasis of PC cells via downregulation of their corresponding target DEGs.

Comparative profiling of miRNA expression in developing seeds of high linoleic and high oleic safflower (Carthamus tinctorius L. plants

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Shijiang eCao

2013-12-01

Full Text Available Vegetable oils high in oleic acid are considered to be advantageous because of their better nutritional value and potential industrial applications. The oleic acid content in the classic safflower oil is normally 10-15% while a natural mutant (ol accumulates elevated oleic acid up to 70% in seed oil. As a part of our investigation into the molecular features of the high oleic (HO trait in safflower we have profiled the microRNA (miRNA populations in developing safflower seeds expressing the ol allele in comparison to the wild type high linoleic (HL safflower using deep sequencing technology. The small RNA populations of the mid-maturity developing embryos of homozygous ol HO and wild type HL safflower had a very similar size distribution pattern, however, only ~16.5% of the unique small RNAs were overlapping in these two genotypes. From these two small RNA populations we have found 55 known miRNAs and identified two candidate novel miRNA families to be likely unique to the developing safflower seeds. Target genes with conserved as well as novel functions were predicted for the conserved miRNAs. We have also identified 13 miRNAs differentially expressed between the HO and HL safflower genotypes. The results may lay a foundation for unravelling the miRNA-mediated molecular processes that regulate oleic acid accumulation in the HO safflower mutant and developmental processes in safflower embryos in general.

MicroRNA-181a* Targets Nanog in a Subpopulation of CD34+ Cells Isolated From Peripheral Blood

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Paul J Mintz

2012-01-01

Full Text Available Exploiting the properties of stem cells by microRNA (miRNA profiling offers an attractive approach to identify new regulators of stem cell fate. Although numerous miRNA have been screened from hematopoietic stem cells (HSC, the targets corresponding to many of these miRNA have not yet been fully elucidated. By miRNA profiling in a subpopulation of CD34+ cells isolated from peripheral blood, we have identified eight clusters of miRNA that were differentially expressed. Further analysis of one of the clusters by bioinformatics revealed that a miRNA, miR-181a*, which is highly expressed in the adherent CD34+ cells, affects the expression levels of Nanog, a stem cell surrogate marker. We show specifically by reporter assay and mutational analysis that miR-181a* targets a seedless 3′ compensatory site in the 3′UTR of Nanog and affects gene expression. We demonstrate that inhibiting miR-181a* upregulates the Nanog expression level, in addition to an increase in alkaline phosphatase activity. Our studies suggest that miR-181a* may be important in controlling the expression level of Nanog in a subpopulation of CD34+ cells.

Establishment of Lipofection for Studying miRNA Function in Human Adipocytes

OpenAIRE

Enlund, Eveliina; Fischer, Simon; Handrick, René; Otte, Kerstin; Debatin, Klaus-Michael; Wabitsch, Martin; Fischer-Posovszky, Pamela

2014-01-01

miRNA dysregulation has recently been linked to human obesity and its related complications such as type 2 diabetes. In order to study miRNA function in human adipocytes, we aimed for the modulation of mature miRNA concentration in these cells. Adipocytes, however, tend to be resistant to transfection and there is often a need to resort to viral transduction or electroporation. Our objective therefore was to identify an efficient, non-viral transfection reagent capable of delivering small RNA...

Salivary extracellular vesicle-associated miRNAs as potential biomarkers in oral squamous cell carcinoma.

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Gai, Chiara; Camussi, Francesco; Broccoletti, Roberto; Gambino, Alessio; Cabras, Marco; Molinaro, Luca; Carossa, Stefano; Camussi, Giovanni; Arduino, Paolo G

2018-04-18

Several studies in the past have investigated the expression of micro RNAs (miRNAs) in saliva as potential biomarkers. Since miRNAs associated with extracellular vesicles (EVs) are known to be protected from enzymatic degradation, we evaluated whether salivary EVs from patients with oral squamous cell carcinoma (OSCC) were enriched with specific subsets of miRNAs. OSCC patients and controls were matched with regards to age, gender and risk factors. Total RNA was extracted from salivary EVs and the differential expression of miRNAs was evaluated by qRT-PCR array and qRT-PCR. The discrimination power of up-regulated miRNAs as biomarkers in OSCC patients versus controls was evaluated by the Receiver Operating Characteristic (ROC) curves. A preliminary qRT-PCR array was performed on samples from 5 OSCC patients and 5 healthy controls whereby a subset of miRNAs were identified that were differentially expressed. On the basis of these results, a cohort of additional 16 patients and 6 controls were analyzed to further confirm the miRNAs that were up-regulated or selectively expressed in the previous pilot study. The following miRNAs: miR-302b-3p and miR-517b-3p were expressed only in EVs from OSCC patients and miR-512-3p and miR-412-3p were up-regulated in salivary EVs from OSCC patients compared to controls with the ROC curve showing a good discrimination power for OSCC diagnosis. The Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway analysis suggested the possible involvement of the miRNAs identified in pathways activated in OSCC. In this work, we suggest that salivary EVs isolated by a simple charge-based precipitation technique can be exploited as a non-invasive source of miRNAs for OSCC diagnosis. Moreover, we have identified a subset of miRNAs selectively enriched in EVs of OSCC patients that could be potential biomarkers.

Role of miRNA-9 in Brain Development

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Balachandar Radhakrishnan

2016-01-01

Full Text Available MicroRNAs (miRNAs are a class of small regulatory RNAs involved in gene regulation. The regulation is effected by either translational inhibition or transcriptional silencing. In vertebrates, the importance of miRNA in development was discovered from mice and zebrafish dicer knockouts. The miRNA-9 (miR-9 is one of the most highly expressed miRNAs in the early and adult vertebrate brain. It has diverse functions within the developing vertebrate brain. In this article, the role of miR-9 in the developing forebrain (telencephalon and diencephalon, midbrain, hindbrain, and spinal cord of vertebrate species is highlighted. In the forebrain, miR-9 is necessary for the proper development of dorsoventral telencephalon by targeting marker genes expressed in the telencephalon. It regulates proliferation in telencephalon by regulating Foxg1, Pax6, Gsh2 , and Meis2 genes. The feedback loop regulation between miR-9 and Nr2e1/Tlx helps in neuronal migration and differentiation. Targeting Foxp1 and Foxp2 , and Map1b by miR-9 regulates the radial migration of neurons and axonal development. In the organizers, miR-9 is inversely regulated by hairy1 and Fgf8 to maintain zona limitans interthalamica and midbrain-hindbrain boundary (MHB. It maintains the MHB by inhibiting Fgf signaling genes and is involved in the neurogenesis of the midbrain-hindbrain by regulating Her genes. In the hindbrain, miR-9 modulates progenitor proliferation and differentiation by regulating Her genes and Elav3. In the spinal cord, miR-9 modulates the regulation of Foxp1 and Onecut1 for motor neuron development. In the forebrain, midbrain, and hindbrain, miR-9 is necessary for proper neuronal progenitor maintenance, neurogenesis, and differentiation. In vertebrate brain development, miR-9 is involved in regulating several region-specific genes in a spatiotemporal pattern.

A bootstrap based analysis pipeline for efficient classification of phylogenetically related animal miRNAs

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Gu Xun

2007-03-01

Full Text Available Abstract Background Phylogenetically related miRNAs (miRNA families convey important information of the function and evolution of miRNAs. Due to the special sequence features of miRNAs, pair-wise sequence identity between miRNA precursors alone is often inadequate for unequivocally judging the phylogenetic relationships between miRNAs. Most of the current methods for miRNA classification rely heavily on manual inspection and lack measurements of the reliability of the results. Results In this study, we designed an analysis pipeline (the Phylogeny-Bootstrap-Cluster (PBC pipeline to identify miRNA families based on branch stability in the bootstrap trees derived from overlapping genome-wide miRNA sequence sets. We tested the PBC analysis pipeline with the miRNAs from six animal species, H. sapiens, M. musculus, G. gallus, D. rerio, D. melanogaster, and C. elegans. The resulting classification was compared with the miRNA families defined in miRBase. The two classifications were largely consistent. Conclusion The PBC analysis pipeline is an efficient method for classifying large numbers of heterogeneous miRNA sequences. It requires minimum human involvement and provides measurements of the reliability of the classification results.