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Sample records for block rna silencing

  1. RNA silencing movement in plants

    Institute of Scientific and Technical Information of China (English)

    Glykeria Mermigka; Frederic Verret; Kriton Kalantidis

    2016-01-01

    Multicellular organisms, like higher plants, need to coordinate their growth and development and to cope with environmental cues. To achieve this, various signal molecules are transported between neighboring cells and distant organs to control the fate of the recipient cells and organs. RNA silencing produces cell non-autonomous signal molecules that can move over short or long distances leading to the sequence specific silencing of a target gene in a well defined area of cells or throughout the entire plant, respectively. The nature of these signal molecules, the route of silencing spread, and the genes involved in their production, movement and reception are discussed in this review. Additionally, a short section on features of silencing spread in animal models is presented at the end of this review.

  2. Strategies underlying RNA silencing suppression by negative strand RNA viruses

    NARCIS (Netherlands)

    Hemmes, J.C.

    2007-01-01

    The research described in this thesis focused on the strategies of negative strand RNA viruses to counteract antiviral RNA silencing. In plants and insects, RNA silencing has been shown to act as a sequence specific antiviral defence mechanism that is characterised by the processing of double strand

  3. Three distinct suppressors of RNA silencing encoded by a 20-kb viral RNA genome

    Science.gov (United States)

    Lu, Rui; Folimonov, Alexey; Shintaku, Michael; Li, Wan-Xiang; Falk, Bryce W.; Dawson, William O.; Ding, Shou-Wei

    2004-11-01

    Viral infection in both plant and invertebrate hosts requires a virus-encoded function to block the RNA silencing antiviral defense. Here, we report the identification and characterization of three distinct suppressors of RNA silencing encoded by the 20-kb plus-strand RNA genome of citrus tristeza virus (CTV). When introduced by genetic crosses into plants carrying a silencing transgene, both p20 and p23, but not coat protein (CP), restored expression of the transgene. Although none of the CTV proteins prevented DNA methylation of the transgene, export of the silencing signal (capable of mediating intercellular silencing spread) was detected only from the F1 plants expressing p23 and not from the CP- or p20-expressing F1 plants, demonstrating suppression of intercellular silencing by CP and p20 but not by p23. Thus, intracellular and intercellular silencing are each targeted by a CTV protein, whereas the third, p20, inhibits silencing at both levels. Notably, CP suppresses intercellular silencing without interfering with intracellular silencing. The novel property of CP suggests a mechanism distinct to p20 and all of the other viral suppressors known to interfere with intercellular silencing and that this class of viral suppressors may not be consistently identified by Agrobacterium coinfiltration because it also induces RNA silencing against the infiltrated suppressor transgene. Our analyses reveal a sophisticated viral counter-defense strategy that targets the silencing antiviral pathway at multiple steps and may be essential for protecting CTV with such a large RNA genome from antiviral silencing in the perennial tree host. RNA interference | citrus tristeza virus | virus synergy | antiviral immunity

  4. Viral counterdefense on RNA silencing : analysis of RNA silencing suppressors from arthropod-borne negative strand RNA plant viruses

    NARCIS (Netherlands)

    Schnettler, E.

    2010-01-01

    This thesis describes that RNA silencing suppressor (RSS) proteins encoded by negative-stranded RNA plant viruses are able to interfere with different RNA silencing pathways in a variety of organisms by interacting with double stranded (ds)RNA molecules. These RSS proteins are able to counteract the

  5. Suppressor of RNA silencing encoded by Rice gall dwarf virus genome segment 11

    Institute of Scientific and Technical Information of China (English)

    LIU FuXiu; ZHAO Qin; RUAN XiaoLei; HE YunWei; LI HuaPing

    2008-01-01

    Rice gall dwarf virus (RGDV) is an important rice pathogen in China and Southeast Asia. However, little is known about the molecular mechanisms of RGDV interactions with plant cells. Here, we have identi-fied an RGDV protein, Pns11, which acts as a suppressor of RNA silencing in coinfiltration assays with the reporter, green fluorescent protein (GFP) in transgenic Nicotiana bentharniana line 16c carrying GFP. Pns11 suppressed local and systemic silencing induced by sense RNA. The spread of mobile FINA si-lencing signals was blocked or inactivated by Pns11. Expression of Pns11 also enhanced Potato virus X pathogenicity in N. benthamiana. This suppressor could reduce, but not eliminate, siRNA in the local and systemic RNA silencing suppression assays, suggesting that Pns11 functions by interfering with initial stages of RNA silencing.

  6. Two classes of short interfering RNA in RNA silencing

    OpenAIRE

    Hamilton, Andrew; Voinnet, Olivier; Chappell, Louise; Baulcombe, David

    2015-01-01

    RNA silencing is a eukaryotic genome defence system that involves processing of double-stranded RNA (dsRNA) into 21–26 nt, short interfering RNA (siRNA). The siRNA mediates suppression of genes corresponding to the dsRNA through targeted RNA degradation. In some plant systems there are additional silencing processes, involving systemic spread of silencing and RNA-directed methylation/transcriptional suppression of homologous genomic DNA. We show here that siRNAs produced in plants from a gree...

  7. Antiviral RNA silencing viral counter defense in plants

    NARCIS (Netherlands)

    Bucher, E.C.

    2006-01-01

    The research described in this thesis centres around the mechanism of RNA silencing in relation to virus-host interaction, an area of increasing importance. It shows how this recently disclosed mechanism can be used to produce virus-resistant plants. Based on the activity of the RNA silencing machin

  8. LNA-antisense rivals siRNA for gene silencing

    DEFF Research Database (Denmark)

    Jepsen, Jan Stenvang; Wengel, Jesper; Stenvang, Jan

    2004-01-01

    Locked nucleic acid (LNA) is a class of nucleic acid analogs possessing unprecedented binding affinity toward complementary DNA and RNA while obeying the Watson-Crick base-pairing rules. For efficient gene silencing in vitro and in vivo, fully modified or chimeric LNA oligonucleotides have been...... applied. LNA oligonucleotides are commercially available, can be transfected using standard techniques, are non-toxic, lead to increased target accessibility, can be designed to activate RNase H, and function in steric block approaches. LNA-Antisense, including gapmer LNA containing a central DNA...... or phosphorothioate-DNA segment flanked by LNA gaps, rivals siRNA as the technology of choice for target validation and therapeutic applications....

  9. Screening and identification of virus-encoded RNA silencing suppressors.

    Science.gov (United States)

    Karjee, Sumona; Islam, Mohammad Nurul; Mukherjee, Sunil K

    2008-01-01

    RNA silencing, including RNA interference, is a novel method of gene regulation and one of the potent host-defense mechanisms against the viruses. In the course of evolution, the viruses have encoded proteins with the potential to suppress the host RNA silencing mechanism as a counterdefense strategy. The virus-encoded RNA silencing suppressors (RSSs) can serve as important biological tools to dissect the detailed RNA silencing pathways and also to evolve the antiviral strategies. Screening and identification of the RSSs are indeed of utmost significance in the field of plant biotechnology. We describe two Green Fluorescent Protein (GFP) reporter-based plant assay systems that rely on two different principles, namely reversal of silencing and enhancement of rolling circle replication (RCR) of geminiviral replicon. These proof-of-concept examples and assay systems could be used to screen various plant, animal, and insect viral ORFs for identification of the RSS activities.

  10. Locus-specific ribosomal RNA gene silencing in nucleolar dominance.

    Directory of Open Access Journals (Sweden)

    Michelle S Lewis

    Full Text Available The silencing of one parental set of rRNA genes in a genetic hybrid is an epigenetic phenomenon known as nucleolar dominance. We showed previously that silencing is restricted to the nucleolus organizer regions (NORs, the loci where rRNA genes are tandemly arrayed, and does not spread to or from neighboring protein-coding genes. One hypothesis is that nucleolar dominance is the net result of hundreds of silencing events acting one rRNA gene at a time. A prediction of this hypothesis is that rRNA gene silencing should occur independent of chromosomal location. An alternative hypothesis is that the regulatory unit in nucleolar dominance is the NOR, rather than each individual rRNA gene, in which case NOR localization may be essential for rRNA gene silencing. To test these alternative hypotheses, we examined the fates of rRNA transgenes integrated at ectopic locations. The transgenes were accurately transcribed in all independent transgenic Arabidopsis thaliana lines tested, indicating that NOR localization is not required for rRNA gene expression. Upon crossing the transgenic A. thaliana lines as ovule parents with A. lyrata to form F1 hybrids, a new system for the study of nucleolar dominance, the endogenous rRNA genes located within the A. thaliana NORs are silenced. However, rRNA transgenes escaped silencing in multiple independent hybrids. Collectively, our data suggest that rRNA gene activation can occur in a gene-autonomous fashion, independent of chromosomal location, whereas rRNA gene silencing in nucleolar dominance is locus-dependent.

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

    NARCIS (Netherlands)

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

    2000-01-01

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

  12. Suppressors of RNA silencing encoded by tomato leaf curl betasatellites

    Indian Academy of Sciences (India)

    Richa Shukla; Sunita Dalal; V G Malathi

    2013-03-01

    Virus encoded RNA-silencing suppressors (RSSs) are the key components evolved by the viruses to counter RNA-silencing defense of plants. Whitefly-transmitted begomoviruses infecting tomato crop code for five different proteins, ORF AC4, ORF AC2 and ORF AV2 in DNA-A component, ORF BV1 in DNA-B and ORF C1 in satellite DNA which are predicted to function as silencing suppressors. In the present study suppressor function of ORF C1 of three betasatellites Tomato leaf curl Bangalore betasatellite ToLCBB-[IN:Hess:08], Cotton leaf curl Multan betasatellite CLCuMB–[IN:Sri:02] and Luffa leaf distortion betasatellite LuLDB-[IN:Lu:04] were examined. Agroinfiltration of GFP-silenced Nicotiana tabaccum cv. Xanthi with the cells expressing C1 protein resulted in reversal of silenced GFP expression. GFP-siRNA level was more than 50-fold lower compared to silenced plants in plants infiltrated with C1 gene from ToLCBB. However, in the case of 35S-C1 CLCuMB and 35S-C1 LuLDB construct, although GFP was expressed, siRNA level was not reduced, indicating that the step at which C1 interfere in RNA-silencing pathway is different.

  13. Synthetic RNA silencing in bacteria - antimicrobial discovery and resistance breaking

    Directory of Open Access Journals (Sweden)

    James E.M. Stach

    2011-09-01

    Full Text Available The increasing incidence and prevalence of antibiotic resistance in bacteria threatens the antibiotic miracle. Conventional antimicrobial drug development has failed to replace the armamentarium needed to combat this problem, and novel solutions are urgently required. Here we review both natural and synthetic RNA silencing and its potential to provide new antibacterials through improved target selection, evaluation and screening. Furthermore, we focus on synthetic RNA silencers as a novel class of antibacterials and review their unique properties.

  14. Gene silencing: Double-stranded RNA mediated mRNA degradation and gene inactivation

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The recent development of gene transfer approaches in plants and animals has revealed that transgene can undergo silencing after integration in the genome. Host genes can also be silenced as a consequence of the presence of a homologous transgene. More and more investigations have demonstrated that doublestranded RNA can silence genes by triggering degradation of homologous RNA in the cytoplasm and by directing methylation of homologous nuclear DNA sequences. Analyses of Arabidopsis mutants and plant viral suppressors of silencing are unraveling RNA-silencing mechanisms and are assessing the role of methylation in transcriptional and posttranscriptional gene silencing. This review will focus on double-stranded RNA mediated mRNA degradation and gene inactivation in plants.

  15. Inhibition of RNA silencing by the coat protein of Pelargonium flower break virus: distinctions from closely related suppressors.

    Science.gov (United States)

    Martínez-Turiño, Sandra; Hernández, Carmen

    2009-02-01

    Viral-derived double-stranded RNAs (dsRNAs) activate RNA silencing, generating small interfering RNAs (siRNAs) which are incorporated into an RNA-induced silencing complex (RISC) that promotes homology-dependent degradation of cognate RNAs. To counteract this, plant viruses express RNA silencing suppressors. Here, we show that the coat protein (CP) of Pelargonium flower break virus (PFBV), a member of the genus Carmovirus, is able to efficiently inhibit RNA silencing. Interestingly, PFBV CP blocked both sense RNA- and dsRNA-triggered RNA silencing and did not preclude generation of siRNAs, which is in contrast with the abilities that have been reported for other carmoviral CPs. We have also found that PFBV CP can bind siRNAs and that this ability correlates with silencing suppression activity and enhancement of potato virus X pathogenicity. Collectively, the results indicate that PFBV CP inhibits RNA silencing by sequestering siRNAs and preventing their incorporation into a RISC, thus behaving similarly to unrelated viral suppressors but dissimilarly to orthologous ones.

  16. Disease-Causing Allele-Specific Silencing by RNA Interference

    Directory of Open Access Journals (Sweden)

    Hirohiko Hohjoh

    2013-04-01

    Full Text Available Small double-stranded RNAs (dsRNAs of approximately 21-nucleotides in size, referred to as small interfering RNA (siRNA duplexes, can induce sequence-specific posttranscriptional gene silencing, or RNA interference (RNAi. Since chemically synthesized siRNA duplexes were found to induce RNAi in mammalian cells, RNAi has become a powerful reverse genetic tool for suppressing the expression of a gene of interest in mammals, including human, and its application has been expanding to various fields. Recent studies further suggest that synthetic siRNA duplexes have the potential for specifically inhibiting the expression of an allele of interest without suppressing the expression of other alleles, i.e., siRNA duplexes likely confer allele-specific silencing. Such gene silencing by RNAi is an advanced technique with very promising applications. In this review, I would like to discuss the potential utility of allele-specific silencing by RNAi as a therapeutic method for dominantly inherited diseases, and describe possible improvements in siRNA duplexes for enhancing their efficacy.

  17. Viral RNA Silencing Suppression: The Enigma of Bunyavirus NSs Proteins

    Directory of Open Access Journals (Sweden)

    Marcio Hedil

    2016-07-01

    Full Text Available The Bunyaviridae is a family of arboviruses including both plant- and vertebrate-infecting representatives. The Tospovirus genus accommodates plant-infecting bunyaviruses, which not only replicate in their plant host, but also in their insect thrips vector during persistent propagative transmission. For this reason, they are generally assumed to encounter antiviral RNA silencing in plants and insects. Here we present an overview on how tospovirus nonstructural NSs protein counteracts antiviral RNA silencing in plants and what is known so far in insects. Like tospoviruses, members of the related vertebrate-infecting bunyaviruses classified in the genera Orthobunyavirus, Hantavirus and Phlebovirus also code for a NSs protein. However, for none of them RNA silencing suppressor activity has been unambiguously demonstrated in neither vertebrate host nor arthropod vector. The second part of this review will briefly describe the role of these NSs proteins in modulation of innate immune responses in mammals and elaborate on a hypothetical scenario to explain if and how NSs proteins from vertebrate-infecting bunyaviruses affect RNA silencing. If so, why this discovery has been hampered so far.

  18. Viral RNA Silencing Suppression: The Enigma of Bunyavirus NSs Proteins.

    Science.gov (United States)

    Hedil, Marcio; Kormelink, Richard

    2016-01-01

    The Bunyaviridae is a family of arboviruses including both plant- and vertebrate-infecting representatives. The Tospovirus genus accommodates plant-infecting bunyaviruses, which not only replicate in their plant host, but also in their insect thrips vector during persistent propagative transmission. For this reason, they are generally assumed to encounter antiviral RNA silencing in plants and insects. Here we present an overview on how tospovirus nonstructural NSs protein counteracts antiviral RNA silencing in plants and what is known so far in insects. Like tospoviruses, members of the related vertebrate-infecting bunyaviruses classified in the genera Orthobunyavirus, Hantavirus and Phlebovirus also code for a NSs protein. However, for none of them RNA silencing suppressor activity has been unambiguously demonstrated in neither vertebrate host nor arthropod vector. The second part of this review will briefly describe the role of these NSs proteins in modulation of innate immune responses in mammals and elaborate on a hypothetical scenario to explain if and how NSs proteins from vertebrate-infecting bunyaviruses affect RNA silencing. If so, why this discovery has been hampered so far. PMID:27455310

  19. CAPN5 gene silencing by short hairpin RNA interference

    OpenAIRE

    Nelson, Nnamdi G; Jessica M Skeie; Muradov, Hakim; Rowell, Hannah A; Seo, Seongjin; Mahajan, Vinit B

    2014-01-01

    Background The purpose of this project was to identify short hairpin RNA (shRNA) sequences that can suppress expression of human CAPN5 in which gain-of-function mutants cause autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV). We created HEK293T cells that stably express an ADNIV disease allele, CAPN5-p.R243L. Transfection protocols were optimized for neuroblastoma SHSY5Y cells. The gene silencing effect of four different shRNA plasmids that target CAPN5 was tested. RNA and...

  20. Tat RNA silencing suppressor activity contributes to perturbation of lymphocyte miRNA by HIV-1

    Directory of Open Access Journals (Sweden)

    Yu Lianbo

    2011-05-01

    Full Text Available Abstract Background MicroRNA (miRNA-mediated RNA silencing is integral to virtually every cellular process including cell cycle progression and response to virus infection. The interplay between RNA silencing and HIV-1 is multifaceted, and accumulating evidence posits a strike-counterstrike interface that alters the cellular environment to favor virus replication. For instance, miRNA-mediated RNA silencing of HIV-1 translation is antagonized by HIV-1 Tat RNA silencing suppressor activity. The activity of HIV-1 accessory proteins Vpr/Vif delays cell cycle progression, which is a process prominently modulated by miRNA. The expression profile of cellular miRNA is altered by HIV-1 infection in both cultured cells and clinical samples. The open question stands of what, if any, is the contribution of Tat RNA silencing suppressor activity or Vpr/Vif activity to the perturbation of cellular miRNA by HIV-1. Results Herein, we compared the perturbation of miRNA expression profiles of lymphocytes infected with HIV-1NL4-3 or derivative strains that are deficient in Tat RNA silencing suppressor activity (Tat K51A substitution or ablated of the vpr/vif open reading frames. Microarrays recapitulated the perturbation of the cellular miRNA profile by HIV-1 infection. The miRNA expression trends overlapped ~50% with published microarray results on clinical samples from HIV-1 infected patients. Moreover, the number of miRNA perturbed by HIV-1 was largely similar despite ablation of Tat RSS activity and Vpr/Vif; however, the Tat RSS mutation lessened HIV-1 downregulation of twenty-two miRNAs. Conclusions Our study identified miRNA expression changes attributable to Tat RSS activity in HIV-1NL4-3. The results accomplish a necessary step in the process to understand the interface of HIV-1 with host RNA silencing activity. The overlap in miRNA expression trends observed between HIV-1 infected CEMx174 lymphocytes and primary cells supports the utility of cultured

  1. Comparative analysis of RNA silencing suppression activities between viral suppressors and an endogenous plant RNA-dependent RNA polymerase.

    Science.gov (United States)

    Yoon, Ju-Yeon; Han, Kyoung-Sik; Park, Han-Yong; Choi, Seung-Kook

    2012-06-01

    RNA silencing is an evolutionarily conserved system that functions as an antiviral mechanism in eukaryotes, including higher plants. To counteract this, several plant viruses express silencing suppressors that inhibit RNA silencing in host plants. Here, we show that both 2b protein from peanut stunt virus (PSV) and a hairpin construct (designated hp-RDR6) that silences endogenous RNA-dependent RNA polymerase 6 (RDR6) strongly suppress RNA silencing. The Agrobacterium infiltration system was used to demonstrate that both PSV 2b and hp-RDR6 suppressed local RNA silencing as strongly as helper component (HC-Pro) from potato virus Y (PVY) and P19 from tomato bush stunt virus (TBSV). The 2b protein from PSV eliminated the small-interfering RNAs (siRNAs) associated with RNA silencing and prevented systemic silencing, similar to 2b protein from cucumber mosaic virus (CMV). On the other hand, hp-RDR6 suppressed RNA silencing by inhibiting the generation of secondary siRNAs. The small coat protein (SCP) of squash mosaic virus (SqMV) also displayed weak suppression activity of RNA silencing. Agrobacterium-mediated gene transfer was used to investigate whether viral silencing suppressors or hp-RDR6 enhanced accumulations of green fluorescence protein (GFP) and β-glucuronidase (GUS) as markers of expression in leaf tissues of Nicotina benthamiana. Expression of both GFP and GUS was significantly enhanced in the presence of PSV 2b or CMV 2b, compared to no suppression or the weak SqMV SCP suppressor. Co-expression with hp-RDR6 also significantly increased the expression of GFP and GUS to levels similar to those induced by PVY HC-Pro and TBSV P19.

  2. Silencing structural and nonstructural genes in baculovirus by RNA interference.

    Science.gov (United States)

    Flores-Jasso, C Fabian; Valdes, Victor Julian; Sampieri, Alicia; Valadez-Graham, Viviana; Recillas-Targa, Felix; Vaca, Luis

    2004-06-01

    We review several aspects of RNAi and gene silencing with baculovirus. We show that the potency of RNAi in Spodoptera frugiperda (Sf21) insect cells correlates well with the efficiency of transfection of the siRNA. Using a fluorescein-labeled siRNA we found that the siRNA localized in areas surrounding the endoplasmic reticulum (ER). Both long (700 nucleotides long) and small ( approximately 25 nucleotides long) interfering RNAs were equally effective in initiating RNA interference (RNAi), and the duration of the interfering effect was indistinguishable. Even though RNAi in Sf21 cells is very effective, in vitro experiments show that these cells fragment the long dsRNA into siRNA poorly, when compared to HEK cells. Finally, we show that in vivo inhibition of baculovirus infection with dsRNA homologous to genes that are essential for baculovirus infectivity depends strongly on the amount of dsRNA used in the assays. Five hundred nanogram of dsRNA directly injected into the haemolymph of insects prevent animal death to over 95%. In control experiments, over 96% of insects not injected with dsRNA or injected with an irrelevant dsRNA died within a week. These results demonstrate the efficiency of dsRNA for in vivo prevention of a viral infection by virus that is very cytotoxic and lytic in animals.

  3. ABCE1 is a highly conserved RNA silencing suppressor.

    Directory of Open Access Journals (Sweden)

    Kairi Kärblane

    Full Text Available ATP-binding cassette sub-family E member 1 (ABCE1 is a highly conserved protein among eukaryotes and archaea. Recent studies have identified ABCE1 as a ribosome-recycling factor important for translation termination in mammalian cells, yeast and also archaea. Here we report another conserved function of ABCE1. We have previously described AtRLI2, the homolog of ABCE1 in the plant Arabidopsis thaliana, as an endogenous suppressor of RNA silencing. In this study we show that this function is conserved: human ABCE1 is able to suppress RNA silencing in Nicotiana benthamiana plants, in mammalian HEK293 cells and in the worm Caenorhabditis elegans. Using co-immunoprecipitation and mass spectrometry, we found a number of potential ABCE1-interacting proteins that might support its function as an endogenous suppressor of RNA interference. The interactor candidates are associated with epigenetic regulation, transcription, RNA processing and mRNA surveillance. In addition, one of the identified proteins is translin, which together with its binding partner TRAX supports RNA interference.

  4. The Enamovirus P0 protein is a silencing suppressor which inhibits local and systemic RNA silencing through AGO1 degradation.

    Science.gov (United States)

    Fusaro, Adriana F; Correa, Regis L; Nakasugi, Kenlee; Jackson, Craig; Kawchuk, Lawrence; Vaslin, Maite F S; Waterhouse, Peter M

    2012-05-10

    The P0 protein of poleroviruses and P1 protein of sobemoviruses suppress the plant's RNA silencing machinery. Here we identified a silencing suppressor protein (SSP), P0(PE), in the Enamovirus Pea enation mosaic virus-1 (PEMV-1) and showed that it and the P0s of poleroviruses Potato leaf roll virus and Cereal yellow dwarf virus have strong local and systemic SSP activity, while the P1 of Sobemovirus Southern bean mosaic virus supresses systemic silencing. The nuclear localized P0(PE) has no discernable sequence conservation with known SSPs, but proved to be a strong suppressor of local silencing and a moderate suppressor of systemic silencing. Like the P0s from poleroviruses, P0(PE) destabilizes AGO1 and this action is mediated by an F-box-like domain. Therefore, despite the lack of any sequence similarity, the poleroviral and enamoviral SSPs have a conserved mode of action upon the RNA silencing machinery. PMID:22361475

  5. The Enamovirus P0 protein is a silencing suppressor which inhibits local and systemic RNA silencing through AGO1 degradation

    International Nuclear Information System (INIS)

    The P0 protein of poleroviruses and P1 protein of sobemoviruses suppress the plant's RNA silencing machinery. Here we identified a silencing suppressor protein (SSP), P0PE, in the Enamovirus Pea enation mosaic virus-1 (PEMV-1) and showed that it and the P0s of poleroviruses Potato leaf roll virus and Cereal yellow dwarf virus have strong local and systemic SSP activity, while the P1 of Sobemovirus Southern bean mosaic virus supresses systemic silencing. The nuclear localized P0PE has no discernable sequence conservation with known SSPs, but proved to be a strong suppressor of local silencing and a moderate suppressor of systemic silencing. Like the P0s from poleroviruses, P0PE destabilizes AGO1 and this action is mediated by an F-box-like domain. Therefore, despite the lack of any sequence similarity, the poleroviral and enamoviral SSPs have a conserved mode of action upon the RNA silencing machinery.

  6. An efficient Potato virus X -based microRNA silencing in Nicotiana benthamiana

    Science.gov (United States)

    Zhao, Jinping; Liu, Qingtao; Hu, Pu; Jia, Qi; Liu, Na; Yin, Kangquan; Cheng, Ye; Yan, Fei; Chen, Jianping; Liu, Yule

    2016-01-01

    Plant microRNAs (miRNAs) play pivotal roles in many biological processes. Although many miRNAs have been identified in various plant species, the functions of these miRNAs remain largely unknown due to the shortage of effective genetic tools to block their functional activity. Recently, miRNA target mimic (TM) technologies have been applied to perturb the activity of specific endogenous miRNA or miRNA families. We previously reported that Tobacco rattle virus (TRV)-based TM expression can successfully mediate virus-based miRNA silencing/suppression (VbMS) in plants. In this study, we show the Potato virus X (PVX)-based TM expression causes strong miRNA silencing in Nicotiana benthamiana. The PVX-based expression of short tandem target mimic (STTMs) against miR165/166 and 159 caused the corresponding phenotype in all infected plants. Thus, a PVX-based VbMS is a powerful method to study miRNA function and may be useful for high-throughput investigation of miRNA function in N. benthamiana. PMID:26837708

  7. Epigeneitc silencing of ribosomal RNA genes by Mybbp1a

    Directory of Open Access Journals (Sweden)

    Tan Bertrand

    2012-06-01

    Full Text Available Abstract Background Transcription of the ribosomal RNA gene repeats by Pol I occurs in the nucleolus and is a fundamental step in ribosome biogenesis and protein translation. Due to tight coordination between ribosome biogenesis and cell proliferation, transcription of rRNA and stable maintenance of rDNA clusters are thought to be under intricate control by intercalated mechanisms, particularly at the epigenetic level. Methods and Results Here we identify the nucleolar protein Myb-binding protein 1a (Mybbp1a as a novel negative regulator of rRNA expression. Suppression of rDNA transcription by Mybbp1a was linked to promoter regulation as illustrated by its binding to the chromatin around the hypermethylated, inactive rDNA gene promoters. Our data further showed that downregulation of Mybbp1a abrogated the local DNA methylation levels and histone marks associated with gene silencing, and altered the promoter occupancy of various factors such UBF and HDACs, consequently leading to elevated rRNA expression. Mechanistically, we propose that Mybbp1a maintains rDNA repeats in a silenced state while in association with the negative epigenetic modifiers HDAC1/2. Conclusions Results from our present work reveal a previously unrecognized co-repressor role of Mybbp1a in rRNA expression. They are further consistent with the scenario that Mybbp1a is an integral constituent of the rDNA epigenetic regulation that underlies the balanced state of rDNA clusters.

  8. AC2 and AC4 proteins of Tomato yellow leaf curl China virus and Tobacco curly shoot virus mediate suppression of RNA silencing

    Institute of Scientific and Technical Information of China (English)

    CUI Xiaofeng; ZHOU Xueping

    2004-01-01

    Tomato yellow leaf curl China virus Y10 isolate (TYLCCNV-Y10) alone could systemically infect host plants such as Nicotiana benthamiana without symptoms. In contrast, Tobacco curly shoot virus Y35 isolate (TbCSV-Y35) alone induces leaf curl symptoms in N. benthamiana. When inoculated into transgenic N. benthamiana plants expressing GFP gene (line 16c), TYLCCNV-Y10 neither reverses the established GFP silencing nor blocks the onset of GFP silencing. In contrast, TbCSV-Y35 can partially reverse the established GFP silencing and block the onset of GFP silencing in new leaves. In the patch co-infiltration assays, the AC2 and AC4 proteins of TYLCCNV-Y10 and TbCSV-Y35 could suppress local GFP silencing and delay systemic GFP silencing, suggesting that they are suppressors of RNA silencing. Comparison of the accumulation levels of GFP mRNA in the co-infiltration patches showed that Y10 AC2 and Y35 AC2 proteins had similar efficiency for suppression of RNA silencing. However, Y35 AC4 protein functioned as a stronger suppressor of RNA silencing than Y10 AC4 protein. Therefore, the pathogenicity difference between TbCSV-Y35 and TYLCCNV-Y10 may be related to the functional difference in their AC4 proteins.

  9. Alfalfa dwarf cytorhabdovirus P protein is a local and systemic RNA silencing supressor which inhibits programmed RISC activity and prevents transitive amplification of RNA silencing.

    Science.gov (United States)

    Bejerman, Nicolás; Mann, Krin S; Dietzgen, Ralf G

    2016-09-15

    Plants employ RNA silencing as an innate defense mechanism against viruses. As a counter-defense, plant viruses have evolved to express RNA silencing suppressor proteins (RSS), which target one or more steps of the silencing pathway. In this study, we show that the phosphoprotein (P) encoded by the negative-sense RNA virus alfalfa dwarf virus (ADV), a species of the genus Cytorhabdovirus, family Rhabdoviridae, is a suppressor of RNA silencing. ADV P has a relatively weak local RSS activity, and does not prevent siRNA accumulation. On the other hand, ADV P strongly suppresses systemic RNA silencing, but does not interfere with the short-distance spread of silencing, which is consistent with its lack of inhibition of siRNA accumulation. The mechanism of suppression appears to involve ADV P binding to RNA-induced silencing complex proteins AGO1 and AGO4 as shown in protein-protein interaction assays when ectopically expressed. In planta, we demonstrate that ADV P likely functions by inhibiting miRNA-guided AGO1 cleavage and prevents transitive amplification by repressing the production of secondary siRNAs. As recently described for lettuce necrotic yellows cytorhabdovirus P, but in contrast to other viral RSS known to disrupt AGO activity, ADV P sequence does not contain any recognizable GW/WG or F-box motifs, which suggests that cytorhabdovirus P proteins may use alternative motifs to bind to AGO proteins. PMID:27543392

  10. Characteristics of RNA silencing in plants: similarities and differences across kingdoms.

    Science.gov (United States)

    Susi, P; Hohkuri, M; Wahlroos, T; Kilby, N J

    2004-01-01

    RNA silencing is a collective term that encompasses the sequence of events that leads to the targeted degradation of cellular mRNA and thus to the silencing of corresponding gene expression. RNA silencing is initiated after introduction into the host genome of a gene that is homologous to an endogenous gene. Transcription of the introduced gene results in the formation of double-stranded RNA (dsRNA) that is cut into smaller dsRNA species termed small interfering RNAs (siRNAs) by an RNaseIII-like enzyme called 'Dicer'. siRNAs associate with a protein complex termed the 'RNA-induced silencing complex' (RISC), which mediates the binding of one strand of siRNAs with mRNAs transcribed from the native 'target' gene. The binding of siRNAs with native gene mRNAs earmarks native gene mRNAs for destruction, resulting in gene silencing. In plants, RNA silencing appears to serve as a defence mechanism against viral pathogens and also to suppress the activity of virus-like mobile genetic elements. In an apparent response to RNA silencing, some plant viruses express suppressors of RNA silencing. RNA silencing also is directly implicated in the regulation of the function(s) of microRNAs, which are the key determinants in an additional cellular mechanism related to the translational repression of genes, the effect of which ultimately impinges on development. The high degree of sequence similarity that exists between genes involved in RNA silencing in widely different organisms underscores the conserved nature of many aspects of the RNA silencing mechanism. However, depending (for example) on the precise nature of the target gene involved, there also are significant differences in the silencing pathways that are engaged by various organisms.

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

    Directory of Open Access Journals (Sweden)

    Adrian Valli

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

  12. Functional gene silencing mediated by chitosan/siRNA nanocomplexes

    Energy Technology Data Exchange (ETDEWEB)

    Ji, A M; Su, D; Che, O; Li, W S; Sun, L; Zhang, Z Y; Xu, F [Department of Pharmaceutical Science, Zhujiang Hospital, Southern Medical University, Guangzhou 510282 (China); Yang, B, E-mail: andrewfxu1998@gmail.co [Department of Chemistry, Indiana University-Bloomington, Bloomington, IN 47405 (United States)

    2009-10-07

    Chitosan/siRNA nanoparticles to knock down FHL2 gene expression were reported in this work. The physicochemical properties such as particle size, surface charge, morphology and complex stability of chitosan nanoparticle-incorporated siRNA were evaluated. Nanoparticles which were formulated with chitosan/siRNA exhibited irregular, lamellar and dendritic structures with a hydrodynamic radius size of about 148 nm and net positive charges with zeta-potential value of 58.5 mV. The knockdown effect of the chitosan/siRNA nanoparticles on gene expression in FHL2 over-expressed human colorectal cancer Lovo cells was investigated. The result showed that FHL2 siRNA formulated within chitosan nanoparticles could knock down about 69.6% FHL2 gene expression, which is very similar to the 68.8% reduced gene expression when siRNA was transfected with liposome Lipofectamine. Western analysis further showed significant FHL-2 protein expression reduced by the chitosan/siRNA nanoparticles. The results also showed that blocking FHL2 expression by siRNA could also inhibit the growth and proliferation of human colorectal cancer Lovo cells. The current results demonstrated that chitosan-based siRNA nanoparticles were a very efficient delivery system for siRNA in vivo as previously reported.

  13. Functional gene silencing mediated by chitosan/siRNA nanocomplexes

    International Nuclear Information System (INIS)

    Chitosan/siRNA nanoparticles to knock down FHL2 gene expression were reported in this work. The physicochemical properties such as particle size, surface charge, morphology and complex stability of chitosan nanoparticle-incorporated siRNA were evaluated. Nanoparticles which were formulated with chitosan/siRNA exhibited irregular, lamellar and dendritic structures with a hydrodynamic radius size of about 148 nm and net positive charges with zeta-potential value of 58.5 mV. The knockdown effect of the chitosan/siRNA nanoparticles on gene expression in FHL2 over-expressed human colorectal cancer Lovo cells was investigated. The result showed that FHL2 siRNA formulated within chitosan nanoparticles could knock down about 69.6% FHL2 gene expression, which is very similar to the 68.8% reduced gene expression when siRNA was transfected with liposome Lipofectamine. Western analysis further showed significant FHL-2 protein expression reduced by the chitosan/siRNA nanoparticles. The results also showed that blocking FHL2 expression by siRNA could also inhibit the growth and proliferation of human colorectal cancer Lovo cells. The current results demonstrated that chitosan-based siRNA nanoparticles were a very efficient delivery system for siRNA in vivo as previously reported.

  14. Evaluation of shRNA-mediated gene silencing by electroporation in LPB fibrosarcoma cells:

    OpenAIRE

    Čemažar, Maja; Kamenšek, Urška; Vidic, Suzana

    2008-01-01

    Background. Silencing oncogenes or other genes that contribute to tumor malignancy and progression offers a promising approach to treating cancer. Specific and efficient silencing of gene expression can be achieved by RNA interference (RNAi) technology using small interfering RNA (siRNA) or short hairpin RNA (shRNA). However, a major challenge in RNAi technology is effective delivery of interfering molecules into target cells. The aim of our study was to evaluate electroporation as a perspect...

  15. The Enamovirus P0 protein is a silencing suppressor which inhibits local and systemic RNA silencing through AGO1 degradation

    Energy Technology Data Exchange (ETDEWEB)

    Fusaro, Adriana F. [University of Sydney, NSW 2006 (Australia); CSIRO Plant Industry, Canberra, P.O. Box 1600, ACT 2601 (Australia); Correa, Regis L. [CSIRO Plant Industry, Canberra, P.O. Box 1600, ACT 2601 (Australia); Depto. de Virologia, IMPPG, UFRJ, 21941-902 (Brazil); Nakasugi, Kenlee; Jackson, Craig [University of Sydney, NSW 2006 (Australia); Kawchuk, Lawrence [Research Centre, Agriculture and Agri-Food Canada, Lethbridge, AB T1J4B1 (Canada); Vaslin, Maite F.S. [Depto. de Virologia, IMPPG, UFRJ, 21941-902 (Brazil); Waterhouse, Peter M., E-mail: peter.waterhouse@sydney.edu.au [University of Sydney, NSW 2006 (Australia); CSIRO Plant Industry, Canberra, P.O. Box 1600, ACT 2601 (Australia)

    2012-05-10

    The P0 protein of poleroviruses and P1 protein of sobemoviruses suppress the plant's RNA silencing machinery. Here we identified a silencing suppressor protein (SSP), P0{sup PE}, in the Enamovirus Pea enation mosaic virus-1 (PEMV-1) and showed that it and the P0s of poleroviruses Potato leaf roll virus and Cereal yellow dwarf virus have strong local and systemic SSP activity, while the P1 of Sobemovirus Southern bean mosaic virus supresses systemic silencing. The nuclear localized P0{sup PE} has no discernable sequence conservation with known SSPs, but proved to be a strong suppressor of local silencing and a moderate suppressor of systemic silencing. Like the P0s from poleroviruses, P0{sup PE} destabilizes AGO1 and this action is mediated by an F-box-like domain. Therefore, despite the lack of any sequence similarity, the poleroviral and enamoviral SSPs have a conserved mode of action upon the RNA silencing machinery.

  16. Post-transcriptional silencing of flavonol synthase mRNA in tobacco leads to fruits with arrested seed set.

    Directory of Open Access Journals (Sweden)

    Monika Mahajan

    Full Text Available Flavonoids are synthesized by phenylpropanoid pathway. They are known to participate in large number of physiological and biochemical processes in plants. Parthenocarpy and male sterility has earlier been reported by silencing chalcone synthase (CHS encoding gene. Silencing of CHS has blocked the synthesis of most of useful flavonoids including flavan-3-ols and flavonols. Also, these studies could not identify whether parthenocarpy/male sterility were due to lack of flavan-3-ols or flavonols or both. Flavonol synthase (FLS is an important enzyme of flavonoid pathway that catalyzes the formation of flavonols. In this article, we propose a novel strategy towards the generation of seedless or less-seeded fruits by downregulation of flavonol biosynthesis in tobacco (Nicotiana tabacum cv Xanthi through post-transcriptional gene silencing (PTGS of FLS encoding mRNA. The FLS silenced lines were observed for 20-80% reduction in FLS encoding gene expression and 25-93% reduction in flavonol (quercetin content. Interestingly, these FLS silenced tobacco lines also showed reduction in their anthocyanidins content. While the content of flavan-3-ols (catechin, epi-catechin and epi-gallocatechin was found to be increased in FLS silenced lines. The delayed flowering in FLS silenced lines could be due to decrease in level of indole acetic acid (IAA at apical region of their shoots. Furthermore, the pollen germination was hampered and pollens were unable to produce functional pollen tube in FLS silenced tobacco lines. Pods of FLS silenced lines contained significantly less number of seeds. The in vitro and in vivo studies where 1 µM quercetin was supplied to germination media, documented the restoration of normal pollen germination and pollen tube growth. This finding identified the role of flavonols particularly quercetin in pollen germination as well as in the regulation of plant fertility. Results also suggest a novel approach towards generation of seedless

  17. Post-transcriptional silencing of flavonol synthase mRNA in tobacco leads to fruits with arrested seed set.

    Science.gov (United States)

    Mahajan, Monika; Ahuja, Paramvir Singh; Yadav, Sudesh Kumar

    2011-01-01

    Flavonoids are synthesized by phenylpropanoid pathway. They are known to participate in large number of physiological and biochemical processes in plants. Parthenocarpy and male sterility has earlier been reported by silencing chalcone synthase (CHS) encoding gene. Silencing of CHS has blocked the synthesis of most of useful flavonoids including flavan-3-ols and flavonols. Also, these studies could not identify whether parthenocarpy/male sterility were due to lack of flavan-3-ols or flavonols or both. Flavonol synthase (FLS) is an important enzyme of flavonoid pathway that catalyzes the formation of flavonols. In this article, we propose a novel strategy towards the generation of seedless or less-seeded fruits by downregulation of flavonol biosynthesis in tobacco (Nicotiana tabacum cv Xanthi) through post-transcriptional gene silencing (PTGS) of FLS encoding mRNA. The FLS silenced lines were observed for 20-80% reduction in FLS encoding gene expression and 25-93% reduction in flavonol (quercetin) content. Interestingly, these FLS silenced tobacco lines also showed reduction in their anthocyanidins content. While the content of flavan-3-ols (catechin, epi-catechin and epi-gallocatechin) was found to be increased in FLS silenced lines. The delayed flowering in FLS silenced lines could be due to decrease in level of indole acetic acid (IAA) at apical region of their shoots. Furthermore, the pollen germination was hampered and pollens were unable to produce functional pollen tube in FLS silenced tobacco lines. Pods of FLS silenced lines contained significantly less number of seeds. The in vitro and in vivo studies where 1 µM quercetin was supplied to germination media, documented the restoration of normal pollen germination and pollen tube growth. This finding identified the role of flavonols particularly quercetin in pollen germination as well as in the regulation of plant fertility. Results also suggest a novel approach towards generation of seedless

  18. Silencing of Inducible Immunoproteasome Subunit Expression by Chemically Modified siRNA and shRNA.

    Science.gov (United States)

    Gvozdeva, Olga V; Prassolov, Vladimir S; Zenkova, Marina A; Vlassov, Valentin V; Chernolovskaya, Elena L

    2016-08-01

    Overexpression of inducible subunits of immunoproteasome is related to pathogenesis of some chronic diseases. Specific inhibition of the immunosubunits may be used for the treatment of these diseases and RNA interference is one of the potent methods used in this area. We designed 2'-O-methyl modified siRNAs with selectively protected nuclease-sensitive sites, which efficiently silence LMP2, LMP7, and MECL-1 genes expression. To provide stable long-lasting inhibition of target genes, short-hairpin RNAs (shRNA) expressed by lentiviral vectors were constructed. Our results demonstrated that chemically modified siRNAs inhibited the expression of target genes with similar efficiency or with efficiency exceeding that of corresponding shRNAs and provide silencing effect for 5 days.

  19. A viral suppressor protein inhibits host RNA silencing by hooking up with Argonautes

    OpenAIRE

    Jin, Hailing; Zhu, Jian-Kang

    2010-01-01

    RNA viruses are particularly vulnerable to RNAi-based defenses in the host, and thus have evolved specific proteins, known as viral suppressors of RNA silencing (VSRs), as a counterdefense. In this issue of Genes & Development, Azevedo and colleagues (pp. 904–915) discovered that P38, the VSR of Turnip crinkle virus, uses its glycine/tryptophane (GW) motifs as an ARGONAUTE (AGO) hook to attract and disarm the host's essential effector of RNA silencing. Several GW motif-containing cellular pro...

  20. On the Grammar of Silence: The Structure of My Undocumented Immigrant Writer's Block

    Science.gov (United States)

    Ledesma, Alberto

    2015-01-01

    In this reflective essay, Alberto Ledesma explores how being undocumented can produce a particular form of writer's block. He argues that there is a pattern of predictable silences and obfuscations inherent in all undocumented immigrant autobiographies that cannot be easily negotiated when undocumented students are asked to write about "their…

  1. Plant responses against invasive nucleic acids: RNA silencing and its suppression by plant viral pathogens

    OpenAIRE

    Alvarado, Veria; Herman B Scholthof

    2009-01-01

    RNA silencing is a common strategy shared by eukaryotic organisms to regulate gene expression, and also operates as a defense mechanism against invasive nucleic acids such as viral transcripts. The silencing pathway is quite sophisticated in higher eukaryotes but the distinct steps and nature of effector complexes vary between and even within species. To counteract this defense mechanism viruses have evolved the ability to encode proteins that suppress silencing to protect their genomes from ...

  2. A viral suppressor protein inhibits host RNA silencing by hooking up with Argonautes

    KAUST Repository

    Jin, Hailing

    2010-05-01

    RNA viruses are particularly vulnerable to RNAi-based defenses in the host, and thus have evolved specific proteins, known as viral suppressors of RNA silencing (VSRs), as a counterdefense. In this issue of Genes & Development, Azevedo and colleagues (pp. 904-915) discovered that P38, the VSR of Turnip crinkle virus, uses its glycine/tryptophane (GW) motifs as an ARGONAUTE (AGO) hook to attract and disarm the host\\'s essential effector of RNA silencing. Several GW motif-containing cellular proteins are known to be important partners of AGOs in RNA silencing effector complexes in yeast, plants, and animals. The GW motif appears to be a versatile and effective tool for regulating the activities of RNA silencing pathways, and the use of GW mimicry to compete for and inhibit host AGOs may be a strategy used by many pathogens to counteract host RNAi-based defenses. © 2010 by Cold Spring Harbor Laboratory Press.

  3. Tobacco mosaic virus movement protein enhances the spread of RNA silencing.

    Directory of Open Access Journals (Sweden)

    Hannes Vogler

    2008-04-01

    Full Text Available Eukaryotic cells restrain the activity of foreign genetic elements, including viruses, through RNA silencing. Although viruses encode suppressors of silencing to support their propagation, viruses may also exploit silencing to regulate host gene expression or to control the level of their accumulation and thus to reduce damage to the host. RNA silencing in plants propagates from cell to cell and systemically via a sequence-specific signal. Since the signal spreads between cells through plasmodesmata like the viruses themselves, virus-encoded plasmodesmata-manipulating movement proteins (MP may have a central role in compatible virus:host interactions by suppressing or enhancing the spread of the signal. Here, we have addressed the propagation of GFP silencing in the presence and absence of MP and MP mutants. We show that the protein enhances the spread of silencing. Small RNA analysis indicates that MP does not enhance the silencing pathway but rather enhances the transport of the signal through plasmodesmata. The ability to enhance the spread of silencing is maintained by certain MP mutants that can move between cells but which have defects in subcellular localization and do not support the spread of viral RNA. Using MP expressing and non-expressing virus mutants with a disabled silencing suppressing function, we provide evidence indicating that viral MP contributes to anti-viral silencing during infection. Our results suggest a role of MP in controlling virus propagation in the infected host by supporting the spread of silencing signal. This activity of MP involves only a subset of its properties implicated in the spread of viral RNA.

  4. ShRNA-mediated gene silencing of β-catenin inhibits growth of human colon cancer cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    AIM: To observe the gene silencing mediated by the specific shRNA targeted against β-catenin and its effect on cell proliferation and cycle distribution in the human colon cancer cell line Colo205.METHODS: Two shRNA plasmid vectors against β-catenin were constructed and transfected into Colo205 cells with LipofectamineTM2000. The down-regulations of β-catenin, c-myc and cyclinD1 expressions were detected by RT-PCR and western blot analysis. The cell proliferation inhibitions were determined by MTT assay and soft agar colony formation assay. The effect of these two β-catenin shRNAs on cell cycle distribution and apoptosis was examined by flow cytometry.RESULTS: These two shRNA vectors targeted against β-catenin efficiently suppressed the expression of β-catenin and its down stream genes, c-myc and cyclinD1. The expression inhibition rates were around 40%-50% either at the mRNA or at the protein level.The shRNA-mediated gene silencing of β-catenin resulted in significant inhibition of cell growth both on the culture plates and in the soft agar. Moreover, the cancer cells showed significant G0/G1 arrest and increased apoptosis at 72 h post transfection due to gene silencing.CONCLUSION: These specific shRNAs targeted against β-catenin could have a gene silencing effect and block the WNT signaling pathway. They could inhibit cell growth, increase apoptosis, and induce cell cycle arrest in Colo205 cells. ShRNA interference against β-catenin is of potential value in gene therapy of colon cancer.

  5. Silencing Huntington's chorea: Is RNA Interference a Potential Cure?

    Directory of Open Access Journals (Sweden)

    Gerlinde A. Metz

    2006-01-01

    Full Text Available In 1872, George Huntington described Huntington's disease as characterized by motor, cognitive and psychiatric impairments. Huntington's disease is a dominant and autosomal mutation on chromosome 4 featuring the insertion of numerous CAG repeats. CAG codes for the amino acid, glutmanine that forms part of the Huntingtin protein (htt. Excess glutamine attachments make htt prone to accumulate in neurons. Three genes can be considered when developing therapies for Huntington's disease. They include targeting the symptoms of the disease, the progression of the disease and the cause of the disease. By using RNA interference (RNAi, the cause of the disease can be targeted. RNAi is a method that could potentially silence the formation of abnormal htt. This paper will discuss how RNAi could potentially cure Huntington's disease, by describing the genetic and proteinomic basis of Huntington's disease, the function of RNAi in Huntington's disease and the problems of benefits of RNAi. Preliminary work using RNAi in transgenic mice has shown a decrease in the behavioural expression of the mutant Huntington gene. There are several limitations associated with using RNAi as a gene therapy. For example, the effects of RNAi are short lived. A transposition system such as Sleeping Beauty can be used to increase the integration of the gene, however, for patients who currently have Huntington's disease, RNAi may potentially be used in combination with drugs or other treatments to target both symptoms and the underlying cause of Huntington's disease. This combination could eventually alleviate many painful symptoms associated with Huntington's disease and could even stop the progressive neurodegeneration of Huntington's disease. This review concludes that a substantial amount of new research is still necessary before RNAi is directly applicable to human patients with Huntington's disease.

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

    Science.gov (United States)

    Pérez-Cañamás, Miryam; Hernández, Carmen

    2015-01-30

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

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

    NARCIS (Netherlands)

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

    2004-01-01

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

  8. Satellite RNAs interfere with the function of viral RNA silencing suppressors

    OpenAIRE

    Wanxia eShen; Phil Chi Khang Au; Bu-Jun eShi; Smith, Neil A.; Dennis, Elizabeth S.; Hui-Shan eGuo; Chang-Yong eZhou; Ming-Bo eWang

    2015-01-01

    Viral satellite RNAs (satRNAs) are small subviral RNAs and depend on the helper virus for replication and spread. satRNAs can attenuate helper virus-induced symptoms, the mechanism of which remains unclear. Here, we show that two virus-encoded suppressors of RNA silencing (VSRs), Cucumber mosaic virus (CMV) 2b and Tombusvirus P19, suppress hairpin RNA (hpRNA)-induced silencing of a β-glucuronidase (GUS) gene in Nicotiana benthamiana. This suppression can be overcome by CMV Y-satellite RNA (Y-...

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

    Science.gov (United States)

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

    2013-03-01

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

  10. Technical advances in trigger-induced RNA interference gene silencing in the parasite Entamoeba histolytica.

    Science.gov (United States)

    Khalil, Mohamed I; Foda, Bardees M; Suresh, Susmitha; Singh, Upinder

    2016-03-01

    Entamoeba histolytica has a robust endogenous RNA interference (RNAi) pathway. There are abundant 27 nucleotide (nt) anti-sense small RNAs (AS sRNAs) that target genes for silencing and the genome encodes many genes involved in the RNAi pathway such as Argonaute proteins. Importantly, an E. histolytica gene with numerous AS sRNAs can function as a "trigger" to induce silencing of a gene that is fused to the trigger. Thus, the amebic RNAi pathway regulates gene expression relevant to amebic biology and has additionally been harnessed as a tool for genetic manipulation. In this study we have further improved the trigger-induced gene silencing method. We demonstrate that rather than using the full-length gene, a short portion of the coding region fused to a trigger is sufficient to induce silencing; the first 537 bp of the E. histolytica rhomboid gene (EhROM1) fused in-frame to the trigger was sufficient to silence EhROM1. We also demonstrated that the trigger method could silence two amebic genes concomitantly; fusion of the coding regions of EhROM1 and transcription factor, EhMyb, in-frame to a trigger gene resulted in both genes being silenced. Alternatively, two genes can be silenced sequentially: EhROM1-silenced parasites with no drug selection plasmid were transfected with trigger-EhMyb, resulting in parasites with both EhROM1 and EhMyb silenced. With all approaches tested, the trigger-mediated silencing was substantive and silencing was maintained despite loss of the G418 selectable marker. All gene silencing was associated with generation of AS sRNAs to the silenced gene. We tested the reversibility of the trigger system using inhibitors of histone modifications but found that the silencing was highly stable. This work represents a technical advance in the trigger gene silencing method in E. histolytica. Approaches that readily silence multiple genes add significantly to the genetic toolkit available to the ameba research community. PMID:26747561

  11. Satellite RNAs interfere with the function of viral RNA silencing suppressors

    Directory of Open Access Journals (Sweden)

    Wanxia eShen

    2015-04-01

    Full Text Available Viral satellite RNAs (satRNAs are small subviral RNAs and depend on the helper virus for replication and spread. satRNAs can attenuate helper virus-induced symptoms, the mechanism of which remains unclear. Here, we show that two virus-encoded suppressors of RNA silencing (VSRs, Cucumber mosaic virus (CMV 2b and Tombusvirus P19, suppress hairpin RNA (hpRNA-induced silencing of a β-glucuronidase (GUS gene in Nicotiana benthamiana. This suppression can be overcome by CMV Y-satellite RNA (Y-Sat via the Y-Sat-derived small interfering RNAs (siRNAs, which bind to the VSRs and displace the bound hpGUS-derived siRNAs. We also show that microRNA target gene expression in N. tabacum was elevated by CMV infection, presumably due to function of the 2b VSR, but this upregulation of microRNA target genes was reversed in the presence of Y-Sat. These results suggest that satRNA infection minimizes the effect of VSRs on host siRNA and microRNA-directed silencing. Our results suggest that the high abundance of satRNA-derived siRNAs contributes to symptom attenuation by binding helper virus-encoded VSRs, minimizing the capacity of the VSRs to bind host siRNA and miRNA and interfere with their function.

  12. Satellite RNAs interfere with the function of viral RNA silencing suppressors.

    Science.gov (United States)

    Shen, Wan-Xia; Au, Phil Chi Khang; Shi, Bu-Jun; Smith, Neil A; Dennis, Elizabeth S; Guo, Hui-Shan; Zhou, Chang-Yong; Wang, Ming-Bo

    2015-01-01

    Viral satellite RNAs (satRNAs) are small subviral RNAs and depend on the helper virus for replication and spread. satRNAs can attenuate helper virus-induced symptoms, the mechanism of which remains unclear. Here, we show that two virus-encoded suppressors of RNA silencing (VSRs), Cucumber mosaic virus (CMV) 2b and Tombusvirus P19, suppress hairpin RNA (hpRNA)-induced silencing of a β-glucuronidase (GUS) gene in Nicotiana benthamiana. This suppression can be overcome by CMV Y-satellite RNA (Y-Sat) via the Y-Sat-derived small interfering RNAs (siRNAs), which bind to the VSRs and displace the bound hpGUS-derived siRNAs. We also show that microRNA target gene expression in N. tabacum was elevated by CMV infection, presumably due to function of the 2b VSR, but this upregulation of microRNA target genes was reversed in the presence of Y-Sat. These results suggest that satRNA infection minimizes the effect of VSRs on host siRNA and microRNA-directed silencing. Our results suggest that the high abundance of satRNA-derived siRNAs contributes to symptom attenuation by binding helper virus-encoded VSRs, minimizing the capacity of the VSRs to bind host siRNA and miRNA and interfere with their function. PMID:25964791

  13. Suppression of RNA silencing by a plant DNA virus satellite requires a host calmodulin-like protein to repress RDR6 expression.

    Directory of Open Access Journals (Sweden)

    Fangfang Li

    2014-02-01

    Full Text Available In plants, RNA silencing plays a key role in antiviral defense. To counteract host defense, plant viruses encode viral suppressors of RNA silencing (VSRs that target different effector molecules in the RNA silencing pathway. Evidence has shown that plants also encode endogenous suppressors of RNA silencing (ESRs that function in proper regulation of RNA silencing. The possibility that these cellular proteins can be subverted by viruses to thwart host defense is intriguing but has not been fully explored. Here we report that the Nicotiana benthamiana calmodulin-like protein Nbrgs-CaM is required for the functions of the VSR βC1, the sole protein encoded by the DNA satellite associated with the geminivirus Tomato yellow leaf curl China virus (TYLCCNV. Nbrgs-CaM expression is up-regulated by the βC1. Transgenic plants over-expressing Nbrgs-CaM displayed developmental abnormities reminiscent of βC1-associated morphological alterations. Nbrgs-CaM suppressed RNA silencing in an Agrobacterium infiltration assay and, when over-expressed, blocked TYLCCNV-induced gene silencing. Genetic evidence showed that Nbrgs-CaM mediated the βC1 functions in silencing suppression and symptom modulation, and was required for efficient virus infection. Moreover, the tobacco and tomato orthologs of Nbrgs-CaM also possessed ESR activity, and were induced by betasatellite to promote virus infection in these Solanaceae hosts. We further demonstrated that βC1-induced Nbrgs-CaM suppressed the production of secondary siRNAs, likely through repressing RNA-DEPENDENT RNA POLYMERASE 6 (RDR6 expression. RDR6-deficient N. benthamiana plants were defective in antiviral response and were hypersensitive to TYLCCNV infection. More significantly, TYLCCNV could overcome host range restrictions to infect Arabidopsis thaliana when the plants carried a RDR6 mutation. These findings demonstrate a distinct mechanism of VSR for suppressing PTGS through usurpation of a host ESR, and

  14. Generation of tobacco lines with widely different reduction in nicotine levels via RNA silencing approaches

    Indian Academy of Sciences (India)

    Peng Wang; Zhifeng Liang; Jia Zeng Wenchao; Wenchao Li; Xiaofen Sun; Zhiqi Miao; Kexuan Tang

    2008-06-01

    Issues related to the nicotine content of tobacco have been public concerns. Several reports have described decreasing nicotine levels by silencing the putrescine N-methyltransferase (PMT) genes, but the reported variations of nicotine levels among transgenic lines are relatively low in general. Here we describe the generation in tobacco (Nicotiana tabacum) lines with widely different, reduced nicotine levels using three kinds of RNA-silencing approaches. The relative efficacies of suppression were compared among the three approaches regarding the aspect of nicotine level in tobacco leaves. By suppressing expression of the PMT genes, over 200 transgenic lines were obtained with nicotine levels reduced by 9.1–96.7%. RNA interference (RNAi) was the most efficient method of reducing the levels of nicotine, whereas cosuppression and antisense methods were less effective. This report gives clues to the efficient generation of plants with a variety of metabolite levels, and the results demonstrate the relative efficiencies of various RNA-silencing methods.

  15. Induction of Silencing Effect of Swedish Mutant Amyloid Precursor Protein by RNA interference

    Institute of Scientific and Technical Information of China (English)

    QIU Xin; CHEN Guohua; PAN Ji'an; CHEN Yu; ZHANG Suming

    2006-01-01

    Over-expression of APP and Swedish mutation could cause some familial early onset AD.In this study, a primary screening was conducted of effective small interference RNAs (siRNAs) targeted wild type APP (APPwt) and Swedish mutant APP (APPswe). One siRNA targeting APPwt and the other siRNA targeting APPswe were designed. All these siRNAs were endogenously expressed by siRNAs expressing plasmids. COS-7 cells were transiently co-transfected with APP-GFP recombinant plasmids and siRNA expression vector. The silencing effect of each siRNA was quantitatively assessed by the level of expression of green fluorescent protein (GFP). It was found that the siRNAs silenced APPwt and APPswe to different degrees. siRNA directed against APPswe was more effective in suppressing the expression of fusion gene of APPswe than that of APPwt. The silencing effect of siRNA directed against APPswe indicating allele-specific silencing property of the siRNAs. Therefore, siRNAs directed against APP play an important role both in the therapeutic study of Alzheimer disease and functional exploration of APP gene.

  16. Short-hairpin RNA-mediated Heat shock protein 90 gene silencing inhibits human breast cancer cell growth in vitro and in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Zuo, Keqiang [Department of General Surgery, Shanghai 10th People' s Hospital, Tongji University School of Medicine, Shanghai 200072 (China); Li, Dan [Department of Nuclear Medicine, Shanghai 10th People' s Hospital, Tongji University School of Medicine, Shanghai 200072 (China); Pulli, Benjamin [Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, 185 Cambridge Street, Boston, MA 02114 (United States); Yu, Fei; Cai, Haidong; Yuan, Xueyu [Department of Nuclear Medicine, Shanghai 10th People' s Hospital, Tongji University School of Medicine, Shanghai 200072 (China); Zhang, Xiaoping, E-mail: zxpsibs@163.com [Department of Nuclear Medicine, Shanghai 10th People' s Hospital, Tongji University School of Medicine, Shanghai 200072 (China); Lv, Zhongwei, E-mail: heyixue163@163.com [Department of Nuclear Medicine, Shanghai 10th People' s Hospital, Tongji University School of Medicine, Shanghai 200072 (China)

    2012-05-04

    Highlights: Black-Right-Pointing-Pointer Hsp90 is over-expressed in human breast cancer. Black-Right-Pointing-Pointer The shRNA-mediated gene silencing of Hsp90 resulted in inhibition of cell growth. Black-Right-Pointing-Pointer Akt and NF-kB were down-regulation after transfection due to Hsp90 silencing. Black-Right-Pointing-Pointer The tumor growth ratio was decline due to Hsp90 silencing. Black-Right-Pointing-Pointer The PCNA expression was down-regulation due to Hsp90 silencing. -- Abstract: Hsp90 interacts with proteins that mediate signaling pathways involved in the regulation of essential processes such as proliferation, cell cycle control, angiogenesis and apoptosis. Hsp90 inhibition is therefore an attractive strategy for blocking abnormal pathways that are crucial for cancer cell growth. In the present study, the role of Hsp90 in human breast cancer MCF-7 cells was examined by stably silencing Hsp90 gene expression with an Hsp90-silencing vector (Hsp90-shRNA). RT-PCR and Western blot analyses showed that Hsp90-shRNA specifically and markedly down-regulated Hsp90 mRNA and protein expression. NF-kB and Akt protein levels were down-regulated in Hsp90-shRNA transfected cells, indicating that Hsp90 knockout caused a reduction of survival factors and induced apoptosis. Treatment with Hsp90-shRNA significantly increased apoptotic cell death and caused cell cycle arrest in the G1/S phase in MCF-7 cells, as shown by flow cytometry. Silencing of Hsp90 also reduced cell viability, as determined by MTT assay. In vivo experiments showed that MCF-7 cells stably transfected with Hsp90-shRNA grew slowly in nude mice as compared with control groups. In summary, the Hsp90-shRNA specifically silenced the Hsp90 gene, and inhibited MCF-7 cell growth in vitro and in vivo. Possible molecular mechanisms underlying the effects of Hsp90-shRNA include the degradation of Hsp90 breast cancer-related client proteins, the inhibition of survival signals and the upregulation of apoptotic

  17. Structural implications into dsRNA binding and RNA silencing suppression by NS3 protein of Rice Hoja Blanca Tenuivirus.

    Science.gov (United States)

    Yang, Xia; Tan, Sook Hwa; Teh, Yee Jin; Yuan, Y Adam

    2011-05-01

    Rice Hoja Blanca Tenuivirus (RHBV), a negative strand RNA virus, has been identified to infect rice and is widely transmitted by the insect vector. NS3 protein encoded by RHBV RNA3 was reported to be a potent RNAi suppressor to counterdefense RNA silencing in plants, insect cells, and mammalian cells. Here, we report the crystal structure of the N-terminal domain of RHBV NS3 (residues 21-114) at 2.0 Å. RHBV NS3 N-terminal domain forms a dimer by two pairs of α-helices in an anti-parallel mode, with one surface harboring a shallow groove at the dimension of 20 Å × 30 Å for putative dsRNA binding. In vitro RNA binding assay and RNA silencing suppression assay have demonstrated that the structural conserved residues located along this shallow groove, such as Arg50, His51, Lys77, and His85, participate in dsRNA binding and RNA silencing suppression. Our results provide the initial structural implications in understanding the RNAi suppression mechanism by RHBV NS3. PMID:21460234

  18. Structure Tuning of Cationic Oligospermine-siRNA Conjugates for Carrier-Free Gene Silencing.

    Science.gov (United States)

    Nothisen, Marc; Bagilet, Jérémy; Behr, Jean-Paul; Remy, Jean-Serge; Kotera, Mitsuharu

    2016-08-01

    Oligospermine-siRNA conjugates are able to induce efficient luciferase gene silencing upon carrier-free transfection. These conjugates are readily accessible by a versatile automated chemistry that we developed using a DMT-spermine phosphoramidite reagent. In this article, we used this chemistry to study a wide range of structural modifications of the oligospermine-siRNA conjugates, i.e., variation of conjugate positions and introduction of chemical modifications to increase nuclease resistance. At first we examined gene silencing activity of a series of siRNA-tris(spermine) conjugates with and without chemical modifications in standard carrier assisted conditions. The three spermine units attached at one of the two ends of the sense strand or at the 3'-end of the antisense strand are compatible with gene silencing activity whereas attachment of spermine units at the 5'-end of the antisense strand abolished the activity. 2'-O-Methylated nucleotides introduced in the sense strand are compatible while not in the antisense strand. Thiophosphate links could be used without activity loss at the 3'-end of both strands and at the 5'-end of the sense strand to conjugate oligospermine. Consequently a series of oligospermine-siRNA conjugates containing 15 to 45 spermines units in various configurations were chosen, prepared, and examined in carrier-free conditions. Attachment of 30 spermine units singly at the 5'-end of the sense strand provides the most potent carrier-free siRNA. Longevity of luciferase gene silencing was studied using oligospermine-siRNA conjugates. Five day long efficiency with more than 80% gene expression knockdown was observed upon transfection without vector. Oligospermine-siRNA conjugates targeting cell-constitutive natural lamin A/C gene were prepared. Efficient gene silencing was observed upon carrier-free transfection of siRNA conjugates containing 20 or 30 spermine residues grafted at the 5'-end of the sense strand. PMID:27398779

  19. RNA-Interference Components Are Dispensable for Transcriptional Silencing of the Drosophila Bithorax-Complex

    KAUST Repository

    Cernilogar, Filippo M.

    2013-06-13

    Background:Beyond their role in post-transcriptional gene silencing, Dicer and Argonaute, two components of the RNA interference (RNAi) machinery, were shown to be involved in epigenetic regulation of centromeric heterochromatin and transcriptional gene silencing. In particular, RNAi mechanisms appear to play a role in repeat induced silencing and some aspects of Polycomb-mediated gene silencing. However, the functional interplay of RNAi mechanisms and Polycomb group (PcG) pathways at endogenous loci remains to be elucidated.Principal Findings:Here we show that the endogenous Dicer-2/Argonaute-2 RNAi pathway is dispensable for the PcG mediated silencing of the homeotic Bithorax Complex (BX-C). Although Dicer-2 depletion triggers mild transcriptional activation at Polycomb Response Elements (PREs), this does not induce transcriptional changes at PcG-repressed genes. Moreover, Dicer-2 is not needed to maintain global levels of methylation of lysine 27 of histone H3 and does not affect PRE-mediated higher order chromatin structures within the BX-C. Finally bioinformatic analysis, comparing published data sets of PcG targets with Argonaute-2-bound small RNAs reveals no enrichment of these small RNAs at promoter regions associated with PcG proteins.Conclusions:We conclude that the Dicer-2/Argonaute-2 RNAi pathway, despite its role in pairing sensitive gene silencing of transgenes, does not have a role in PcG dependent silencing of major homeotic gene cluster loci in Drosophila. © 2013 Cernilogar et al.

  20. RNA-mediated gene silencing in the cereal fungal pathogen Cochliobolus sativus.

    Science.gov (United States)

    Leng, Yueqiang; Wu, Chengxiang; Liu, Zhaohui; Friesen, Timothy L; Rasmussen, Jack B; Zhong, Shaobin

    2011-04-01

    A high-throughput RNA-mediated gene silencing system was developed for Cochliobolus sativus (anamorph: Bipolaris sorokiniana), the causal agent of spot blotch, common root rot and black point in barley and wheat. The green fluorescent protein gene (GFP) and the proteinaceous host-selective toxin gene (ToxA) were first introduced into C. sativus via the polyethylene glycol (PEG)-mediated transformation method. Transformants with a high level of expression of GFP or ToxA were generated. A silencing vector (pSGate1) based on the Gateway cloning system was developed and used to construct RNA interference (RNAi) vectors. Silencing of GFP and ToxA in the transformants was demonstrated by transformation with the RNAi construct expressing hairpin RNA (hpRNA) of the target gene. The polyketide synthase gene (CsPKS1), involved in melanin biosynthesis pathways in C. sativus, was also targeted by transformation with the RNAi vector (pSGate1-CsPKS1) encoding hpRNA of the CsPKS1 gene. The transformants with pSGate1-CsPKS1 exhibited an albino phenotype or reduced melanization, suggesting effective silencing of the endogenous CsPKS1 in C. sativus. Sectors exhibiting the wild-type phenotype of the fungus appeared in some of the CsPKS1-silenced transformants after subcultures as a result of inactivation or deletions of the RNAi transgene. The gene silencing system established provides a useful tool for functional genomics studies in C. sativus and other filamentous fungi.

  1. Viral suppressors of RNA interference impair RNA silencing induced by a Semliki Forest virus replicon in tick cells

    NARCIS (Netherlands)

    Garcia, S.; Billecocq, A.; Crance, J.M.; Prins, M.W.; Garin, D.; Bouloy, M.

    2006-01-01

    It was recently shown that infection of ISE6 tick cells by a recombinant Semliki Forest virus (SFV) expressing a heterologous gene induced small interfering RNAs (siRNAs) and silencing of the gene. To gain information on RNA interference (RNAi) in ticks, three known viral inhibitors that act in diff

  2. Applications of RNA interference-based gene silencing in animal agriculture.

    Science.gov (United States)

    Long, Charles R; Tessanne, Kimberly J; Golding, Michael C

    2010-01-01

    Classical genetic selection, recently aided by genomic selection tools, has been successful in achieving remarkable progress in livestock improvement. However, genetic selection has led to decreased genetic diversity and, in some cases, acquisition of undesirable traits. In order to meet the increased demands of our expanding population, new technologies and practices must be developed that contend with zoonotic and animal disease, environmental impacts of large farming operations and the increased food and fibre production needed to feed and clothe our society. Future increases in productivity may be dependent upon the acquisition of genetic traits not currently encoded by the genomes of animals used in standard agricultural practice, thus making classical genetic selection impossible. Genetic engineering of livestock is commonly used to produce pharmaceuticals or to impart enhanced production characteristics to animals, but has also demonstrated its usefulness in producing animals with disease resistance. However, significant challenges remain because it has been more difficult to produce animals in which specific genes have been removed. It is now possible to modify livestock genomes to block expression of endogenous and exogenous genes (such as those expressed following virus infection). In the present review, we discuss mechanisms of silencing gene expression via the biology of RNA interference (RNAi), the technology of activating the RNAi pathway and the application of this technology to enhance livestock production through increased production efficiency and prevention of disease. An increased demand for sustainable food production is at the forefront of scientific challenges and RNAi technology will undoubtedly play a key role.

  3. Gene silencing by RNA interference in the house dust mite, Dermatophagoides pteronyssinus.

    Science.gov (United States)

    Marr, Edward J; Sargison, Neil D; Nisbet, Alasdair J; Burgess, Stewart T G

    2015-12-01

    This is the first report of gene silencing by RNA interference (RNAi) in the European house dust mite, Dermatophagoides pteronyssinus, Trouessart, 1897. Using a non-invasive immersion method first developed for the honey bee mite, Varroa destructor, a significant reduction in the expression of D. pteronyssinus glutathione-S-transferase mu-class 1 enzyme (DpGST-mu1) was achieved following overnight immersion in double stranded RNA encoding DpGST-mu1. Although no detrimental phenotypic changes were observed following silencing, this technique can now be used to address fundamental physiological questions and assess the potential therapeutic benefit in silencing D. pteronyssinus target genes in selected domestic situations of high human-mite interface. PMID:26212476

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

    Institute of Scientific and Technical Information of China (English)

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

    2010-01-01

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

  5. Dynamics and Complexity in a Time Delay Model of RNA Silencing with Periodic Forcing

    Directory of Open Access Journals (Sweden)

    Svetoslav Nikolov

    2008-10-01

    Full Text Available Simple periodic behavior and occurrence of complex oscillatory phenomena underlie of a large number of biochemical system models. In many cases the transition from stable to simple/complex oscillatory behavior can be connected with the appearance of abnormal process likes as cancer. In this paper we propose a time delay model of RNA silencing (also known as RNA interference with periodic forcing. In organisms with RNA silencing, each cell has a miniature "immune system" able to generate and amplify specific responses to a variety of gene transcripts. The consequences of a time delay on the dynamics of this model are analysed using Hopf's theorem. Our analytical calculations predict that time delay acts as a key bifurcation parameter. From the accomplished numerical results, it becomes clear that model has complexity oscillatory behavior when the amplitude of periodic force (i.e. the confusion in the target mRNA synthesis is large.

  6. The NS3 protein of rice hoja blanca virus suppresses RNA silencing in mammalian cells

    NARCIS (Netherlands)

    Schnettler, E.; Hemmes, J.C.; Goldbach, R.W.; Prins, M.W.

    2008-01-01

    The NS3 protein of the tenuivirus rice hoja blanca virus (RHBV) has previously been shown to represent the viral RNA interference (RNAi) suppressor and is active in both plant and insect cells by binding short interfering RNAs (siRNAs) in vitro. Using a firefly luciferase-based silencing assay it is

  7. The Ebola virus VP35 protein is a suppressor of RNA silencing

    NARCIS (Netherlands)

    Haasnoot, J.; Vries, de W.; Geutjes, E.J.; Prins, M.W.; Haan, de P.; Berkhout, B.

    2007-01-01

    RNA silencing or interference (RNAi) is a gene regulation mechanism in eukaryotes that controls cell differentiation and developmental processes via expression of microRNAs. RNAi also serves as an innate antiviral defence response in plants, nematodes, and insects. This antiviral response is trigger

  8. Transporting silence : Design of carriers for siRNA to angiogenic endothelium

    NARCIS (Netherlands)

    Mixson, AJ; Ansari, AM; Fens, MHAM; Tang, QQ; Zhou, Q; Molema, G; Lu, PY; Scaria, PV; Storm, G; Woodle, MC

    2005-01-01

    The recently developed siRNA oligonucleotides are an attractive alternative to antisense as a therapeutic modality because of their robust, gene selective silencing of drug target protein expression. To achieve therapeutic success, however, several hurdles must be overcome including rapid clearance,

  9. Interplays between Soil-Borne Plant Viruses and RNA Silencing-Mediated Antiviral Defense in Roots

    Science.gov (United States)

    Andika, Ida Bagus; Kondo, Hideki; Sun, Liying

    2016-01-01

    Although the majority of plant viruses are transmitted by arthropod vectors and invade the host plants through the aerial parts, there is a considerable number of plant viruses that infect roots via soil-inhabiting vectors such as plasmodiophorids, chytrids, and nematodes. These soil-borne viruses belong to diverse families, and many of them cause serious diseases in major crop plants. Thus, roots are important organs for the life cycle of many viruses. Compared to shoots, roots have a distinct metabolism and particular physiological characteristics due to the differences in development, cell composition, gene expression patterns, and surrounding environmental conditions. RNA silencing is an important innate defense mechanism to combat virus infection in plants, but the specific information on the activities and molecular mechanism of RNA silencing-mediated viral defense in root tissue is still limited. In this review, we summarize and discuss the current knowledge regarding RNA silencing aspects of the interactions between soil-borne viruses and host plants. Overall, research evidence suggests that soil-borne viruses have evolved to adapt to the distinct mechanism of antiviral RNA silencing in roots.

  10. Virus-Induced Gene Silencing as a Tool for Delivery of dsRNA into Plants

    OpenAIRE

    sprotocols

    2014-01-01

    Authors: Meenu Padmanabhan and Savithramma P. Dinesh-Kumar1 Corresponding author ([](mailto:)) ### INTRODUCTION The inherent RNA silencing mechanism in plants has been effectively manipulated as a tool for the targeted down-regulation of genes. Numerous methods have been employed to initiate this homology-based RNA degradation process, but all rely on the activity of double-stranded RNAs (dsRNAs) corresponding to th...

  11. An siRNA-based method for efficient silencing of gene expression in mature brown adipocytes.

    Science.gov (United States)

    Isidor, Marie S; Winther, Sally; Basse, Astrid L; Petersen, M Christine H; Cannon, Barbara; Nedergaard, Jan; Hansen, Jacob B

    2016-01-01

    Brown adipose tissue is a promising therapeutic target for opposing obesity, glucose intolerance and insulin resistance. The ability to modulate gene expression in mature brown adipocytes is important to understand brown adipocyte function and delineate novel regulatory mechanisms of non-shivering thermogenesis. The aim of this study was to optimize a lipofection-based small interfering RNA (siRNA) transfection protocol for efficient silencing of gene expression in mature brown adipocytes. We determined that a critical parameter was to deliver the siRNA to mature adipocytes by reverse transfection, i.e. transfection of non-adherent cells. Using this protocol, we effectively knocked down both high- and low-abundance transcripts in a model of mature brown adipocytes (WT-1) as well as in primary mature mouse brown adipocytes. A functional consequence of the knockdown was confirmed by an attenuated increase in uncoupled respiration (thermogenesis) in response to β-adrenergic stimulation of mature WT-1 brown adipocytes transfected with uncoupling protein 1 siRNA. Efficient gene silencing was also obtained in various mouse and human white adipocyte models (3T3-L1, primary mouse white adipocytes, hMADS) with the ability to undergo "browning." In summary, we report an easy and versatile reverse siRNA transfection protocol to achieve specific silencing of gene expression in various models of mature brown and browning-competent white adipocytes, including primary cells.

  12. A versatile assay for the identification of RNA silencing suppressors based on complementation of viral movement.

    Science.gov (United States)

    Powers, Jason G; Sit, Tim L; Qu, Feng; Morris, T Jack; Kim, Kook-Hyung; Lommel, Steven A

    2008-07-01

    The cell-to-cell movement of Turnip crinkle virus (TCV) in Nicotiana benthamiana requires the presence of its coat protein (CP), a known suppressor of RNA silencing. RNA transcripts of a TCV construct containing a reporter gene (green fluorescent protein) (TCV-sGFP) in place of the CP open reading frame generated foci of three to five cells. TCV CP delivered in trans by Agrobacterium tumefaciens infiltration potentiated movement of TCV-sGFP and increased foci diameter, on average, by a factor of four. Deletion of the TCV movement proteins in TCV-sGFP (construct TCVDelta92-sGFP) abolished the movement complementation ability of TCV CP. Other known suppressors of RNA silencing from a wide spectrum of viruses also complemented the movement of TCV-sGFP when delivered in trans by Agrobacterium tumefaciens. These include suppressors from nonplant viruses with no known plant movement function, demonstrating that this assay is based solely on RNA silencing suppression. While the TCV-sGFP construct is primarily used as an infectious RNA transcript, it was also subcloned for direct expression from Agrobacterium tumefaciens for simple quantification of suppressor activity based on fluorescence levels in whole leaves. Thus, this system provides the flexibility to assay for suppressor activity in either the cytoplasm or nucleus, depending on the construct employed. PMID:18533829

  13. An siRNA-based method for efficient silencing of gene expression in mature brown adipocytes.

    Science.gov (United States)

    Isidor, Marie S; Winther, Sally; Basse, Astrid L; Petersen, M Christine H; Cannon, Barbara; Nedergaard, Jan; Hansen, Jacob B

    2016-01-01

    Brown adipose tissue is a promising therapeutic target for opposing obesity, glucose intolerance and insulin resistance. The ability to modulate gene expression in mature brown adipocytes is important to understand brown adipocyte function and delineate novel regulatory mechanisms of non-shivering thermogenesis. The aim of this study was to optimize a lipofection-based small interfering RNA (siRNA) transfection protocol for efficient silencing of gene expression in mature brown adipocytes. We determined that a critical parameter was to deliver the siRNA to mature adipocytes by reverse transfection, i.e. transfection of non-adherent cells. Using this protocol, we effectively knocked down both high- and low-abundance transcripts in a model of mature brown adipocytes (WT-1) as well as in primary mature mouse brown adipocytes. A functional consequence of the knockdown was confirmed by an attenuated increase in uncoupled respiration (thermogenesis) in response to β-adrenergic stimulation of mature WT-1 brown adipocytes transfected with uncoupling protein 1 siRNA. Efficient gene silencing was also obtained in various mouse and human white adipocyte models (3T3-L1, primary mouse white adipocytes, hMADS) with the ability to undergo "browning." In summary, we report an easy and versatile reverse siRNA transfection protocol to achieve specific silencing of gene expression in various models of mature brown and browning-competent white adipocytes, including primary cells. PMID:27386153

  14. MicroRNA silencing in primates: towards development of novel therapeutics

    DEFF Research Database (Denmark)

    Petri, Andreas; Lindow, Morten; Kauppinen, Sakari

    2009-01-01

    MicroRNAs (miRNA) comprise an abundant class of small noncoding RNAs that act as important posttranscriptional regulators of gene expression. Accumulating evidence showing that aberrantly expressed miRNAs play important roles in human cancers underscores them as potential targets for therapeutic ...... intervention. Recent reports on efficient miRNA silencing in rodents and nonhuman primates using high-affinity targeting by chemically modified antisense oligonucleotides highlight the utility of such compounds in the development of miRNA-based cancer therapeutics....

  15. Development of RNA Interference Trigger-Mediated Gene Silencing in Entamoeba invadens.

    Science.gov (United States)

    Suresh, Susmitha; Ehrenkaufer, Gretchen; Zhang, Hanbang; Singh, Upinder

    2016-04-01

    Entamoeba histolytica, a protozoan parasite, is an important human pathogen and a leading parasitic cause of death. The organism has two life cycle stages, trophozoites, which are responsible for tissue invasion, and cysts, which are involved in pathogen transmission. Entamoeba invadens is the model system to study Entamoeba developmental biology, as high-grade regulated encystation and excystation are readily achievable. However, the lack of gene-silencing tools in E. invadens has limited the molecular studies that can be performed. Using the endogenous RNA interference (RNAi) pathway in Entamoeba, we developed an RNAi-based trigger gene-silencing approach inE. invadens We demonstrate that a gene's coding region that has abundant antisense small RNAs (sRNAs) can trigger silencing of a gene that is fused to it. The trigger fusion leads to the generation of abundant antisense sRNAs that map to the target gene, with silencing occurring independently of trigger location at the 5' or 3' end of a gene. Gene silencing is stably maintained during development, including encystation and excystation. We have used this approach to successfully silence two E. invadens genes: a putative rhomboid protease gene and a SHAQKY family Myb gene. The Myb gene is upregulated during oxidative stress and development, and its downregulation led, as predicted, to decreased viability under oxidative stress and decreased cyst formation. Thus, the RNAi trigger silencing method can be used to successfully investigate the molecular functions of genes inE. invadens Dissection of the molecular basis of Entamoeba stage conversion is now possible, representing an important technical advance for the system. PMID:26787723

  16. Development of RNA Interference Trigger-Mediated Gene Silencing in Entamoeba invadens.

    Science.gov (United States)

    Suresh, Susmitha; Ehrenkaufer, Gretchen; Zhang, Hanbang; Singh, Upinder

    2016-04-01

    Entamoeba histolytica, a protozoan parasite, is an important human pathogen and a leading parasitic cause of death. The organism has two life cycle stages, trophozoites, which are responsible for tissue invasion, and cysts, which are involved in pathogen transmission. Entamoeba invadens is the model system to study Entamoeba developmental biology, as high-grade regulated encystation and excystation are readily achievable. However, the lack of gene-silencing tools in E. invadens has limited the molecular studies that can be performed. Using the endogenous RNA interference (RNAi) pathway in Entamoeba, we developed an RNAi-based trigger gene-silencing approach inE. invadens We demonstrate that a gene's coding region that has abundant antisense small RNAs (sRNAs) can trigger silencing of a gene that is fused to it. The trigger fusion leads to the generation of abundant antisense sRNAs that map to the target gene, with silencing occurring independently of trigger location at the 5' or 3' end of a gene. Gene silencing is stably maintained during development, including encystation and excystation. We have used this approach to successfully silence two E. invadens genes: a putative rhomboid protease gene and a SHAQKY family Myb gene. The Myb gene is upregulated during oxidative stress and development, and its downregulation led, as predicted, to decreased viability under oxidative stress and decreased cyst formation. Thus, the RNAi trigger silencing method can be used to successfully investigate the molecular functions of genes inE. invadens Dissection of the molecular basis of Entamoeba stage conversion is now possible, representing an important technical advance for the system.

  17. MicroRNA-Dependent Transcriptional Silencing of Transposable Elements in Drosophila Follicle Cells.

    Science.gov (United States)

    Mugat, Bruno; Akkouche, Abdou; Serrano, Vincent; Armenise, Claudia; Li, Blaise; Brun, Christine; Fulga, Tudor A; Van Vactor, David; Pélisson, Alain; Chambeyron, Séverine

    2015-05-01

    RNA interference-related silencing mechanisms concern very diverse and distinct biological processes, from gene regulation (via the microRNA pathway) to defense against molecular parasites (through the small interfering RNA and the Piwi-interacting RNA pathways). Small non-coding RNAs serve as specificity factors that guide effector proteins to ribonucleic acid targets via base-pairing interactions, to achieve transcriptional or post-transcriptional regulation. Because of the small sequence complementarity required for microRNA-dependent post-transcriptional regulation, thousands of microRNA (miRNA) putative targets have been annotated in Drosophila. In Drosophila somatic ovarian cells, genomic parasites, such as transposable elements (TEs), are transcriptionally repressed by chromatin changes induced by Piwi-interacting RNAs (piRNAs) that prevent them from invading the germinal genome. Here we show, for the first time, that a functional miRNA pathway is required for the piRNA-mediated transcriptional silencing of TEs in this tissue. Global miRNA depletion, caused by tissue- and stage-specific knock down of drosha (involved in miRNA biogenesis), AGO1 or gawky (both responsible for miRNA activity), resulted in loss of TE-derived piRNAs and chromatin-mediated transcriptional de-silencing of TEs. This specific TE de-repression was also observed upon individual titration (by expression of the complementary miRNA sponge) of two miRNAs (miR-14 and miR-34) as well as in a miR-14 loss-of-function mutant background. Interestingly, the miRNA defects differentially affected TE- and 3' UTR-derived piRNAs. To our knowledge, this is the first indication of possible differences in the biogenesis or stability of TE- and 3' UTR-derived piRNAs. This work is one of the examples of detectable phenotypes caused by loss of individual miRNAs in Drosophila and the first genetic evidence that miRNAs have a role in the maintenance of genome stability via piRNA-mediated TE repression.

  18. Self-incompatibility: Smi silences through a novel sRNA pathway.

    Science.gov (United States)

    Finnegan, E Jean; Liang, Dacheng; Wang, Ming-Bo

    2011-05-01

    Self-incompatibility in Brassicaceae is determined by the interaction between S-Locus Protein 11 (SP11) on the pollen and S-receptor kinase (SRK) in the stigma. Pollen from heterozygotes generally displays products of both SP11 alleles, but in some heterozygotes SP11 expression is monoallelic, with one allele (SP11(R)) being silenced by promoter methylation. An exciting development in understanding the mechanism behind monoallelic silencing came recently when Y. Tarutani et al. [Nature 2010;466:983-986] identified a 24-nucleotide sRNA (termed Smi) derived from a non-coding gene within the dominant S-haplotype, and suggested that Smi directs promoter methylation. We propose that rather than having a direct effect on DNA methylation, Smi is the first step in a novel cis-acting siRNA pathway that directs widespread monoallelic SP11(R) promoter methylation.

  19. The NS3 protein of rice hoja blanca virus suppresses RNA silencing in mammalian cells.

    Science.gov (United States)

    Schnettler, Esther; Hemmes, Hans; Goldbach, Rob; Prins, Marcel

    2008-01-01

    The NS3 protein of the tenuivirus rice hoja blanca virus (RHBV) has previously been shown to represent the viral RNA interference (RNAi) suppressor and is active in both plant and insect cells by binding short interfering RNAs (siRNAs) in vitro. Using a firefly luciferase-based silencing assay it is described here that NS3 is also active in mammalian cells. This activity is independent of the inducer molecule used. Using either synthetic siRNAs or a short hairpin RNA construct, NS3 was able to significantly suppress the RNAi-mediated silencing of luciferase expression in both monkey (Vero) and human (HEK293) cells. These results support the proposed mode of action of NS3 to act by sequestering siRNAs, the key molecules of the RNAi pathway conserved in all eukaryotes. The possible applications of this protein in modulating RNAi and investigating the proposed antiviral RNAi response in mammalian cell systems are discussed. PMID:18089758

  20. Efficient gene silencing in metastatic tumor by siRNA formulated in surface-modified nanoparticles.

    Science.gov (United States)

    Li, Shyh-Dar; Chono, Sumio; Huang, Leaf

    2008-02-18

    We have developed a nanoparticle (NP) formulation for systemically delivering siRNA into metastatic tumors. The NP, composed of nucleic acids, a polycationic peptide and cationic liposome, was prepared in a self-assembling process. The NP was then modified by PEG-lipid containing a targeting ligand, anisamide, and thus was decorated for targeting sigma receptor expressing B16F10 tumor. The activity of the targeted NP was compared with the naked NP (no PEGylation) and non-targeted NP (no ligand). The delivery efficiency of the targeted NP was 4-fold higher than the non-targeted NP and could be competed by excess free ligand. Luciferase siRNA was used to evaluate the gene silencing activity in the B16F10 cells, which were stably transduced with a luciferase gene. The gene silencing activity of the targeted NP was significantly higher than the other formulations and lasted for 4 days. While confocal microscopy showed that the naked NP provided no tissue selectivity and non-targeted NP was ineffective for tumor uptake, the targeted NP effectively penetrated the lung metastasis, but not the liver. It resulted in 70-80% gene silencing in the metastasis model after a single i.v. injection (150 microg siRNA/kg). This effective formulation also showed very little immunotoxicity.

  1. Silencing microRNA-155 ameliorates experimental autoimmune encephalomyelitis.

    Science.gov (United States)

    Murugaiyan, Gopal; Beynon, Vanessa; Mittal, Akanksha; Joller, Nicole; Weiner, Howard L

    2011-09-01

    IFN-γ-producing Th1 and IL-17-producing Th17 cells are the key participants in various autoimmune diseases, including multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). Although both of these T cell subsets are known to be regulated by specific transcription factors and cytokines, the role of microRNAs that control these two inflammatory T cell subsets and whether targeting microRNAs can have therapeutic effects are not known. In this study, we show that microRNA-155 (Mir-155) expression is elevated in CD4(+) T cells during EAE, and Mir-155(-/-) mice had a delayed course and reduced severity of disease and less inflammation in the CNS. The attenuation of EAE in Mir-155(-/-) mice was associated with a decrease in Th1 and Th17 responses in the CNS and peripheral lymphoid organs. The T cell-intrinsic function of Mir-155(-/-) was demonstrated by the resistance of Mir-155(-/-) CD4(+) T cell-repleted Rag-1(-/-) mice to EAE. Finally, we found that anti-Mir-155 treatment reduced clinical severity of EAE when given before and after the appearance of clinical symptoms. These findings demonstrate that Mir-155 confers susceptibility to EAE by affecting inflammatory T cell responses and identify Mir-155 as a new target for therapeutic intervention in multiple sclerosis. PMID:21788439

  2. S100A4 silencing blocks invasive ability of esophageal squamous cell carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Dong Chen; Xue-Feng Zheng; Ze-You Yang; Dong-Xiao Liu; Guo-You Zhang; Xue-Long Jiao; Hui Zhao

    2012-01-01

    AIM:To investigate a potential role of S100A4 in esophagus squamous cell carcinoma metastasis (ESCCs).METHODS:Expression of S100A4 and E-cadherin were analyzed in frozen sections from ESCCs (metastasis,n =28; non-metastasis,n =20) by reverse transcription-polymerase chain reaction,quantitative polymerase chain reaction and immunohistochemistry.To explore the influence of S100A4 on esophageal cancer invasion and metastasis,S100A4 was overexpressed or silenced by S100A4 siRNA in TE-13 or Eca-109 cells in vitro and in vivo.RESULTS:We found the mRNA and protein levels of S100A4 expression in ESCCs was significantly upregulated,and more importantly,that expression of S100A4 and E cadherin are strongly negatively correlated in patients who had metastasis.It was indicated that overexpression of S100A4 in TE-13 and Eca-109 cells downregulates the expression of E-cadherin,leading to increased cell migration in vitro,whereas knockdown of S100A4 inhibited cell migration and upregulation of E-cadherin expression.Moreover,the loss of cell metastatic potential was rescued by overexpression of E-cadherin completely.In addition,nude mice inoculated with S100A4 siRNA-transfected cells exhibited a significantly decreased invasion ability in vivo.CONCLUSION:S100A4 may be involved in ESCC progression by regulate E-cadherin expression,vectorbased RNA interference targeting S100A4 is a potential therapeutic method for human ESCC.

  3. Comprehensive protein-based artificial microRNA screens for effective gene silencing in plants.

    Science.gov (United States)

    Li, Jian-Feng; Chung, Hoo Sun; Niu, Yajie; Bush, Jenifer; McCormack, Matthew; Sheen, Jen

    2013-05-01

    Artificial microRNA (amiRNA) approaches offer a powerful strategy for targeted gene manipulation in any plant species. However, the current unpredictability of amiRNA efficacy has limited broad application of this promising technology. To address this, we developed epitope-tagged protein-based amiRNA (ETPamir) screens, in which target mRNAs encoding epitope-tagged proteins were constitutively or inducibly coexpressed in protoplasts with amiRNA candidates targeting single or multiple genes. This design allowed parallel quantification of target proteins and mRNAs to define amiRNA efficacy and mechanism of action, circumventing unpredictable amiRNA expression/processing and antibody unavailability. Systematic evaluation of 63 amiRNAs in 79 ETPamir screens for 16 target genes revealed a simple, effective solution for selecting optimal amiRNAs from hundreds of computational predictions, reaching ∼100% gene silencing in plant cells and null phenotypes in transgenic plants. Optimal amiRNAs predominantly mediated highly specific translational repression at 5' coding regions with limited mRNA decay or cleavage. Our screens were easily applied to diverse plant species, including Arabidopsis thaliana, tobacco (Nicotiana benthamiana), tomato (Solanum lycopersicum), sunflower (Helianthus annuus), Catharanthus roseus, maize (Zea mays) and rice (Oryza sativa), and effectively validated predicted natural miRNA targets. These screens could improve plant research and crop engineering by making amiRNA a more predictable and manageable genetic and functional genomic technology.

  4. GENE SILENCING BY PARENTAL RNA INTERFERENCE IN THE GREEN RICE LEAFHOPPER, Nephotettix cincticeps (HEMIPTERA: CICADELLIDAE).

    Science.gov (United States)

    Matsumoto, Yukiko; Hattori, Makoto

    2016-03-01

    RNA interference (RNAi) has been widely used for investigating gene function in many nonmodel insect species. Parental RNAi causes gene knockdown in the next generation through the administration of double-strand RNA (dsRNA) to the mother generation. In this study, we demonstrate that parental RNAi mediated gene silencing is effective in determining the gene function of the cuticle and the salivary glands in green rice leafhopper (GRH), Nephotettix cincticeps (Uhler). Injection of dsRNA of NcLac2 (9 ng/female) to female parents caused a strong knockdown of laccase-2 gene of first instar nymphs, which eventually led to high mortality rates and depigmentation of side lines on the body. The effects of parental RNAi on the mortality of the nymphs were maintained through 12-14 days after the injections. We also confirmed the effectiveness of parental RNAi induced silencing on the gene expressed in the salivary gland, the gene product of which is passed from instar to instar. The parental RNAi method can be used to examine gene function by phenotyping many offspring nymphs with injection of dsRNA into a small number of parent females, and may be applicable to high-efficiency determination of gene functions in this species.

  5. Exonuclease-mediated degradation of nascent RNA silences genes linked to severe malaria.

    Science.gov (United States)

    Zhang, Qingfeng; Siegel, T Nicolai; Martins, Rafael M; Wang, Fei; Cao, Jun; Gao, Qi; Cheng, Xiu; Jiang, Lubin; Hon, Chung-Chau; Scheidig-Benatar, Christine; Sakamoto, Hiroshi; Turner, Louise; Jensen, Anja T R; Claes, Aurelie; Guizetti, Julien; Malmquist, Nicholas A; Scherf, Artur

    2014-09-18

    Antigenic variation of the Plasmodium falciparum multicopy var gene family enables parasite evasion of immune destruction by host antibodies. Expression of a particular var subgroup, termed upsA, is linked to the obstruction of blood vessels in the brain and to the pathogenesis of human cerebral malaria. The mechanism determining upsA activation remains unknown. Here we show that an entirely new type of gene silencing mechanism involving an exonuclease-mediated degradation of nascent RNA controls the silencing of genes linked to severe malaria. We identify a novel chromatin-associated exoribonuclease, termed PfRNase II, that controls the silencing of upsA var genes by marking their transcription start site and intron-promoter regions leading to short-lived cryptic RNA. Parasites carrying a deficient PfRNase II gene produce full-length upsA var transcripts and intron-derived antisense long non-coding RNA. The presence of stable upsA var transcripts overcomes monoallelic expression, resulting in the simultaneous expression of both upsA and upsC type PfEMP1 proteins on the surface of individual infected red blood cells. In addition, we observe an inverse relationship between transcript levels of PfRNase II and upsA-type var genes in parasites from severe malaria patients, implying a crucial role of PfRNase II in severe malaria. Our results uncover a previously unknown type of post-transcriptional gene silencing mechanism in malaria parasites with repercussions for other organisms. Additionally, the identification of RNase II as a parasite protein controlling the expression of virulence genes involved in pathogenesis in patients with severe malaria may provide new strategies for reducing malaria mortality. PMID:25043062

  6. Synthetic Polymer Hybridization with DNA and RNA Directs Nanoparticle Loading, Silencing Delivery, and Aptamer Function.

    Science.gov (United States)

    Zhou, Zhun; Xia, Xin; Bong, Dennis

    2015-07-22

    We report herein discrete triplex hybridization of DNA and RNA with polyacrylates. Length-monodisperse triazine-derivatized polymers were prepared on gram-scale by reversible addition-fragmentation chain-transfer polymerization. Despite stereoregio backbone heterogeneity, the triazine polymers bind T/U-rich DNA or RNA with nanomolar affinity upon mixing in a 1:1 ratio, as judged by thermal melts, circular dichroism, gel-shift assays, and fluorescence quenching. We call these polyacrylates "bifacial polymer nucleic acids" (bPoNAs). Nucleic acid hybridization with bPoNA enables DNA loading onto polymer nanoparticles, siRNA silencing delivery, and can further serve as an allosteric trigger of RNA aptamer function. Thus, bPoNAs can serve as tools for both non-covalent bioconjugation and structure-function nucleation. It is anticipated that bPoNAs will have utility in both bio- and nanotechnology. PMID:26138550

  7. Salicylic acid and gentisic acid induce RNA silencing-related genes and plant resistance to RNA pathogens.

    Science.gov (United States)

    Campos, Laura; Granell, Pablo; Tárraga, Susana; López-Gresa, Pilar; Conejero, Vicente; Bellés, José María; Rodrigo, Ismael; Lisón, Purificación

    2014-04-01

    We have observed that treatments with salicylic acid (SA) or gentisic acid (GA) induced resistance to RNA pathogens such as ToMV and CEVd in tomato and Gynura auriantiaca, respectively. Accumulation of SA and GA has been found to occur in plants infected by these pathogens, thus pointing out a possible defence role of both molecules. To study the molecular basis of the observed induced resistance to RNA pathogens the induction of silencing-related genes by SA and GA was considered. For that purpose, we searched for tomato genes which were orthologous to those described in Arabidopsis thaliana, such as AtDCL1, AtDCL2, AtDCL4, AtRDR1, AtRDR2 and AtRDR6, and we tracked their induction in tomato along virus and viroid infections. We observed that CEVd significantly induced all these genes in tomato, with the exception of ToRDR6, being the induction of ToDCL4 the most outstanding. Regarding the ToMV asymptomatic infection, with the exception of ToRDR2, we observed a significant induction of all the indicated silencing-related genes, being ToDCL2 the most induced gene. Subsequently, we analyzed their transcriptional activation by SA and at the time when ToMV was inoculated on plants. ToDCL2, ToRDR1 and ToRDR2 were significantly induced by both SA and GA, whereas ToDCL1 was only induced by SA. Such an induction resulted more effective by SA treatment, which is in agreement with the stronger SA-induced resistance observed. Our results suggest that the observed delay in the RNA pathogen accumulation could be due to the pre-induction of RNA silencing-related genes by SA or GA.

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

    Science.gov (United States)

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

    2009-01-01

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

  9. Bifunctional oligodeoxynucleotide/antagomiR constructs: evaluation of a new tool for microRNA silencing.

    Science.gov (United States)

    Ziegler, Saskia; Eberle, Mariel Esther; Wölfle, Sabine J; Heeg, Klaus; Bekeredjian-Ding, Isabelle

    2013-12-01

    MicroRNAs (miRNAs) are fine-tuners in cellular processes, including those of the immune response. To study their functions and effects in immune cells, it is necessary to achieve specific silencing of individual miRNAs. To date, introduction of antisense microRNAs (antagomiRs) into primary cells is based on electroporation, lipofection, and viral vectors. However, these techniques often compromise viability, proliferative capacity, and differentiation. Furthermore, efficiency varies depending on the cell type and some are not suitable for in vivo approaches. To overcome these limitations we exploited the property of phosphorothioate (PTO)-modified DNA oligodeoxynucleotides (ODN) to enter cells with high efficacy: we developed and evaluated ODN/antagomiR constructs that consist of a PTO-ODN carrier covalently linked to a fully methylated antagomiR RNA sequence. Using these constructs, we achieved transfection efficiency of approximately 99% in leukocytes-in particular, in B lymphocytes that are hard to transfect with other methods. Our data demonstrate that miRNA silencing by the antagomiR portion of the constructs was specific and efficient, which could be further confirmed by an increase in target protein under silencing conditions. The constructs were successfully tested in human B cells, plasmacytoid dendritic cells, monocytes, and monocyte-derived dendritic cells, thus demonstrating their versatility. Moreover, introduction of stimulatory CpG sequences into the ODN portion conveys immune stimulatory quality when intended. Thus, bifunctional ODN/antagomiR constructs represent a highly efficient, versatile, and easy-to-handle tool to manipulate cellular miRNA expression levels and to allow the subsequent investigation of specific miRNA functions. PMID:24236889

  10. Enhanced transgene expression in sugarcane by co-expression of virus-encoded RNA silencing suppressors.

    Directory of Open Access Journals (Sweden)

    San-Ji Gao

    Full Text Available Post-transcriptional gene silencing is commonly observed in polyploid species and often poses a major limitation to plant improvement via biotechnology. Five plant viral suppressors of RNA silencing were evaluated for their ability to counteract gene silencing and enhance the expression of the Enhanced Yellow Fluorescent Protein (EYFP or the β-glucuronidase (GUS reporter gene in sugarcane, a major sugar and biomass producing polyploid. Functionality of these suppressors was first verified in Nicotiana benthamiana and onion epidermal cells, and later tested by transient expression in sugarcane young leaf segments and protoplasts. In young leaf segments co-expressing a suppressor, EYFP reached its maximum expression at 48-96 h post-DNA introduction and maintained its peak expression for a longer time compared with that in the absence of a suppressor. Among the five suppressors, Tomato bushy stunt virus-encoded P19 and Barley stripe mosaic virus-encoded γb were the most efficient. Co-expression with P19 and γb enhanced EYFP expression 4.6-fold and 3.6-fold in young leaf segments, and GUS activity 2.3-fold and 2.4-fold in protoplasts compared with those in the absence of a suppressor, respectively. In transgenic sugarcane, co-expression of GUS and P19 suppressor showed the highest accumulation of GUS levels with an average of 2.7-fold more than when GUS was expressed alone, with no detrimental phenotypic effects. The two established transient expression assays, based on young leaf segments and protoplasts, and confirmed by stable transgene expression, offer a rapid versatile system to verify the efficiency of RNA silencing suppressors that proved to be valuable in enhancing and stabilizing transgene expression in sugarcane.

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

    Energy Technology Data Exchange (ETDEWEB)

    Dugan, L

    2006-02-08

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

  12. Selective silencing of gene target expression by siRNA expression plasmids in human cervical cancer cells.

    Science.gov (United States)

    Peralta-Zaragoza, Oscar; De-la-O-Gómez, Faustino; Deas, Jessica; Fernández-Tilapa, Gloria; Fierros-Zárate, Geny Del Socorro; Gómez-Cerón, Claudia; Burguete-García, Ana; Torres-Poveda, Kirvis; Bermúdez-Morales, Victor Hugo; Rodríguez-Dorantes, Mauricio; Pérez-Plasencia, Carlos; Madrid-Marina, Vicente

    2015-01-01

    RNA interference is a natural mechanism to silence post-transcriptional gene expression in eukaryotic cells in which microRNAs act to cleave or halt the translation of target mRNAs at specific target sequences. Mature microRNAs, 19-25 nucleotides in length, mediate their effect at the mRNA level by inhibiting translation, or inducing cleavage of the mRNA target. This process is directed by the degree of complementary nucleotides between the microRNAs and the target mRNA; perfect complementary base pairing induces cleavage of mRNA, whereas several mismatches lead to translational arrest. Biological effects of microRNAs can be manipulated through the use of small interference RNAs (siRNAs) generated by chemical synthesis, or by cloning in molecular vectors. The cloning of a DNA insert in a molecular vector that will be transcribed into the corresponding siRNAs is an approach that has been developed using siRNA expression plasmids. These vectors contain DNA inserts designed with software to generate highly efficient siRNAs which will assemble into RNA-induced silencing complexes (RISC), and silence the target mRNA. In addition, the DNA inserts may be contained in cloning cassettes, and introduced in other molecular vectors. In this chapter we describe an attractive technology platform to silence cellular gene expression using specific siRNA expression plasmids, and evaluate its biological effect on target gene expression in human cervical cancer cells. PMID:25348304

  13. siRNA epidermal growth factor receptor silencing in U251 glioma cells

    Institute of Scientific and Technical Information of China (English)

    Chunsheng Kang; Zhiyong Zhang; Zhifan Jia; Qiang Huang; Guangxiu Wang; Mingzhe Qiu; Peiyu Pu

    2008-01-01

    BACKGROUND: Dicer, a large multidomain ribonuclease, is responsible for processing double-stranded RNAs (dsRNAs) to 20-bp-long small interfering RNAs (siRNAs), which act as effectors during RNA interference (RNAi). OBJECTIVE: To observe the efficacy of siRNA cocktails generated by recombinant human Dicer on the down-regulation of epidermal growth factor receptor (EGFR) expression in human glioma cells. DESIGN, TIME AND SETTING: The following in vitro experiment was performed at the Department of Neurosurgery, Tianjin Medical University General Hospital and Laboratory of Neuro-Oncology, Tianjin Neurological Institute. MATERIALS: Mini-RNA isolation kit, human placenta complimentary DNA (cDNA) was produced by Tiangen Biotech (Beijing, China), human glioblastoma U251-MG cells were produced by the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. METHODS: A PCR product from the human EGFR, which corresponded to the tyrosine kinase domain of the 3'-end fragment, was used as the T7-promotor for in vitro transcription, siRNA cocktails were generated by in vitro dicing of double stranded RNA. A total of 500, 250 and 125 μg siRNA cocktails were transiently transfected into U251 glioma cells through the use of the GeneSilencer. MAIN OUTCOME MEASURE: Expression of EGFR was detected by real-time PCR. RESULTS: The total PCR product of the human EGFR, corresponding to the tyrosine kinase domain, is approximately 680 bp in length. The PCR transcriptants included GCC leader sequences and a T7 promoter sequence, with a fragment of EGFR cDNA at the center. The T7 promoter was prepared for in vitro transcription of dsRNA. After dicing for 24 hours, the 21-nt siRNA cocktails were verified by 4% agarose gel. The difference between threshold cycle of a sample assay and threshold cycle of the corresponding endogenous reference (△ Ct) among parental U251 cells and cells transfected with different doses of siRNA cocktails were determined to be 3.06, 7.35, and 10

  14. Efficient transformation and artificial miRNA gene silencing in Lemna minor.

    Science.gov (United States)

    Cantó-Pastor, A; Mollá-Morales, A; Ernst, E; Dahl, W; Zhai, J; Yan, Y; Meyers, B C; Shanklin, J; Martienssen, R

    2015-01-01

    Despite rapid doubling time, simple architecture and ease of metabolic labelling, a lack of genetic tools in the Lemnaceae (duckweed) has impeded the full implementation of this organism as a model for biological research. Here, we present technologies to facilitate high-throughput genetic studies in duckweed. We developed a fast and efficient method for producing Lemna minor stable transgenic fronds via Agrobacterium-mediated transformation and regeneration from tissue culture. Additionally, we engineered an artificial microRNA (amiRNA) gene silencing system. We identified a Lemna gibba endogenous miR166 precursor and used it as a backbone to produce amiRNAs. As a proof of concept we induced the silencing of CH42, a magnesium chelatase subunit, using our amiRNA platform. Expression of CH42 in transgenic L. minor fronds was significantly reduced, which resulted in reduction of chlorophyll pigmentation. The techniques presented here will enable tackling future challenges in the biology and biotechnology of Lemnaceae. PMID:24989135

  15. Post-Transcriptional Silencing of Flavonol Synthase mRNA in Tobacco Leads to Fruits with Arrested Seed Set

    OpenAIRE

    Monika Mahajan; Paramvir Singh Ahuja; Sudesh Kumar Yadav

    2011-01-01

    Flavonoids are synthesized by phenylpropanoid pathway. They are known to participate in large number of physiological and biochemical processes in plants. Parthenocarpy and male sterility has earlier been reported by silencing chalcone synthase (CHS) encoding gene. Silencing of CHS has blocked the synthesis of most of useful flavonoids including flavan-3-ols and flavonols. Also, these studies could not identify whether parthenocarpy/male sterility were due to lack of flavan-3-ols or flavonols...

  16. The 2b protein of Asparagus virus 2 functions as an RNA silencing suppressor against systemic silencing to prove functional synteny with related cucumoviruses.

    Science.gov (United States)

    Shimura, Hanako; Masuta, Chikara; Yoshida, Naoto; Sueda, Kae; Suzuki, Masahiko

    2013-08-01

    Asparagus virus 2 (AV-2) is a member of the genus Ilarvirus in the family Bromoviridae. We cloned the coat protein (CP) and the 2b protein (2b) genes of AV-2 isolates from asparagus plants from various regions and found that the sequence for CP and for 2b was highly conserved among the isolates, suggesting that AV-2 from around the world is almost identical. We then made an AV-2 infectious clone by simultaneous inoculation with in vitro transcripts of RNAs 1-3 of AV-2 and in vitro-synthesized CP, which is necessary for initial infection. Because 2b of cucumoviruses in Bromoviridae can suppress systemic silencing as well as local silencing, we analyzed whether there is functional synteny of 2b between AV-2 and cucumovirus. Using the AV-2 infectious clone, we here provided first evidence that Ilarvirus 2b functions as an RNA silencing suppressor; AV-2 2b has suppressor activity against systemic silencing but not local silencing.

  17. Efficient oncogene silencing and metastasis inhibition via systemic delivery of siRNA.

    Science.gov (United States)

    Li, Shyh-Dar; Chono, Sumio; Huang, Leaf

    2008-05-01

    The selective delivery of small interfering RNA (siRNA) to metastatic tumors remains a challenging task. We have developed a nanoparticle (NP) formulation composed of siRNA, a carrier DNA, a polycationic peptide, and cationic liposomes. The NP was obtained by a self-assembling process, followed by surface modification with a polyethylene glycol (PEG)-conjugated ligand, anisamide. The NP was PEGylated and a ligand was presented to target sigma receptor-expressing murine melanoma cells, B16F10. The lung metastasis model was established by intravenous (i.v.) injection of the B16F10 cells into C57BL/6 mice. A mixture of siRNA against MDM2, c-myc, and vascular endothelial growth factor (VEGF) co-formulated in the targeted NP caused simultaneous silencing of each of the oncogenes in the metastatic nodules. Two consecutive i.v. injections of siRNA in the targeted NP significantly reduced the lung metastasis (approximately 70-80%) at a relatively low dose (0.45 mg/kg), whereas free siRNA and the nontargeted NP showed little effect. This targeted NP formulation significantly prolonged the mean survival time of the animals by 30% as compared to the untreated controls. At the therapeutic dose, the targeted NP showed little local and systemic immunotoxicity and did not decrease the body weight or damage the major organs.

  18. Artificial microRNAs (amiRNAs) engineering - On how microRNA-based silencing methods have affected current plant silencing research.

    Science.gov (United States)

    Sablok, Gaurav; Pérez-Quintero, Alvaro L; Hassan, Mehedi; Tatarinova, Tatiana V; López, Camilo

    2011-03-18

    In recent years, endogenous microRNAs have been described as important regulators of gene expression in eukaryotes. Artificial microRNAs (amiRNAs) represent a recently developed miRNA-based strategy to silence endogenous genes. amiRNAs can be created by exchanging the miRNA/miRNA(∗) sequence within a miRNA precursor with a sequence designed to match the target gene, this is possible as long as the secondary RNA structure of the precursor is kept intact. In this review, we summarize the basic methodologies to design amiRNAs and detail their applications in plants genetic functional studies as well as their potential for crops genetic improvement. PMID:21329663

  19. The potyviral suppressor of RNA silencing confers enhanced resistance to multiple pathogens

    International Nuclear Information System (INIS)

    Helper component-protease (HC-Pro) is a plant viral suppressor of RNA silencing, and transgenic tobacco expressing HC-Pro has increased susceptibility to a broad range of viral pathogens. Here we report that these plants also exhibit enhanced resistance to unrelated heterologous pathogens. Tobacco mosaic virus (TMV) infection of HC-Pro-expressing plants carrying the N resistance gene results in fewer and smaller lesions compared to controls without HC-Pro. The resistance to TMV is compromised but not eliminated by expression of nahG, which prevents accumulation of salicylic acid (SA), an important defense signaling molecule. HC-Pro-expressing plants are also more resistant to tomato black ring nepovirus (TBRV) and to the oomycete Peronospora tabacina. Enhanced TBRV resistance is SA-independent, whereas the response to P. tabacina is associated with early induction of markers characteristic of SA-dependent defense. Thus, a plant viral suppressor of RNA silencing enhances resistance to multiple pathogens via both SA-dependent and SA-independent mechanisms

  20. Analysis of the siRNA-Mediated Gene Silencing Process Targeting Three Homologous Genes Controlling Soybean Seed Oil Quality.

    Directory of Open Access Journals (Sweden)

    Sha Lu

    Full Text Available In the past decade, RNA silencing has gained significant attention because of its success in genomic scale research and also in the genetic improvement of crop plants. However, little is known about the molecular basis of siRNA processing in association with its target transcript. To reveal this process for improving hpRNA-mediated gene silencing in crop plants, the soybean GmFAD3 gene family was chosen as a test model. We analyzed RNAi mutant soybean lines in which three members of the GmFAD3 gene family were silenced. The silencing levels of FAD3A, FAD3B and FAD3C were correlated with the degrees of sequence homology between the inverted repeat of hpRNA and the GmFAD3 transcripts in the RNAi lines. Strikingly, transgenes in two of the three RNAi lines were heavily methylated, leading to a dramatic reduction of hpRNA-derived siRNAs. Small RNAs corresponding to the loop portion of the hairpin transcript were detected while much lower levels of siRNAs were found outside of the target region. siRNAs generated from the 318-bp inverted repeat were found to be diced much more frequently at stem sequences close to the loop and associated with the inferred cleavage sites on the target transcripts, manifesting "hot spots". The top candidate hpRNA-derived siRNA share certain sequence features with mature miRNA. This is the first comprehensive and detailed study revealing the siRNA-mediated gene silencing mechanism in crop plants using gene family GmFAD3 as a test model.

  1. Analysis of the siRNA-Mediated Gene Silencing Process Targeting Three Homologous Genes Controlling Soybean Seed Oil Quality.

    Science.gov (United States)

    Lu, Sha; Yin, Xiaoyan; Spollen, William; Zhang, Ning; Xu, Dong; Schoelz, James; Bilyeu, Kristin; Zhang, Zhanyuan J

    2015-01-01

    In the past decade, RNA silencing has gained significant attention because of its success in genomic scale research and also in the genetic improvement of crop plants. However, little is known about the molecular basis of siRNA processing in association with its target transcript. To reveal this process for improving hpRNA-mediated gene silencing in crop plants, the soybean GmFAD3 gene family was chosen as a test model. We analyzed RNAi mutant soybean lines in which three members of the GmFAD3 gene family were silenced. The silencing levels of FAD3A, FAD3B and FAD3C were correlated with the degrees of sequence homology between the inverted repeat of hpRNA and the GmFAD3 transcripts in the RNAi lines. Strikingly, transgenes in two of the three RNAi lines were heavily methylated, leading to a dramatic reduction of hpRNA-derived siRNAs. Small RNAs corresponding to the loop portion of the hairpin transcript were detected while much lower levels of siRNAs were found outside of the target region. siRNAs generated from the 318-bp inverted repeat were found to be diced much more frequently at stem sequences close to the loop and associated with the inferred cleavage sites on the target transcripts, manifesting "hot spots". The top candidate hpRNA-derived siRNA share certain sequence features with mature miRNA. This is the first comprehensive and detailed study revealing the siRNA-mediated gene silencing mechanism in crop plants using gene family GmFAD3 as a test model.

  2. Tobacco rattle virus vector: A rapid and transient means of silencing manduca sexta genes by plant mediated RNA interference.

    Directory of Open Access Journals (Sweden)

    Pavan Kumar

    Full Text Available BACKGROUND: RNAi can be achieved in insect herbivores by feeding them host plants stably transformed to express double stranded RNA (dsRNA of selected midgut-expressed genes. However, the development of stably transformed plants is a slow and laborious process and here we developed a rapid, reliable and transient method. We used viral vectors to produce dsRNA in the host plant Nicotiana attenuata to transiently silence midgut genes of the plant's lepidopteran specialist herbivore, Manduca sexta. To compare the efficacy of longer, undiced dsRNA for insect gene silencing, we silenced N. attenuata's dicer genes (NaDCL1- 4 in all combinations in a plant stably transformed to express dsRNA targeting an insect gene. METHODOLOGY/PRINCIPAL FINDINGS: Stable transgenic N. attenuata plants harboring a 312 bp fragment of MsCYP6B46 in an inverted repeat orientation (ir-CYP6B46 were generated to produce CYP6B46 dsRNA. After consuming these plants, transcripts of CYP6B46 were significantly reduced in M. sexta larval midguts. The same 312 bp cDNA was cloned in an antisense orientation into a TRV vector and Agro-infiltrated into N. attenuata plants. When larvae ingested these plants, similar reductions in CYP6B46 transcripts were observed without reducing transcripts of the most closely related MsCYP6B45. We used this transient method to rapidly silence the expression of two additional midgut-expressed MsCYPs. CYP6B46 transcripts were further reduced in midguts, when the larvae fed on ir-CYP6B46 plants transiently silenced for two combinations of NaDCLs (DCL1/3/4 and DCL2/3/4 and contained higher concentrations of longer, undiced CYP6B46 dsRNA. CONCLUSIONS: Both stable and transient expression of CYP6B46 dsRNA in host plants provides a specific and robust means of silencing this gene in M. sexta larvae, but the transient system is better suited for high throughput analyses. Transiently silencing NaDCLs in ir-CYP6B46 plants increased the silencing of MsCYP6B46

  3. Epitope-tagged protein-based artificial miRNA screens for optimized gene silencing in plants.

    Science.gov (United States)

    Li, Jian-Feng; Zhang, Dandan; Sheen, Jen

    2014-04-01

    Artificial miRNA (amiRNA) technology offers highly specific gene silencing in diverse plant species. The principal challenge in amiRNA application is to select potent amiRNAs from hundreds of bioinformatically designed candidates to enable maximal target gene silencing at the protein level. To address this issue, we developed the epitope-tagged protein-based amiRNA (ETPamir) screens, in which single or multiple potential target genes encoding epitope-tagged proteins are constitutively or inducibly coexpressed with individual amiRNA candidates in plant protoplasts. Accumulation of tagged proteins, detected by immunoblotting with commercial tag antibodies, inversely and quantitatively reflects amiRNA efficacy in vivo. The core procedure, from protoplast isolation to identification of optimal amiRNA, can be completed in 2-3 d. The ETPamir screens circumvent the limited availability of plant antibodies and the complexity of plant amiRNA silencing at target mRNA and/or protein levels. The method can be extended to verify predicted target genes for endogenous plant miRNAs.

  4. Effect of silencing of ATM expression by siRNA on radiosensitivity of human lung adenocarcinoma A549 cells

    International Nuclear Information System (INIS)

    Objective: To investigate the effect of silencing of ataxia-telangiectasia mutated (ATM) expression by plasmid-mediated RNA interference on the radiosensitivity of human lung adenocarcinoma A549 cells. Methods: Eukaryotic expression plasmid containing ATM small interfering RNA (siRNA) (pSilencer2.1-ATM), as well as pSilencer2.1-nonspecific, was constructed.Lung adenocarcinoma A549 cells were divided into positive group, negative group,and control group to be transfected with pSilencer2.1-ATM, pSilencer2.1-nonspecific, and no plasmid, respectively. The mRNA and protein expression of ATM was measured by RT-PCR and Western blot, respectively. The change in cell radiosensitivity was observed by colony-forming assay. Cell cycle and cell apoptosis were analyzed by flow cytometry. Results: The eukaryotic expression plasmid containing ATM siRNA was successfully constructed. The RT-PCR and Western blot demonstrated that the expression of ATM was down-regulated in the positive group. The sensitization enhancement ratios (D0 ratios) for the positive group and negative group were 1.50 and 1.01, respectively. The flow cytometry revealed that the proportions of A549 cells in G1 and G2/M phases were significantly lower in the positive group than in the control group (51.27% vs 61.85%, P = 0.012; 6.34% vs 10.91%, P = 0.008) and that the apoptosis rate was significantly higher in the positive group than in the control group and negative group (49.31% vs 13.58%, P = 0.000; 49.31% vs 13.17%, P = 0.000). Conclusions: Silencing of ATM expression may increase the radiosensitivity of human lung adenocarcinoma A549 cells, probably by affecting the cell cycle and promoting cell apoptosis. (authors)

  5. Naturally occurring endo-siRNA silences LINE-1 retrotransposons in human cells through DNA methylation.

    Science.gov (United States)

    Chen, Long; Dahlstrom, Jane E; Lee, Sung-Hun; Rangasamy, Danny

    2012-07-01

    Long interspersed nuclear element 1 (LINE-1) retrotransposons are mutagens that are capable of generating deleterious mutations by inserting themselves into genes and affecting gene function in the human genome. In normal cells, the activity of LINE-1 retrotransposon is mostly repressed, maintaining a stable genome structure. In contrast, cancer cells are characterized by aberrant expression of LINE-1 retrotransposons, which, in principle, have the potential to contribute to genomic instability. The mechanistic pathways that regulate LINE-1 expression remain unclear. Using deep-sequencing small RNA analysis, we identified a subset of differentially expressed endo-siRNAs that directly regulate LINE-1 expression. Detailed analyses suggest that these endo-siRNAs are significantly depleted in human breast cancer cells compared with normal breast cells. The overexpression of these endo-siRNAs in cancer cells markedly silences endogenous LINE-1 expression through increased DNA methylation of the LINE-1 5'-UTR promoter. The finding that endo-siRNAs can silence LINE-1 activity through DNA methylation suggests that a functional link exists between the expression of endo-siRNAs and LINE-1 retrotransposons in human cells.

  6. Growth Inhibition of Head and Neck Squamous Cell Carcinoma Cells by sgRNA Targeting the Cyclin D1 mRNA Based on TRUE Gene Silencing

    OpenAIRE

    Iizuka, Satoshi; Oridate, Nobuhiko; Nashimoto, Masayuki; Fukuda, Satoshi; Tamura, Masato

    2014-01-01

    Head and neck squamous cell carcinoma (HNSCC) exhibits increased expression of cyclin D1 (CCND1). Previous studies have shown a correlation between poor prognosis of HNSCC and cyclin D1 overexpression. tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing) is one of the RNA-mediated gene expression control technologies that have therapeutic potential. This technology is based on a unique enzymatic property of mammalian tRNase ZL, which is that it can cleave any target RNA at any...

  7. Mastering Dendrimer Self-Assembly for Efficient siRNA Delivery: From Conceptual Design to In Vivo Efficient Gene Silencing.

    Science.gov (United States)

    Chen, Chao; Posocco, Paola; Liu, Xiaoxuan; Cheng, Qiang; Laurini, Erik; Zhou, Jiehua; Liu, Cheng; Wang, Yang; Tang, Jingjie; Col, Valentina Dal; Yu, Tianzhu; Giorgio, Suzanne; Fermeglia, Maurizio; Qu, Fanqi; Liang, Zicai; Rossi, John J; Liu, Minghua; Rocchi, Palma; Pricl, Sabrina; Peng, Ling

    2016-07-01

    Self-assembly is a fundamental concept and a powerful approach in molecular science. However, creating functional materials with the desired properties through self-assembly remains challenging. In this work, through a combination of experimental and computational approaches, the self-assembly of small amphiphilic dendrons into nanosized supramolecular dendrimer micelles with a degree of structural definition similar to traditional covalent high-generation dendrimers is reported. It is demonstrated that, with the optimal balance of hydrophobicity and hydrophilicity, one of the self-assembled nanomicellar systems, totally devoid of toxic side effects, is able to deliver small interfering RNA and achieve effective gene silencing both in cells - including the highly refractory human hematopoietic CD34(+) stem cells - and in vivo, thus paving the way for future biomedical implementation. This work presents a case study of the concept of generating functional supramolecular dendrimers via self-assembly. The ability of carefully designed and gauged building blocks to assemble into supramolecular structures opens new perspectives on the design of self-assembling nanosystems for complex and functional applications. PMID:27244195

  8. Soilborne wheat mosaic virus (SBWMV 19K protein belongs to a class of cysteine rich proteins that suppress RNA silencing

    Directory of Open Access Journals (Sweden)

    Howard Amanda

    2005-03-01

    Full Text Available Abstract Amino acid sequence analyses indicate that the Soilborne wheat mosaic virus (SBWMV 19K protein is a cysteine-rich protein (CRP and shares sequence homology with CRPs derived from furo-, hordei-, peclu- and tobraviruses. Since the hordei- and pecluvirus CRPs were shown to be pathogenesis factors and/or suppressors of RNA silencing, experiments were conducted to determine if the SBWMV 19K CRP has similar activities. The SBWMV 19K CRP was introduced into the Potato virus X (PVX viral vector and inoculated to tobacco plants. The SBWMV 19K CRP aggravated PVX-induced symptoms and restored green fluorescent protein (GFP expression to GFP silenced tissues. These observations indicate that the SBWMV 19K CRP is a pathogenicity determinant and a suppressor of RNA silencing.

  9. Platinum Interference with siRNA Non-seed Regions Fine-Tunes Silencing Capacity

    DEFF Research Database (Denmark)

    Hedman, Hanna K; Kirpekar, Finn; Elmroth, Sofi K C

    2011-01-01

    Knowledge concerning the molecular mechanisms governing the influence of non-coding RNAs on protein production has emerged rapidly during the past decade. Today, two main research areas can be identified, one oriented toward the use of artificially introduced siRNAs for manipulation of gene...... of the platination sites, i.e., with altered degradation patterns compared to native RNAs. The MALDI-MS method was successfully used to further identify and characterize platinated RNA, with the naturally occurring platinum isotopic patterns serving as sensitive fingerprints for metalated sites. Expression assays...... all confirm biological activity of antisense-platinated siRNAs, here with platination sites located outside of the seed region. A significant reduction of silencing capacity was observed as a general trend, however. Of the two complexes studied, oxaliplatin exhibits the larger influence, thus...

  10. RNA polymerase V targets transcriptional silencing components to promoters of protein-coding genes.

    Science.gov (United States)

    Zheng, Qi; Rowley, M Jordan; Böhmdorfer, Gudrun; Sandhu, Davinder; Gregory, Brian D; Wierzbicki, Andrzej T

    2013-01-01

    Transcriptional gene silencing controls transposons and other repetitive elements through RNA-directed DNA methylation (RdDM) and heterochromatin formation. A key component of the Arabidopsis RdDM pathway is ARGONAUTE4 (AGO4), which associates with siRNAs to mediate DNA methylation. Here, we show that AGO4 preferentially targets transposable elements embedded within promoters of protein-coding genes. This pattern of AGO4 binding cannot be simply explained by the sequences of AGO4-bound siRNAs; instead, AGO4 binding to specific gene promoters is also mediated by long non-coding RNAs (lncRNAs) produced by RNA polymerase V. lncRNA-mediated AGO4 binding to gene promoters directs asymmetric DNA methylation to these genomic regions and is involved in regulating the expression of targeted genes. Finally, AGO4 binding overlaps sites of DNA methylation affected by the biotic stress response. Based on these findings, we propose that the targets of AGO4-directed RdDM are regulatory units responsible for controlling gene expression under specific environmental conditions.

  11. The Promise and Challenge of Therapeutic MicroRNA Silencing in Diabetes and Metabolic Diseases.

    Science.gov (United States)

    Sethupathy, Praveen

    2016-06-01

    MicroRNAs (miRNAs) are small, non-coding, RNA molecules that regulate gene expression. They have a long evolutionary history and are found in plants, viruses, and animals. Although initially discovered in 1993 in Caenorhabditis elegans, they were not appreciated as widespread and abundant gene regulators until the early 2000s. Studies in the last decade have found that miRNAs confer phenotypic robustness in the face of environmental perturbation, may serve as diagnostic and prognostic indicators of disease, underlie the pathobiology of a wide array of complex disorders, and represent compelling therapeutic targets. Pre-clinical studies in animal models have demonstrated that pharmacologic manipulation of miRNAs, mostly in the liver, can modulate metabolic phenotypes and even reverse the course of insulin resistance and diabetes. There is cautious optimism in the field about miRNA-based therapies for diabetes, several of which are already in various stages of clinical trials. This review will highlight both the promise and the most pressing challenges of therapeutic miRNA silencing in diabetes and related conditions. PMID:27112956

  12. Growth inhibition of head and neck squamous cell carcinoma cells by sgRNA targeting the cyclin D1 mRNA based on TRUE gene silencing.

    Directory of Open Access Journals (Sweden)

    Satoshi Iizuka

    Full Text Available Head and neck squamous cell carcinoma (HNSCC exhibits increased expression of cyclin D1 (CCND1. Previous studies have shown a correlation between poor prognosis of HNSCC and cyclin D1 overexpression. tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing is one of the RNA-mediated gene expression control technologies that have therapeutic potential. This technology is based on a unique enzymatic property of mammalian tRNase ZL, which is that it can cleave any target RNA at any desired site by recognizing a pre-tRNA-like complex formed between the target RNA and an artificial small guide RNA (sgRNA. In this study, we designed several sgRNAs targeting human cyclin D1 mRNA to examine growth inhibition of HNSCC cells. Transfection of certain sgRNAs decreased levels of cyclin D1 mRNA and protein in HSC-2 and HSC-3 cells, and also inhibited their proliferation. The combination of these sgRNAs and cisplatin showed more than additive inhibition of cancer cell growth. These findings demonstrate that TRUE gene silencing of cyclin D1 leads to inhibition of the growth of HNSCC cells and suggest that these sgRNAs alone or combined with cisplatin may be a useful new therapy for HNSCCs.

  13. Growth inhibition of head and neck squamous cell carcinoma cells by sgRNA targeting the cyclin D1 mRNA based on TRUE gene silencing.

    Science.gov (United States)

    Iizuka, Satoshi; Oridate, Nobuhiko; Nashimoto, Masayuki; Fukuda, Satoshi; Tamura, Masato

    2014-01-01

    Head and neck squamous cell carcinoma (HNSCC) exhibits increased expression of cyclin D1 (CCND1). Previous studies have shown a correlation between poor prognosis of HNSCC and cyclin D1 overexpression. tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing) is one of the RNA-mediated gene expression control technologies that have therapeutic potential. This technology is based on a unique enzymatic property of mammalian tRNase ZL, which is that it can cleave any target RNA at any desired site by recognizing a pre-tRNA-like complex formed between the target RNA and an artificial small guide RNA (sgRNA). In this study, we designed several sgRNAs targeting human cyclin D1 mRNA to examine growth inhibition of HNSCC cells. Transfection of certain sgRNAs decreased levels of cyclin D1 mRNA and protein in HSC-2 and HSC-3 cells, and also inhibited their proliferation. The combination of these sgRNAs and cisplatin showed more than additive inhibition of cancer cell growth. These findings demonstrate that TRUE gene silencing of cyclin D1 leads to inhibition of the growth of HNSCC cells and suggest that these sgRNAs alone or combined with cisplatin may be a useful new therapy for HNSCCs. PMID:25437003

  14. A Pooled shRNA Screen Identifies Rbm15, Spen, and Wtap as Factors Required for Xist RNA-Mediated Silencing

    Directory of Open Access Journals (Sweden)

    Benoit Moindrot

    2015-07-01

    Full Text Available X-chromosome inactivation is the process that evolved in mammals to equalize levels of X-linked gene expression in XX females relative to XY males. Silencing of a single X chromosome in female cells is mediated by the non-coding RNA Xist. Although progress has been made toward identifying factors that function in the maintenance of X inactivation, the primary silencing factors are largely undefined. We developed an shRNA screening strategy to produce a ranked list of candidate primary silencing factors. Validation experiments performed on several of the top hits identified the SPOC domain RNA binding proteins Rbm15 and Spen and Wtap, a component of the m6A RNA methyltransferase complex, as playing an important role in the establishment of Xist-mediated silencing. Localization analysis using super-resolution 3D-SIM microscopy demonstrates that these factors co-localize with Xist RNA within the nuclear matrix subcompartment, consistent with a direct interaction.

  15. DsRNA-mediated silencing of Nudix hydrolase in Trichinella spiralis inhibits the larval invasion and survival in mice.

    Science.gov (United States)

    Zhang, Shuai Bing; Jiang, Peng; Wang, Zhong Quan; Long, Shao Rong; Liu, Ruo Dan; Zhang, Xi; Yang, Wei; Ren, Hui Jun; Cui, Jing

    2016-03-01

    The aim of this study was to investigate the functions of Trichinella spiralis Nudix hydrolase (TsNd) during the larval invasion of intestinal epithelial cells (IECs), development and survival in host by RNAi. The TsNd-specific double-stranded RNA (dsRNA) was designed to silence the expression of TsNd in T. spiralis larvae. DsRNA were delivered to the larvae by soaking incubation or electroporation. Silencing effect of TsNd transcription and expression was determined by real-time PCR and Western blotting, respectively. The infectivity of larvae treated with dsRNA was investigated by the in vitro larval invasion of IECs and experimental infection in mice. After being soaked with 40 ng/μl of dsRNA-TsNd, the transcription and expression level of TsNd gene was inhibited 65.8% and 56.4%, respectively. After being electroporated with 40 ng/μl of dsRNA-TsNd, the transcription and expression level of TsNd gene was inhibited 74.2% and 58.2%, respectively. Silencing TsNd expression by both soaking and electroporation inhibited significantly the larval invasion of IECs in a dose-dependent manner (r1 = -0.96798, r2 = -0.98707). Compared with the mice inoculated with untreated larvae, mice inoculated with larvae soaked with TsNd dsRNA displayed a 49.9% reduction in adult worms and 39.9% reduction in muscle larvae, while mice inoculated with larvae electroporated with TsNd dsRNA displayed a 83.4% reduction in adult worms and 69.5% reduction in muscle larvae, indicating that electroporation has a higher efficiency than soaking in inhibiting the larval development and survival in mice. Our results showed that silencing TsNd expression in T. spiralis inhibited significantly the larval invasion and survival in host. PMID:26778819

  16. Prolonged efficiency of siRNA-mediated gene silencing in primary cultures of human preadipocytes and adipocytes

    OpenAIRE

    Lee, Mi-Jeong; Pickering, R. Taylor; Puri, Vishwajeet

    2013-01-01

    Objective Primary human preadipocytes and differentiated adipocytes in culture are valuable cell culture systems to study adipogenesis and adipose function in relation to human adipose biology. To use these systems for mechanistic studies, we studied siRNA-mediated knockdown of genes for its effectiveness. Design and Methods Methods were developed to effectively deliver siRNA to for gene silencing in primary preadipocytes isolated from human subcutaneous adipose tissue and newly-differentiate...

  17. DICER-LIKE2 plays a primary role in transitive silencing of transgenes in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Sizolwenkosi Mlotshwa

    Full Text Available Dicer-like (DCL enzymes play a pivotal role in RNA silencing in plants, processing the long double-stranded RNA (dsRNA that triggers silencing into the primary short interfering RNAs (siRNAs that mediate it. The siRNA population can be augmented and silencing amplified via transitivity, an RNA-dependent RNA polymerase (RDR-dependent pathway that uses the target RNA as substrate to generate secondary siRNAs. Here we report that Arabidopsis DCL2-but not DCL4-is required for transitivity in cell-autonomous, post-transcriptional silencing of transgenes. An insertion mutation in DCL2 blocked sense transgene-induced silencing and eliminated accumulation of the associated RDR-dependent siRNAs. In hairpin transgene-induced silencing, the dcl2 mutation likewise eliminated accumulation of secondary siRNAs and blocked transitive silencing, but did not block silencing mediated by primary siRNAs. Strikingly, in all cases, the dcl2 mutation eliminated accumulation of all secondary siRNAs, including those generated by other DCL enzymes. In contrast, mutations in DCL4 promoted a dramatic shift to transitive silencing in the case of the hairpin transgene and enhanced silencing induced by the sense transgene. Suppression of hairpin and sense transgene silencing by the P1/HC-Pro and P38 viral suppressors was associated with elimination of secondary siRNA accumulation, but the suppressors did not block processing of the stem of the hairpin transcript into primary siRNAs. Thus, these viral suppressors resemble the dcl2 mutation in their effects on siRNA biogenesis. We conclude that DCL2 plays an essential, as opposed to redundant, role in transitive silencing of transgenes and may play a more important role in silencing of viruses than currently thought.

  18. Epigenetic silencing of monoallelically methylated miRNA loci in precancerous colorectal lesions.

    Science.gov (United States)

    Menigatti, M; Staiano, T; Manser, C N; Bauerfeind, P; Komljenovic, A; Robinson, M; Jiricny, J; Buffoli, F; Marra, G

    2013-01-01

    Epigenetic silencing of protein-encoding genes is common in early-stage colorectal tumorigenesis. Less is known about the methylation-mediated silencing of genes encoding microRNAs (miRNAs), which are also important epigenetic modulators of gene expression. Using quantitative PCR, we identified 56 miRNAs that were expressed in normal colorectal mucosa and in HT29 colorectal cancer cells treated with demethylating agents but not in untreated HT29 cells, suggesting that they probably undergo methylation-induced silencing during colorectal tumorigenesis. One of these, miR-195, had recently been reported to be underexpressed in colorectal cancers and to exert tumor-suppressor effects in colorectal cancer cells. We identified the transcription start site (TSS) for primary miRNA (pri-miR)-497/195, the primary precursor that yields miR-195 and another candidate on our list, miR-497, and a single CpG island upstream to the TSS, which controls expression of both miRNAs. Combined bisulfite restriction analysis and bisulfite genomic sequencing studies revealed monoallelic methylation of this island in normal colorectal mucosa (50/50 samples) and full methylation in most colorectal adenomas (38/50; 76%). The hypermethylated precancerous lesions displayed significantly downregulated expression of both miRNAs. Similar methylation patterns were observed at two known imprinted genes, MEG3 and GNAS-AS1, which encode several of the 56 miRNAs on our list. Imprinting at these loci was lost in over half the adenomas (62% at MEG3 and 52% at GNAS-AS1). Copy-number alterations at MEG3, GNAS-AS1 and pri-miR-497/195, which are frequent in colorectal cancers, were less common in adenomas and confined to tumors displaying differential methylation at the involved locus. Our data show that somatically acquired, epigenetic changes at monoallelically methylated regions encoding miRNAs are relatively frequent in sporadic colorectal adenomas and might contribute to the onset and progression of

  19. The RNA silencing enzyme RNA polymerase v is required for plant immunity.

    Directory of Open Access Journals (Sweden)

    Ana López

    2011-12-01

    Full Text Available RNA-directed DNA methylation (RdDM is an epigenetic control mechanism driven by small interfering RNAs (siRNAs that influence gene function. In plants, little is known of the involvement of the RdDM pathway in regulating traits related to immune responses. In a genetic screen designed to reveal factors regulating immunity in Arabidopsis thaliana, we identified NRPD2 as the OVEREXPRESSOR OF CATIONIC PEROXIDASE 1 (OCP1. NRPD2 encodes the second largest subunit of the plant-specific RNA Polymerases IV and V (Pol IV and Pol V, which are crucial for the RdDM pathway. The ocp1 and nrpd2 mutants showed increases in disease susceptibility when confronted with the necrotrophic fungal pathogens Botrytis cinerea and Plectosphaerella cucumerina. Studies were extended to other mutants affected in different steps of the RdDM pathway, such as nrpd1, nrpe1, ago4, drd1, rdr2, and drm1drm2 mutants. Our results indicate that all the mutants studied, with the exception of nrpd1, phenocopy the nrpd2 mutants; and they suggest that, while Pol V complex is required for plant immunity, Pol IV appears dispensable. Moreover, Pol V defective mutants, but not Pol IV mutants, show enhanced disease resistance towards the bacterial pathogen Pseudomonas syringae DC3000. Interestingly, salicylic acid (SA-mediated defenses effective against PsDC3000 are enhanced in Pol V defective mutants, whereas jasmonic acid (JA-mediated defenses that protect against fungi are reduced. Chromatin immunoprecipitation analysis revealed that, through differential histone modifications, SA-related defense genes are poised for enhanced activation in Pol V defective mutants and provide clues for understanding the regulation of gene priming during defense. Our results highlight the importance of epigenetic control as an additional layer of complexity in the regulation of plant immunity and point towards multiple components of the RdDM pathway being involved in plant immunity based on genetic evidence

  20. AGO6 functions in RNA-mediated transcriptional gene silencing in shoot and root meristems in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Changho Eun

    Full Text Available RNA-directed DNA methylation (RdDM is a small interfering RNA (siRNA-mediated epigenetic modification that contributes to transposon silencing in plants. RdDM requires a complex transcriptional machinery that includes specialized RNA polymerases, named Pol IV and Pol V, as well as chromatin remodelling proteins, transcription factors, RNA binding proteins, and other plant-specific proteins whose functions are not yet clarified. In Arabidopsis thaliana, DICER-LIKE3 and members of the ARGONAUTE4 group of ARGONAUTE (AGO proteins are involved, respectively, in generating and using 24-nt siRNAs that trigger methylation and transcriptional gene silencing of homologous promoter sequences. AGO4 is the main AGO protein implicated in the RdDM pathway. Here we report the identification of the related AGO6 in a forward genetic screen for mutants defective in RdDM and transcriptional gene silencing in shoot and root apical meristems in Arabidopsis thaliana. The identification of AGO6, and not AGO4, in our screen is consistent with the primary expression of AGO6 in shoot and root growing points.

  1. The siRNA-mediated silencing of Trichinella spiralis nudix hydrolase results in reduction of larval infectivity.

    Science.gov (United States)

    Wang, Zhong Quan; Zhang, Shuai Bing; Jiang, Peng; Liu, Ruo Dan; Long, Shao Rong; Zhang, Xi; Ren, Hui Jun; Cui, Jing

    2015-09-01

    Previous studies showed that Trichinella spiralis Nudix hydrolase (TsNd) bound to intestinal epithelial cells (IECs), and vaccination of mice with rTsNd or TsNd DNA produced a partial protective immunity against T. spiralis infection. In this study, three TsNd specific small interfering RNA (siRNA) were designed to silence the expression of TsNd in T. spiralis larvae. SiRNAs were delivered to the larvae by electroporation. Silencing effect of TsNd transcription and expression was determined by real-time PCR and Western blotting, respectively. The infectivity of the larvae treated with siRNA was investigated by the in vitro larval invasion of IECs and experimental infection in mice. The results showed that siRNAs were efficiently delivered into T. spiralis larvae through electroporation. Real-time PCR and Western blotting showed that transcription and expression level of TsNd gene was inhibited 73.3 and 76.7 %, respectively, after being electroporated with 2 μM of siRNA-275 for 1 day. Silencing TsNd expression inhibited significantly the larval invasion of IECs (P < 0.01) and was in a dose-dependent manner (r = -0.97941). The mice with infected larvae treated with TsNd siRNA displayed a 63.6 % reduction in intestinal adult worms and 68.8 % reduction in muscle larval burden compared with mice infected with control siRNA-treated larvae. Our results showed that silencing TsNd expression in T. spiralis significantly reduced the larval infectivity and survival in host. PMID:26231837

  2. Loss of dsRNA-based gene silencing in Entamoeba histolytica: implications for approaches to genetic analysis.

    Science.gov (United States)

    MacFarlane, Ryan C; Singh, Upinder

    2008-06-01

    The ability to regulate gene expression in the protozoan parasite Entamoeba histolytica is critical in determining gene function. We previously published that expression of dsRNA specific to E. histolytica serine threonine isoleucine rich protein (EhSTIRP) resulted in reduction of gene expression [MacFarlane, R.C., Singh, U., 2007. Identification of an Entamoeba histolytica serine, threonine, isoleucine, rich protein with roles in adhesion and cytotoxicity. Eukaryotic Cell 6, 2139-2146]. However, after approximately one year of continuous drug selection, the expression of EhSTIRP reverted to wild-type levels. We confirmed that the parasites (i) contained the appropriate dsRNA plasmid, (ii) were not contaminated with other plasmids, (iii) the drug selectable marker was functional, and (iv) sequenced the dsRNA portion of the construct. This work suggests that in E. histolytica long term cultivation of parasites expressing dsRNA can lead to the loss of dsRNA based silencing through the selection of "RNAi" negative parasites. Thus, users of the dsRNA silencing approach should proceed with caution and regularly confirm gene down regulation. The development and use of constructs for inducible expression of dsRNA may help alleviate this potential problem. PMID:18346737

  3. Gene Overexpression and RNA Silencing Tools for the Genetic Manipulation of the S-(+-Abscisic Acid Producing Ascomycete Botrytis cinerea

    Directory of Open Access Journals (Sweden)

    Zhong-Tao Ding

    2015-05-01

    Full Text Available The phytopathogenic ascomycete Botrytis cinerea produces several secondary metabolites that have biotechnical significance and has been particularly used for S-(+-abscisic acid production at the industrial scale. To manipulate the expression levels of specific secondary metabolite biosynthetic genes of B. cinerea with Agrobacterium tumefaciens-mediated transformation system, two expression vectors (pCBh1 and pCBg1 with different selection markers and one RNA silencing vector, pCBSilent1, were developed with the In-Fusion assembly method. Both expression vectors were highly effective in constitutively expressing eGFP, and pCBSilent1 effectively silenced the eGFP gene in B. cinerea. Bcaba4, a gene suggested to participate in ABA biosynthesis in B. cinerea, was then targeted for gene overexpression and RNA silencing with these reverse genetic tools. The overexpression of bcaba4 dramatically induced ABA formation in the B. cinerea wild type strain Bc-6, and the gene silencing of bcaba4 significantly reduced ABA-production in an ABA-producing B. cinerea strain.

  4. The RNA-binding Protein TDP-43 Selectively Disrupts MicroRNA-1/206 Incorporation into the RNA-induced Silencing Complex*♦

    Science.gov (United States)

    King, Isabelle N.; Yartseva, Valeria; Salas, Donaldo; Kumar, Abhishek; Heidersbach, Amy; Ando, D. Michael; Stallings, Nancy R.; Elliott, Jeffrey L.; Srivastava, Deepak; Ivey, Kathryn N.

    2014-01-01

    MicroRNA (miRNA) maturation is regulated by interaction of particular miRNA precursors with specific RNA-binding proteins. Following their biogenesis, mature miRNAs are incorporated into the RNA-induced silencing complex (RISC) where they interact with mRNAs to negatively regulate protein production. However, little is known about how mature miRNAs are regulated at the level of their activity. To address this, we screened for proteins differentially bound to the mature form of the miR-1 or miR-133 miRNA families. These muscle-enriched, co-transcribed miRNA pairs cooperate to suppress smooth muscle gene expression in the heart. However, they also have opposing roles, with the miR-1 family, composed of miR-1 and miR-206, promoting myogenic differentiation, whereas miR-133 maintains the progenitor state. Here, we describe a physical interaction between TDP-43, an RNA-binding protein that forms aggregates in the neuromuscular disease, amyotrophic lateral sclerosis, and the miR-1, but not miR-133, family. Deficiency of the TDP-43 Drosophila ortholog enhanced dmiR-1 activity in vivo. In mammalian cells, TDP-43 limited the activity of both miR-1 and miR-206, but not the miR-133 family, by disrupting their RISC association. Consistent with TDP-43 dampening miR-1/206 activity, protein levels of the miR-1/206 targets, IGF-1 and HDAC4, were elevated in TDP-43 transgenic mouse muscle. This occurred without corresponding Igf-1 or Hdac4 mRNA increases and despite higher miR-1 and miR-206 expression. Our findings reveal that TDP-43 negatively regulates the activity of the miR-1 family of miRNAs by limiting their bioavailability for RISC loading and suggest a processing-independent mechanism for differential regulation of miRNA activity. PMID:24719334

  5. Oral cancer cells may rewire alternative metabolic pathways to survive from siRNA silencing of metabolic enzymes

    International Nuclear Information System (INIS)

    Cancer cells may undergo metabolic adaptations that support their growth as well as drug resistance properties. The purpose of this study is to test if oral cancer cells can overcome the metabolic defects introduced by using small interfering RNA (siRNA) to knock down their expression of important metabolic enzymes. UM1 and UM2 oral cancer cells were transfected with siRNA to transketolase (TKT) or siRNA to adenylate kinase (AK2), and Western blotting was used to confirm the knockdown. Cellular uptake of glucose and glutamine and production of lactate were compared between the cancer cells with either TKT or AK2 knockdown and those transfected with control siRNA. Statistical analysis was performed with student T-test. Despite the defect in the pentose phosphate pathway caused by siRNA knockdown of TKT, the survived UM1 or UM2 cells utilized more glucose and glutamine and secreted a significantly higher amount of lactate than the cells transferred with control siRNA. We also demonstrated that siRNA knockdown of AK2 constrained the proliferation of UM1 and UM2 cells but similarly led to an increased uptake of glucose/glutamine and production of lactate by the UM1 or UM2 cells survived from siRNA silencing of AK2. Our results indicate that the metabolic defects introduced by siRNA silencing of metabolic enzymes TKT or AK2 may be compensated by alternative feedback metabolic mechanisms, suggesting that cancer cells may overcome single defective pathways through secondary metabolic network adaptations. The highly robust nature of oral cancer cell metabolism implies that a systematic medical approach targeting multiple metabolic pathways may be needed to accomplish the continued improvement of cancer treatment

  6. The bifunctional abiotic stress signalling regulator and endogenous RNA silencing suppressor FIERY1 is required for lateral root formation

    KAUST Repository

    Chen, Hao

    2010-09-28

    The Arabidopsis FIERY1 (FRY1) locus was originally identified as a negative regulator of stress-responsive gene expression and later shown to be required for suppression of RNA silencing. In this study we discovered that the FRY1 locus also regulates lateral root formation. Compared with the wild type, fry1 mutant seedlings generated significantly fewer lateral roots under normal growth conditions and also exhibited a dramatically reduced sensitivity to auxin in inducing lateral root initiation. Using transgenic plants that overexpress a yeast homolog of FRY1 that possesses only the 3\\', 5\\'-bisphosphate nucleotidase activity but not the inositol 1-phosphatase activity, we demonstrated that the lateral root phenotypes in fry1 result from loss of the nucleotidase activity. Furthermore, a T-DNA insertion mutant of another RNA silencing suppressor, XRN4 (but not XRN2 or XRN3), which is an exoribonuclease that is inhibited by the substrate of the FRY1 3\\', 5\\'-bisphosphate nucleotidase, exhibits similar lateral root defects. Although fry1 and xrn4 exhibited reduced sensitivity to ethylene, our experiments demonstrated that restoration of ethylene sensitivity in the fry1 mutant is not sufficient to rescue the lateral root phenotypes of fry1. Our results indicate that RNA silencing modulated by FRY1 and XRN4 plays an important role in shaping root architecture. © 2010 Blackwell Publishing Ltd.

  7. The NS3 protein of Rice hoja blanca tenuivirus suppresses RNA silencing in plant and insect hosts by efficiently binding both siRNAs and miRNAs.

    Science.gov (United States)

    Hemmes, Hans; Lakatos, Lóránt; Goldbach, Rob; Burgyán, József; Prins, Marcel

    2007-07-01

    RNA silencing plays a key role in antiviral defense as well as in developmental processes in plants and insects. Negative strand RNA viruses such as the plant virus Rice hoja blanca tenuivirus (RHBV) replicate in plants and in their insect transmission vector. Like most plant-infecting viruses, RHBV encodes an RNA silencing suppressor, the NS3 protein, and here it is demonstrated that this protein is capable of suppressing RNA silencing in both plants and insect cells. Biochemical analyses showed that NS3 efficiently binds siRNA as well as miRNA molecules. Binding of NS3 is greatly influenced by the size of small RNA molecules, as 21 nucleotide (nt) siRNA molecules are bound > 100 times more efficiently than 26 nt species. Competition assays suggest that the activity of NS3 is based on binding to siRNAs prior to strand separation during the assembly of the RNA-induced silencing complex. In addition, NS3 has a high affinity for miRNA/miRNA* duplexes, indicating that its activity might also interfere with miRNA-regulated gene expression in both insects and plants. PMID:17513697

  8. An SGS3-like protein functions in RNA-directed DNA methylation and transcriptional gene silencing in Arabidopsis

    KAUST Repository

    Zheng, Zhimin

    2010-01-06

    RNA-directed DNA methylation (RdDM) is an important epigenetic mechanism for silencing transgenes and endogenous repetitive sequences such as transposons. The RD29A promoter-driven LUCIFERASE transgene and its corresponding endogenous RD29A gene are hypermethylated and silenced in the Arabidopsis DNA demethylase mutant ros1. By screening for second-site suppressors of ros1, we identified the RDM12 locus. The rdm12 mutation releases the silencing of the RD29A-LUC transgene and the endogenous RD29A gene by reducing the promoter DNA methylation. The rdm12 mutation also reduces DNA methylation at endogenous RdDM target loci, including transposons and other repetitive sequences. In addition, the rdm12 mutation affects the levels of small interfering RNAs (siRNAs) from some of the RdDM target loci. RDM12 encodes a protein with XS and coiled-coil domains, and is similar to SGS3, which is a partner protein of RDR6 and can bind to double-stranded RNAs with a 5′ overhang, and is required for several post-transcriptional gene silencing pathways. Our results show that RDM12 is a component of the RdDM pathway, and suggest that RdDM may involve double-stranded RNAs with a 5′ overhang and the partnering between RDM12 and RDR2. © 2010 Blackwell Publishing Ltd.

  9. Elicitation of hypersensitive responses in Nicotiana glutinosa by the suppressor of RNA silencing protein P0 from poleroviruses.

    Science.gov (United States)

    Wang, Ken-Der; Empleo, Roman; Nguyen, Tan Tri V; Moffett, Peter; Sacco, Melanie Ann

    2015-06-01

    Plant disease resistance (R) proteins that confer resistance to viruses recognize viral gene products with diverse functions, including viral suppressors of RNA silencing (VSRs). The P0 protein from poleroviruses is a VSR that targets the ARGONAUTE1 (AGO1) protein for degradation, thereby disrupting RNA silencing and antiviral defences. Here, we report resistance against poleroviruses in Nicotiana glutinosa directed against Turnip yellows virus (TuYV) and Potato leafroll virus (PLRV). The P0 proteins from TuYV (P0(T) (u) ), PLRV (P0(PL) ) and Cucurbit aphid-borne yellows virus (P0(CA) ) were found to elicit a hypersensitive response (HR) in N. glutinosa accession TW59, whereas other accessions recognized P0(PL) only. Genetic analysis showed that recognition of P0(T) (u) by a resistance gene designated RPO1 (Resistance to POleroviruses 1) is inherited as a dominant allele. Expression of P0 from a Potato virus X (PVX) expression vector transferred recognition to the recombinant virus on plants expressing RPO1, supporting P0 as the unique Polerovirus factor eliciting resistance. The induction of HR required a functional P0 protein, as P0(T) (u) mutants with substitutions in the F-box motif that abolished VSR activity were unable to elicit HR. We surmised that the broad P0 recognition seen in TW59 and the requirement for the F-box protein motif could indicate detection of P0-induced AGO1 degradation and disruption of RNA silencing; however, other viral silencing suppressors, including the PVX P25 that also causes AGO1 degradation, failed to elicit HR in N. glutinosa. Investigation of P0 elicitation of RPO1 could provide insight into P0 activities within the cell that trigger resistance.

  10. Expression of geminiviral AC2 RNA silencing suppressor changes sugar and jasmonate responsive gene expression in transgenic tobacco plants

    Directory of Open Access Journals (Sweden)

    Soitamo Arto J

    2012-11-01

    Full Text Available Abstract Background RNA-silencing is a conserved gene regulation and surveillance machinery, which in plants, is also used as major defence mechanism against viruses. Various virus-specific dsRNA structures are recognized by the silencing machinery leading to degradation of the viral RNAs or, as in case of begomoviruses, to methylation of their DNA genomes. Viruses produce specific RNA silencing suppressor (RSS proteins to prevent these host defence mechanisms, and as these interfere with the silencing machinery they also disturb the endogenous silencing reactions. In this paper, we describe how expression of AC2 RSS, derived from African cassava mosaic geminivirus changes transcription profile in tobacco (Nicotiana tabacum leaves and in flowers. Results Expression of AC2 RSS in transgenic tobacco plants induced clear phenotypic changes both in leaves and in flowers. Transcriptomes of these plants were strongly altered, with total of 1118 and 251 differentially expressed genes in leaves and flowers, respectively. The three most up-regulated transcript groups were related to stress, cell wall modifications and signalling, whereas the three most down-regulated groups were related to translation, photosynthesis and transcription. It appears that many of the gene expression alterations appeared to be related to enhanced biosynthesis of jasmonate and ethylene, and consequent enhancement of the genes and pathways that are regulated by these hormones, or to the retrograde signalling caused by the reduced photosynthetic activity and sugar metabolism. Comparison of these results to a previous transcriptional profiling of HC-Pro RSS-expressing plants revealed that some of same genes were induced by both RSSs, but their expression levels were typically higher in AC2 than in HC-Pro RSS expressing plants. All in all, a large number of transcript alterations were found to be specific to each of the RSS expressing transgenic plants. Conclusions AC2 RSS in

  11. Reversal of microRNA-150 silencing disadvantages crizotinib-resistant NPM-ALK(+) cell growth.

    Science.gov (United States)

    Hoareau-Aveilla, Coralie; Valentin, Thibaud; Daugrois, Camille; Quelen, Cathy; Mitou, Géraldine; Quentin, Samuel; Jia, Jinsong; Spicuglia, Salvatore; Ferrier, Pierre; Ceccon, Monica; Giuriato, Sylvie; Gambacorti-Passerini, Carlo; Brousset, Pierre; Lamant, Laurence; Meggetto, Fabienne

    2015-09-01

    The regulatory microRNA miR-150 is involved in the development of hemopathies and is downregulated in T-lymphomas, such as anaplastic large-cell lymphoma (ALCL) tumors. ALCL is defined by the presence or absence of translocations that activate the anaplastic lymphoma kinase (ALK), with nucleophosmin-ALK (NPM-ALK) fusions being the most common. Here, we compared samples of primary NPM-ALK(+) and NPM-ALK(-) ALCL to investigate the role of miR-150 downstream of NPM-ALK. Methylation of the MIR150 gene was substantially elevated in NPM-ALK(+) biopsies and correlated with reduced miR-150 expression. In NPM-ALK(+) cell lines, DNA hypermethylation-mediated miR-150 repression required ALK-dependent pathways, as ALK inhibition restored miR-150 expression. Moreover, epigenetic silencing of miR-150 was due to the activation of STAT3, a major downstream substrate of NPM-ALK, in cooperation with DNA methyltransferase 1 (DNMT1). Accordingly, miR-150 repression was turned off following treatment with the DNMT inhibitor, decitabine. In murine NPM-ALK(+) xenograft models, miR-150 upregulation induced antineoplastic activity. Treatment of crizotinib-resistant NPM-ALK(+) KARPAS-299-CR06 cells with decitabine or ectopic miR-150 expression reduced viability and growth. Altogether, our results suggest that hypomethylating drugs, alone or in combination with other agents, may benefit ALK(+) patients harboring tumors resistant to crizotinib and other anti-ALK tyrosine kinase inhibitors (TKIs). Moreover, these results support further work on miR-150 in these and other ALK(+) malignancies.

  12. Reversal of microRNA-150 silencing disadvantages crizotinib-resistant NPM-ALK(+) cell growth

    Science.gov (United States)

    Hoareau-Aveilla, Coralie; Valentin, Thibaud; Daugrois, Camille; Quelen, Cathy; Mitou, Géraldine; Quentin, Samuel; Jia, Jinsong; Spicuglia, Salvatore; Ferrier, Pierre; Ceccon, Monica; Giuriato, Sylvie; Gambacorti-Passerini, Carlo; Brousset, Pierre; Lamant, Laurence; Meggetto, Fabienne

    2015-01-01

    The regulatory microRNA miR-150 is involved in the development of hemopathies and is downregulated in T-lymphomas, such as anaplastic large-cell lymphoma (ALCL) tumors. ALCL is defined by the presence or absence of translocations that activate the anaplastic lymphoma kinase (ALK), with nucleophosmin-ALK (NPM-ALK) fusions being the most common. Here, we compared samples of primary NPM-ALK(+) and NPM-ALK(–) ALCL to investigate the role of miR-150 downstream of NPM-ALK. Methylation of the MIR150 gene was substantially elevated in NPM-ALK(+) biopsies and correlated with reduced miR-150 expression. In NPM-ALK(+) cell lines, DNA hypermethylation–mediated miR-150 repression required ALK-dependent pathways, as ALK inhibition restored miR-150 expression. Moreover, epigenetic silencing of miR-150 was due to the activation of STAT3, a major downstream substrate of NPM-ALK, in cooperation with DNA methyltransferase 1 (DNMT1). Accordingly, miR-150 repression was turned off following treatment with the DNMT inhibitor, decitabine. In murine NPM-ALK(+) xenograft models, miR-150 upregulation induced antineoplastic activity. Treatment of crizotinib-resistant NPM-ALK(+) KARPAS-299-CR06 cells with decitabine or ectopic miR-150 expression reduced viability and growth. Altogether, our results suggest that hypomethylating drugs, alone or in combination with other agents, may benefit ALK(+) patients harboring tumors resistant to crizotinib and other anti-ALK tyrosine kinase inhibitors (TKIs). Moreover, these results support further work on miR-150 in these and other ALK(+) malignancies. PMID:26258416

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

    International Nuclear Information System (INIS)

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

  14. Expression of RNA interference triggers from an oncolytic herpes simplex virus results in specific silencing in tumour cells in vitro and tumours in vivo

    International Nuclear Information System (INIS)

    Delivery of small interfering RNA (siRNA) to tumours remains a major obstacle for the development of RNA interference (RNAi)-based therapeutics. Following the promising pre-clinical and clinical results with the oncolytic herpes simplex virus (HSV) OncoVEXGM-CSF, we aimed to express RNAi triggers from oncolytic HSV, which although has the potential to improve treatment by silencing tumour-related genes, was not considered possible due to the highly oncolytic properties of HSV. To evaluate RNAi-mediated silencing from an oncolytic HSV backbone, we developed novel replicating HSV vectors expressing short-hairpin RNA (shRNA) or artificial microRNA (miRNA) against the reporter genes green fluorescent protein (eGFP) and β-galactosidase (lacZ). These vectors were tested in non-tumour cell lines in vitro and tumour cells that are moderately susceptible to HSV infection both in vitro and in mice xenografts in vivo. Silencing was assessed at the protein level by fluorescent microscopy, x-gal staining, enzyme activity assay, and western blotting. Our results demonstrate that it is possible to express shRNA and artificial miRNA from an oncolytic HSV backbone, which had not been previously investigated. Furthermore, oncolytic HSV-mediated delivery of RNAi triggers resulted in effective and specific silencing of targeted genes in tumour cells in vitro and tumours in vivo, with the viruses expressing artificial miRNA being comprehensibly more effective. This preliminary data provide the first demonstration of oncolytic HSV-mediated expression of shRNA or artificial miRNA and silencing of targeted genes in tumour cells in vitro and in vivo. The vectors developed in this study are being adapted to silence tumour-related genes in an ongoing study that aims to improve the effectiveness of oncolytic HSV treatment in tumours that are moderately susceptible to HSV infection and thus, potentially improve response rates seen in human clinical trials

  15. RNA interference-mediated silencing of speckle-type POZ protein promotes apoptosis of renal cell cancer cells

    Directory of Open Access Journals (Sweden)

    Liu X

    2016-04-01

    Full Text Available Xiaoxia Liu, Guiling Sun, Xiuju Sun Department of Nephrology, Affiliated Hospital of Weifang Medical University, Weifang, People’s Republic of China Abstract: This study aimed to investigate the effects of silencing the speckle-type POZ protein (SPOP gene on renal cell cancer (RCC cells and to explore its possible mechanism. The A498 and ACHN RCC cells were transfected with small interference RNA (siRNA-SPOP by lipofection methods. The silencing efficiency was monitored by quantitative real-time polymerase chain reaction and Western blot. The effects of SPOP silencing on cell apoptosis, cell viability, colony formation ability, cell migration ability, and chemosensitivity to Sorafenib were assessed by flow cytometry, an MTT assay, a colony formation assay, a trans-well migration assay, and a CCK-8 assay, respectively. Its effects on the expression of several cytokines were determined by a protein microarray. Relevant signaling pathways were also analyzed. Compared with the control group, the cell apoptosis rate was significantly higher; the cell viability, the colony formation, and migration ability were significantly decreased in the siRNA-SPOP group. The protein microarray screening showed that the expression of vascular endothelial growth factor receptor, matrix metallopeptidase-9, vascular cell adhesion molecule-1, and stromal cell-derived factor-1 in the siRNA group was significantly decreased and that the expression of granulocyte–macrophage colony-stimulating factor and E-cadherin was significantly increased (P<0.05. The relevant signaling pathways were the integrin-mediated cell surface interactions pathway and extracellular matrix organization signal pathway. SPOP gene silencing induced cell apoptosis, decreased cell viability, colony formation, and migration ability, and elevated the drug sensitivity in the RCC cells. A possible mechanism is that silencing SPOP induces the differential expression of E-cadherin, vascular endothelial

  16. Gene silencing of the tick protective antigens, Bm86, Bm91 and subolesin, in the one-host tick Boophilus microplus by RNA interference.

    Science.gov (United States)

    Nijhof, Ard M; Taoufik, Amar; de la Fuente, José; Kocan, Katherine M; de Vries, Erik; Jongejan, Frans

    2007-05-01

    The use of RNA interference (RNAi) to assess gene function has been demonstrated in several three-host tick species but adaptation of RNAi to the one-host tick, Boophilus microplus, has not been reported. We evaluated the application of RNAi in B. microplus and the effect of gene silencing on three tick-protective antigens: Bm86, Bm91 and subolesin. Gene-specific double-stranded (dsRNA) was injected into two tick stages, freshly molted unfed and engorged females, and specific gene silencing was confirmed by real time PCR. Gene silencing occurred in injected unfed females after they were allowed to feed. Injection of dsRNA into engorged females caused gene silencing in the subsequently oviposited eggs and larvae that hatched from these eggs, but not in adults that developed from these larvae. dsRNA injected into engorged females could be detected by quantitative real-time RT-PCR in eggs 14 days from the beginning of oviposition, demonstrating that unprocessed dsRNA was incorporated in the eggs. Eggs produced by engorged females injected with subolesin dsRNA were abnormal, suggesting that subolesin may play a role in embryonic development. The injection of dsRNA into engorged females to obtain gene-specific silencing in eggs and larvae is a novel method which can be used to study gene function in tick embryogenesis. PMID:17196597

  17. siRNA-Mediated Silencing of doublesex during Female Development of the Dengue Vector Mosquito Aedes aegypti.

    Science.gov (United States)

    Mysore, Keshava; Sun, Longhua; Tomchaney, Michael; Sullivan, Gwyneth; Adams, Haley; Piscoya, Andres S; Severson, David W; Syed, Zainulabeuddin; Duman-Scheel, Molly

    2015-11-01

    The development of sex-specific traits, including the female-specific ability to bite humans and vector disease, is critical for vector mosquito reproduction and pathogen transmission. Doublesex (Dsx), a terminal transcription factor in the sex determination pathway, is known to regulate sex-specific gene expression during development of the dengue fever vector mosquito Aedes aegypti. Here, the effects of developmental siRNA-mediated dsx silencing were assessed in adult females. Targeting of dsx during A. aegypti development resulted in decreased female wing size, a correlate for body size, which is typically larger in females. siRNA-mediated targeting of dsx also resulted in decreased length of the adult female proboscis. Although dsx silencing did not impact female membrane blood feeding or mating behavior in the laboratory, decreased fecundity and fertility correlated with decreased ovary length, ovariole length, and ovariole number in dsx knockdown females. Dsx silencing also resulted in disruption of olfactory system development, as evidenced by reduced length of the female antenna and maxillary palp and the sensilla present on these structures, as well as disrupted odorant receptor expression. Female lifespan, a critical component of the ability of A. aegypti to transmit pathogens, was also significantly reduced in adult females following developmental targeting of dsx. The results of this investigation demonstrate that silencing of dsx during A. aegypti development disrupts multiple sex-specific morphological, physiological, and behavioral traits of adult females, a number of which are directly or indirectly linked to mosquito reproduction and pathogen transmission. Moreover, the olfactory phenotypes observed connect Dsx to development of the olfactory system, suggesting that A. aegypti will be an excellent system in which to further assess the developmental genetics of sex-specific chemosensation. PMID:26544686

  18. siRNA-Mediated Silencing of doublesex during Female Development of the Dengue Vector Mosquito Aedes aegypti.

    Directory of Open Access Journals (Sweden)

    Keshava Mysore

    2015-11-01

    Full Text Available The development of sex-specific traits, including the female-specific ability to bite humans and vector disease, is critical for vector mosquito reproduction and pathogen transmission. Doublesex (Dsx, a terminal transcription factor in the sex determination pathway, is known to regulate sex-specific gene expression during development of the dengue fever vector mosquito Aedes aegypti. Here, the effects of developmental siRNA-mediated dsx silencing were assessed in adult females. Targeting of dsx during A. aegypti development resulted in decreased female wing size, a correlate for body size, which is typically larger in females. siRNA-mediated targeting of dsx also resulted in decreased length of the adult female proboscis. Although dsx silencing did not impact female membrane blood feeding or mating behavior in the laboratory, decreased fecundity and fertility correlated with decreased ovary length, ovariole length, and ovariole number in dsx knockdown females. Dsx silencing also resulted in disruption of olfactory system development, as evidenced by reduced length of the female antenna and maxillary palp and the sensilla present on these structures, as well as disrupted odorant receptor expression. Female lifespan, a critical component of the ability of A. aegypti to transmit pathogens, was also significantly reduced in adult females following developmental targeting of dsx. The results of this investigation demonstrate that silencing of dsx during A. aegypti development disrupts multiple sex-specific morphological, physiological, and behavioral traits of adult females, a number of which are directly or indirectly linked to mosquito reproduction and pathogen transmission. Moreover, the olfactory phenotypes observed connect Dsx to development of the olfactory system, suggesting that A. aegypti will be an excellent system in which to further assess the developmental genetics of sex-specific chemosensation.

  19. Silencing MicroRNA-155 Attenuates Cardiac Injury and Dysfunction in Viral Myocarditis via Promotion of M2 Phenotype Polarization of Macrophages

    OpenAIRE

    Yingying Zhang; Mengying Zhang; Xueqin Li; Zongsheng Tang; Xiangmin Wang; Min Zhong; Qifeng Suo; Yao Zhang; Kun Lv

    2016-01-01

    Macrophage infiltration is a hallmark feature of viral myocarditis. As studies have shown that microRNA-155 regulates the differentiation of macrophages, we aimed to investigate the role of microRNA-155 in VM. We report that silencing microRNA-155 protects mice from coxsackievirus B3 induced myocarditis. We found that microRNA-155 expression was upregulated and localized primarily in heart-infiltrating macrophages and CD4+ T lymphocytes during acute myocarditis. In contrast with wildtype (WT)...

  20. RNA interference silencing of CHS greatly alters the growth pattern of apple (Malus x domestica).

    Science.gov (United States)

    Dare, Andrew P; Hellens, Roger P

    2013-08-01

    Plants produce a vast array of phenolic compounds which are essential for their survival on land. One major class of polyphenols are the flavonoids and their formation is dependent on the enzyme chalcone synthase (CHS). In a recent study we silenced the CHS genes of apple (Malus × domestica Borkh.) and observed a loss of pigmentation in the fruit skin, flowers and stems. More surprisingly, highly silenced lines were significantly reduced in size, with small leaves and shortened internode lengths. Chemical analysis also revealed that the transgenic shoots contained greatly reduced concentrations of flavonoids which are known to modulate auxin flow. An auxin transport study verified this, with an increased auxin transport in the CHS-silenced lines. Overall, these findings suggest that auxin transport in apple has adapted to take place in the presence of high endogenous concentrations of flavonoids. Removal of these compounds therefore results in abnormal auxin movement and a highly disrupted growth pattern.

  1. DNA methylation mediated silencing of microRNA-145 is a potential prognostic marker in patients with lung adenocarcinoma

    OpenAIRE

    Wenjie Xia; Qiang Chen; Jie Wang; Qixing Mao; Gaochao Dong; Run Shi; YanYan Zheng; Lin Xu; Feng Jiang

    2015-01-01

    The molecular mechanism of down-regulated microRNA-145 (miR-145) expression in lung adenocarcinoma (LAC) remains largely unknown. We hypothesized that aberrant hyper-methylation of the CpG sites silenced the expression of miR-145 in LAC. In consideration of its pivotal role in LAC development and progression, we also evaluated the clinical utility of miR-145 as a prognostic marker. We assessed the DNA methylation status of the miR-145 promoter region in 20 pairs of LAC and the matched non-tum...

  2. Transient down-regulation of the RNA silencing machinery increases efficiency of Agrobacterium-mediated transformation of Arabidopsis.

    Science.gov (United States)

    Bilichak, Andriy; Yao, Youli; Kovalchuk, Igor

    2014-06-01

    Agrobacterium tumefaciens is a plant pathogen that is widely used in plant transformation. As the process of transgenesis includes the delivery of single-stranded T-DNA molecule, we hypothesized that transformation rate may negatively correlate with the efficiency of the RNA-silencing machinery. Using mutants compromised in either the transcriptional or post-transcriptional gene-silencing pathways, two inhibitors of stable transformation were revealed-AGO2 and NRPD1a. Furthermore, an immunoprecipitation experiment has shown that NRPD1, a subunit of Pol IV, directly interacts with Agrobacterium T-DNA in planta. Using the Tobacco rattle virus (TRV)--based virus-induced gene silencing (VIGS) technique, we demonstrated that the transient down-regulation of the expression of either AGO2 or NRPD1a genes in reproductive organs of Arabidopsis, leads to an increase in transformation rate. We observed a 6.0- and 3.5-fold increase in transformation rate upon transient downregulation of either AGO2 or NRPD1a genes, respectively. This is the first report demonstrating the increase in the plant transformation rate via VIGS-mediated transient down-regulation of the components of epigenetic machinery in reproductive tissue.

  3. The NS3 protein of Rice hoja blanca tenuivirus suppresses RNA silencing in plant and insect hosts by efficiently binding both siRNAs and miRNAs

    NARCIS (Netherlands)

    Hemmes, J.C.; Lakatos, L.; Goldbach, R.W.; Burgyan, J.; Prins, M.W.

    2007-01-01

    RNA silencing plays a key role in antiviral defense as well as in developmental processes in plants and insects. Negative strand RNA viruses such as the plant virus Rice hoja blanca tenuivirus (RHBV) replicate in plants and in their insect transmission vector. Like most plant-infecting viruses, RHBV

  4. RNA沉默——植物基因组免疫的安全防线%RNA silencing, the fundamental security strategy of genomic immunity: a review

    Institute of Scientific and Technical Information of China (English)

    董丽; 郭惠珊

    2012-01-01

    The 21-24 nucleotides small RNA that generated from double strands RNA can trigger two types of epigenetic gene silencing in plants. One is Post-Transcriptional Gene Silencing (PTGS), characterized by cleavage of homologous mRNA in cytoplasm. Transcriptional Gene Silencing (TGS) is another one, in which transcription inhibition is obtained through small RNA-directed DNA methylation of homologous promoter region. Here we summarized the relationship and differences between PTGS and TGS, the current achievement in the study of RNA silencing spreading, as well as the discrepancy of exogenous and endogenous gene silencing, and discussed the underlying reasons in the end.%在植物中,起始于双链RNA的长度为21~24个核苷酸的小RNA,能够诱发两种形式的表观遗传学基因沉默.转录后基因沉默(Post-transcriptional gene silencing,PTGS),可以介导细胞质中同源mRNA的降解;转录基因沉默(Transcriptional gene silencing,TGS),则主要通过小RNA介导同源启动子区的DNA甲基化,从而抑制基因转录.文中针对PTGS和TGS通路的区别与联系、沉默传递研究的现状及内源与外源基因沉默的异同点等问题进行探讨.

  5. MicroRNA-mediated NBS1 Gene Silence and Its Effects on Telomerase Activation in Hela Cells

    Institute of Scientific and Technical Information of China (English)

    CAO Sun-qiong; REN Chang-shan

    2008-01-01

    Objective:To research the silence of NBS1 after transfection microRNA expressing eukaryotic recombinants and the changes of telomerase activation in teiomerase-positive cell line Hela.Methods:According to the sequence of NBS1 mRNA,the NBS1 pre-microRNA was designed and synthesized,then cloned into the GFP reporter pcDNA6.2-GW/EmGFP-miR vector and transfected into Hela cells.The integrity of the insert fragment was verified through colony PCR and sequencing analysis.The NBS1 gene expression of NBS1 microRNA recombinants was detected by Real-Time PCR and western blot.Telomerase activity in Hela cells was assayed by TRAP-PCR-EB.Results:Sequences of insert fragment in microRNA expressing recombinants were correct.The NBS1 gene expression was decreased,and the telomerase activation of Hela cell reduced.Conclusion:NBS1 microRNA inhibits NBS1 gene expression,and depresses telomerase activation of Hela cells.This confirms that there is relevance between NBS1 gene and telomerase activity.

  6. MicroRNA-dependent development revealed by RNA interference-mediated gene silencing of LmDicer1 in the migratory locust

    Institute of Scientific and Technical Information of China (English)

    Yan-Li Wang; Mei-Ling Yang; Feng Jiang; Jian-Zhen Zhang; Le Kang

    2013-01-01

    MicroRNAs(miRNAs)are small noncoding RNAs,which participate in many biological processes.The small RNA transcriptome in the migratory locust has been characterized and 50 conserved miRNA families and 185 potential locust-specific miRNA family candidates have been identified using high-throughput sequencing.However,it is unclear whether miRNAs influence a wide variety of locusts' biological processes,such as growth or development.In insects,Dicer 1 ribonuclease transforms miRNA precursors into mature miRNAs.Thus,using systemic RNA interference(RNAi)to silence the expression of Dicerl in the migratory locust,Locusta migratoria,we reduced miRNA contents in the locust and disrupted two types of molt(nymph-nymph,and nymph-adult).The RNAi of LmDicerl also resulted in a high mortality in L.migratora.Our study revealed that LmDicerl was essential for miRNA regulation and development of L.migratoria.These results further support our notion that LmDicerl could serve as an excellent target for developing novel strategies for controlling this important insect pest.

  7. Filaggrin silencing by shRNA directly impairs the skin barrier function of normal human epidermal keratinocytes and then induces an immune response

    Energy Technology Data Exchange (ETDEWEB)

    Dang, N.N. [Department of Dermatology, Jinan Central Hospital Affiliated to Shandong University, Jinan, Shandong Province (China); College of Life Science, Shandong Normal University, Jinan, Shandong Province (China); Pang, S.G. [Department of Endocrinology, Jinan Central Hospital Affiliated to Shandong University, Jinan, Shandong Province (China); Song, H.Y. [Department of Dermatology, Jinan Central Hospital Affiliated to Shandong University, Jinan, Shandong Province (China); An, L.G. [College of Life Science, Shandong Normal University, Jinan, Shandong Province (China); Ma, X.L. [Central Laboratory, Jinan Central Hospital Affiliated to Shandong University, Jinan, Shandong Province (China)

    2014-11-14

    The objective of this study was to investigate whether a single defect in skin barrier function simulated by filaggrin silencing could induce Th2-predominant inflammation. Filaggrin gene expression was silenced in cultured normal human epidermal keratinocytes (NHEKs) using small hairpin RNA (shRNA, GTTGGCTCAAGCATATTATTT). The efficacy of silencing was confirmed by polymerase chain reaction (PCR) and Western blotting. Filaggrin-silenced cells (LV group), shRNA control cells (NC group), and noninfected cells (Blank group) were evaluated. The expression of cornified cell envelope-related proteins, including cytokeratin (CK)-5, -10, -14, loricrin, involucrin, and transglutaminase (TGM)-1, was detected by Western blotting. Interleukins (IL)-2, IL-4, IL-5, IL-12p70, IL-13, and interferon-gamma (IFN-γ) were detected by enzyme-linked immunosorbent assay (ELISA). After filaggrin was successfully silenced by shRNA, the expressions of CK-5, -10, -14, involucrin, and TGM-1 in NHEKs were significantly downregulated compared to the Blank and NC groups (P<0.05 or P<0.01); only loricrin expression was markedly upregulated (P<0.01). Filaggrin silencing also resulted in significant increases of IL-2, IL-4, IL-5, and IL-13 (P<0.05 or P<0.01), and significant decreases of IL-12p70 and IFN-γ (P<0.01) compared with cells in the Blank and NC groups. Filaggrin silencing impaired normal skin barrier function mainly by targeting the cornified cell envelope. The immune response after filaggrin silencing was characterized by Th2 cells, mainly because of the inhibition of IFN-γ expression. Lack of filaggrin may directly impair skin barrier function and then further induce the immune response.

  8. Endomembrane-associated RSD-3 is important for RNAi induced by extracellular silencing RNA in both somatic and germ cells of Caenorhabditis elegans

    Science.gov (United States)

    Imae, Rieko; Dejima, Katsufumi; Kage-Nakadai, Eriko; Arai, Hiroyuki; Mitani, Shohei

    2016-01-01

    RNA silencing signals in C. elegans spread among cells, leading to RNAi throughout the body. During systemic spread of RNAi, membrane trafficking is thought to play important roles. Here, we show that RNAi Spreading Defective-3 (rsd-3), which encodes a homolog of epsinR, a conserved ENTH (epsin N-terminal homology) domain protein, generally participates in cellular uptake of silencing RNA. RSD-3 is previously thought to be involved in systemic RNAi only in germ cells, but we isolated several deletion alleles of rsd-3, and found that these mutants are defective in the spread of silencing RNA not only into germ cells but also into somatic cells. RSD-3 is ubiquitously expressed, and intracellularly localized to the trans-Golgi network (TGN) and endosomes. Tissue-specific rescue experiments indicate that RSD-3 is required for importing silencing RNA into cells rather than exporting from cells. Structure/function analysis showed that the ENTH domain alone is sufficient, and membrane association of the ENTH domain is required, for RSD-3 function in systemic RNAi. Our results suggest that endomembrane trafficking through the TGN and endosomes generally plays an important role in cellular uptake of silencing RNA. PMID:27306325

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

    Institute of Scientific and Technical Information of China (English)

    Hong-Tao Liu; Chun-Lei Lu

    2013-01-01

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

  10. The silence of MUC2 mRNA induced by promoter hypermethylation associated with HBV in Hepatocellular Carcinoma

    Directory of Open Access Journals (Sweden)

    Ling Yang

    2013-01-01

    Full Text Available Abstract Background To evaluate the promoter methylation status of MUC2 gene and mRNA expression in patients with hepatocellular carcinoma. Methods We analyzed MUC2 methylation by MSP, and MUC2 mRNA by real-time PCR in 74 HCC. Results MUC2 mRNA were lower in HCC tissues (Mean -ΔCt = −4.70 than that in Non-HCC tissues (Mean -ΔCt = −2.98. Expression of MUC2 was elevated in only 23 (31.08% of the 74 HCC patients. MUC2 promoter was hypermethylated in 62.2% (46/74 of HCCs, and in only 18.9% (14/74 of non-tumor samples. MUC2 mRNA were lower in HCC patients with hypermethylation (Mean -ΔΔCt = −2.25 than those with demethylation (Mean -ΔΔCt = −0.22, and there is a decreased tendency for MUC2 mRNA in HCC patients with promoter hypermethylation (p = 0.011. There was a significantly correlation found between MUC2 mRNA and HBV and AFP in HCC. The loss of MUC2 mRNA and hypermethylation could be poor prognostic factors. After treated by 5-Aza-CdR and TSA, we found that MUC2 mRNA induced significantly in 7721, Huh7 and HepG2 cells. Conclusion The results suggested that MUC2 mRNA silenced by promoter hypermethylation is associated with high levels HBV in HCC.

  11. Pentatricopeptide repeats: Modular blocks for building RNA-binding proteins

    OpenAIRE

    Filipovska, Aleksandra; Rackham, Oliver

    2013-01-01

    Pentatricopeptide repeat (PPR) proteins control diverse aspects of RNA metabolism across the eukaryotic domain. Recent computational and structural studies have provided new insights into how they recognize RNA, and show that the recognition is sequence-specific and modular. The modular code for RNA-binding by PPR proteins holds great promise for the engineering of new tools to target RNA and identifying RNAs bound by natural PPR proteins.

  12. RNA interference blocking the apoptosis in HEK293 cells induced by overexpression of alpha-synuclein

    Institute of Scientific and Technical Information of China (English)

    Tao Chen; Beisha Tang; Xiaoping Liao; Guoqiang Wen; Xinxiang Yan; Jifeng Guo; Yuhu Zhang; Feng Ouyang; Zhigang Long; Li Cao; Jing Li

    2009-01-01

    BACKGROUND: Overexpression of o-synuclein can induce cell apoptosis. RNA interference (RNAi)may block specific gene function and cause gene silencing.OBJECTIVE: To construct a specific and effective RNAi plasmid for the a-synuclein gene and investigate if RNAi can block apoptosis in HEK293 cells, induced by overexpression of wild-type α-synuclein.DESIGN, TIME AND SETTING: A contrast experiment based on genetically engineered cytobiology was performed at the State Key Lab of Medical Genetics of China, Xiangya Medical College of Central South University, between October 2004 and October 2008.MATERIALS: HEK293 cells and pBSHH1 plasmid were provided by the State Key Lab of Medical Genetics of China; OligDNA sequence by Sagon Bioengineering Company, Shanghai;Lipofectamine 2000 by Invitrogen, USA;α-synuclein monoclonal antibody, Hoechst 33258, and MTT by Sigma, USA; Horseradish peroxidase-coupled goat anti-rat luG by KPL, USA; FACSan flow cytometry by BD, USA.METHODS: Four target sites were used to construct hairpin RNA pBSHH1 vectors-pSYNi-1,pSYNi-2, pSYNi-3 and pSYNi-4-which were cloned in the pBSHH1 plasmid. HEK293 cells were transfected using Lipofectamine 2000. In addition, a non-transfect group and a negative plasmid transfect group were established. The cultured HEK293 cells were processed as follows:transfection of blank plasmid (blank control group), transfection of α-synuclein-pEGFP and RNAi negative vector (negative control group), and transfection of a-synuclein-pEGFP and pSYNi-1 (transfection group). Cells in all groups were transfected with Lipofectamine 2000 for 48 hours.MAIN OUTCOME MEASURES: Expression of α-synuclein mRNA and protein were detected by RT-PCR and Western blot. Cell morphology was observed under an inverted fluorescence microscope; cell viability was measured using MTT method; and cell apoptosis was determined with Annexin V-PE flow cytometry.RESULTS: a-synuclein mRNA and protein expressions were significantly decreased in the pSYNi-1

  13. A novel sweet potato potyvirus open reading frame (ORF) is expressed via polymerase slippage and suppresses RNA silencing

    Science.gov (United States)

    Untiveros, Milton; Olspert, Allan; Artola, Katrin

    2016-01-01

    Summary The single‐stranded, positive‐sense RNA genome of viruses in the genus Potyvirus encodes a large polyprotein that is cleaved to yield 10 mature proteins. The first three cleavage products are P1, HCpro and P3. An additional short open reading frame (ORF), called pipo, overlaps the P3 region of the polyprotein ORF. Four related potyviruses infecting sweet potato (Ipomoea batatas) are predicted to contain a third ORF, called pispo, which overlaps the 3′ third of the P1 region. Recently, pipo has been shown to be expressed via polymerase slippage at a conserved GA6 sequence. Here, we show that pispo is also expressed via polymerase slippage at a GA6 sequence, with higher slippage efficiency (∼5%) than at the pipo site (∼1%). Transient expression of recombinant P1 or the ‘transframe’ product, P1N‐PISPO, in Nicotiana benthamiana suppressed local RNA silencing (RNAi), but only P1N‐PISPO inhibited short‐distance movement of the silencing signal. These results reveal that polymerase slippage in potyviruses is not limited to pipo expression, but can be co‐opted for the evolution and expression of further novel gene products. PMID:26757490

  14. Evidence for a piwi-dependent RNA silencing of the gypsy endogenous retrovirus by the Drosophila melanogaster flamenco gene.

    Science.gov (United States)

    Sarot, Emeline; Payen-Groschêne, Geneviève; Bucheton, Alain; Pélisson, Alain

    2004-03-01

    In Drosophila melanogaster, the endogenous retrovirus gypsy is repressed by the functional alleles (restrictive) of an as-yet-uncloned heterochromatic gene called flamenco. Using gypsy-lacZ transcriptional fusions, we show here that this repression takes place not only in the follicle cells of restrictive ovaries, as was previously observed, but also in restrictive larval female gonads. Analyses of the role of gypsy cis-regulatory sequences in the control of gypsy expression are also presented. They rule out the hypothesis that gypsy would contain a single binding region for a putative Flamenco repressor. Indeed, the ovarian expression of a chimeric yp3-lacZ construct was shown to become sensitive to the Flamenco regulation when any of three different 5'-UTR gypsy sequences (ranging from 59 to 647 nucleotides) was incorporated into the heterologous yp3-lacZ transcript. The piwi mutation, which is known to affect RNA-mediated homology-dependent transgene silencing, was also shown to impede the repression of gypsy in restrictive female gonads. Finally, a RNA-silencing model is also supported by the finding in ovaries of short RNAs (25-27 nucleotides long) homologous to sequences from within the gypsy 5'-UTR. PMID:15082550

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

    Institute of Scientific and Technical Information of China (English)

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

    2011-01-01

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

  16. Recombinant adenovirus-mediated shRNA silencing of midkine gene in BxPC-3 cells

    Institute of Scientific and Technical Information of China (English)

    Mingyue Xiong; Kunzheng Wang

    2009-01-01

    Objective:To investigate the silencing effects of recombinant adenovirus Ad-shRNA-MK on midkine(MK) gene in pancreatic cancer cells. Methods:Ad-shRNA-MK was used to infect pancreatic cancer BxPC-3 cells. Assays were conducted for knockdown of the MK gene on the day of infection and on the 1a, 3rd, 5th, 7th, and 9th days post-infection by using immunocytochemistry, real-time RT-PCR, and Western blot analysis. Results:The adenoviral Ad-shRNA-PTN was constructed successfully, and infection was confirmed by electron microscopic observation. By using real-time RT-PCR, the inhibition rates of MK mRNA expression in the BxPC-3 cells were 20%, 80%, 55%, and 23% on the 1st, 3rd, 5th, and 7th days post-infection. Immunocytochemistry and Western blot analysis confirmed this effect at the gene product level. Conclusion:Efficient and specific knockdown of MK in pancreatic cancer cells by adenoviral Ad-shRNA-PTN is a potentially powerful tool for the study of gene therapy of pancreatic cancer nerve infiltration.

  17. Efficient CRISPR-Mediated Post-Transcriptional Gene Silencing in a Hyperthermophilic Archaeon Using Multiplexed crRNA Expression

    Directory of Open Access Journals (Sweden)

    Ziga Zebec

    2016-10-01

    Full Text Available CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats-mediated RNA degradation is catalyzed by a type III system in the hyperthermophilic archaeon Sulfolobus solfataricus. Earlier work demonstrated that the system can be engineered to target specifically mRNA of an endogenous host reporter gene, namely the β-galactosidase in S. solfataricus. Here, we investigated the effect of single and multiple spacers targeting the mRNA of a second reporter gene, α-amylase, at the same, and at different, locations respectively, using a minimal CRISPR (miniCR locus supplied on a viral shuttle vector. The use of increasing numbers of spacers reduced mRNA levels at progressively higher levels, with three crRNAs (CRISPR RNAs leading to ∼ 70–80% reduction, and five spacers resulting in an α-amylase gene knockdown of > 90% measured on both mRNA and protein activity levels. Our results indicate that this technology can be used to increase or modulate gene knockdown for efficient post-transcriptional gene silencing in hyperthermophilic archaea, and potentially also in other organisms.

  18. Colonic gene silencing using siRNA-loaded calcium phosphate/PLGA nanoparticles ameliorates intestinal inflammation in vivo.

    Science.gov (United States)

    Frede, Annika; Neuhaus, Bernhard; Klopfleisch, Robert; Walker, Catherine; Buer, Jan; Müller, Werner; Epple, Matthias; Westendorf, Astrid M

    2016-01-28

    Cytokines and chemokines are predominant players in the progression of inflammatory bowel diseases. While systemic neutralization of these players with antibodies works well in some patients, serious contraindications and side effects have been reported. Therefore, the local interference of cytokine signaling mediated by siRNA-loaded nanoparticles might be a promising new therapeutic approach. In this study, we produced multi-shell nanoparticles consisting of a calcium phosphate (CaP) core coated with siRNA directed against pro-inflammatory mediators, encapsulated into poly(d,l-lactide-co-glycolide acid) (PLGA), and coated with a final outer layer of polyethyleneimine (PEI), for the local therapeutic treatment of colonic inflammation. In cell culture, siRNA-loaded CaP/PLGA nanoparticles exhibited a rapid cellular uptake, almost no toxicity, and an excellent in vitro gene silencing efficiency. Importantly, intrarectal application of these nanoparticles loaded with siRNA directed against TNF-α, KC or IP-10 to mice suffering from dextran sulfate sodium (DSS)-induced colonic inflammation led to a significant decrease of the target genes in colonic biopsies and mesenteric lymph nodes which was accompanied with a distinct amelioration of intestinal inflammation. Thus, this study provides evidence that the specific and local modulation of the inflammatory response by CaP/PLGA nanoparticle-mediated siRNA delivery could be a promising approach for the treatment of intestinal inflammation.

  19. Development of Nano- and Microparticle Technologies for Targeted Gene Silencing through RNA Interference Manipulation of the Immune Response in Inflammatory Lung Disease

    OpenAIRE

    Kelly, Ciara

    2011-01-01

    RNA Interference (RNAi) allows specific and potent knockdown of target genes and interest now lies beyond its use as a molecular biology tool and in its potential as a therapeutic to mediate gene silencing in diseased cells. Targeted local delivery of small interfering RNA (siRNA) to the lungs via inhalation offers a unique opportunity to treat a range of previously unbeatable or poorly controlled respiratory conditions. Alveolar macrophages are the first line of defence against inhaled toxin...

  20. Quantitative measurement of delivery and gene silencing activities of siRNA polyplexes containing pyridylthiourea-grafted polyethylenimines.

    Science.gov (United States)

    Pinel, Sophie; Aman, Emmanuel; Erblang, Felix; Dietrich, Jonathan; Frisch, Benoit; Sirman, Julien; Kichler, Antoine; Sibler, Annie-Paule; Dontenwill, Monique; Schaffner, Florence; Zuber, Guy

    2014-05-28

    The activity of synthetic interfering nucleic acids (siRNAs) relies on the capacity of delivery systems to efficiently transport nucleic acids into the cytosol of target cells. The pyridylthiourea-grafted 25KDa polyethylenimine (πPEI) is an excellent carrier for siRNA delivery into cells and it was extensively investigated in this report. Quantification of the siRNA-mediated gene silencing efficiency indicated that the πPEI specific delivery activity at the cell level may be measured and appears relatively constant in various cell lines. Delivery experiments assaying inhibitors of various entry pathways or concanamycin A, an inhibitor of the H(+)/ATPase vacuolar pump showed that the πPEI/siRNA polyplexes did not require any specific entry mode but strongly relied on vacuolar acidification for functional siRNA delivery. Next, πPEI polyplexes containing a siRNA targeting the transcription factor HIF-1α, known to be involved in tumor progression, were locally injected into mice xenografted with a human glioblastoma. A 55% reduction of the level of the target mRNA was observed at doses comparable to those used in vitro when the πPEI delivery activity was calculated per cell. Altogether, our study underscores the usefulness of "simple"/rough cationic polymers for siRNA delivery despite their intrinsic limitations. The study underscores as well as that bottom-up strategies make sense. The in vitro experiments can precede in vivo administration and be of high value for selection of the carrier with enhanced specific delivery activity and parallel other research aiming at improving synthetic delivery systems for resilience in the blood and for enhanced tissue-targeting capacity. PMID:24631860

  1. Genetic variability and evolutionary implications of RNA silencing suppressor genes in RNA1 of sweet potato chlorotic stunt virus isolates infecting sweetpotato and related wild species.

    Directory of Open Access Journals (Sweden)

    Arthur K Tugume

    Full Text Available BACKGROUND: The bipartite single-stranded RNA genome of Sweet potato chlorotic stunt virus (SPCSV, genus Crinivirus; Closteroviridae encodes a Class 1 RNase III (RNase3, a putative hydrophobic protein (p7 and a 22-kDa protein (p22 from genes located in RNA1. RNase3 and p22 suppress RNA silencing, the basal antiviral defence mechanism in plants. RNase3 is sufficient to render sweetpotato (Ipomoea batatas virus-susceptible and predisposes it to development of severe diseases following infection with unrelated virus. The incidence, strains and gene content of SPCSV infecting wild plant species have not been studied. METHODOLOGY/PRINCIPAL FINDINGS: Thirty SPCSV isolates were characterized from 10 wild Ipomoea species, Hewittia sublobata or Lepistemon owariensis (family Convolvulaceae in Uganda and compared with 34 local SPCSV isolates infecting sweetpotatoes. All isolates belonged to the East African (EA strain of SPCSV and contained RNase3 and p7, but p22 was not detected in six isolates. The three genes showed only limited genetic variability and the proteins were under purifying selection. SPCSV isolates lacking p22 synergized with Sweet potato feathery mottle virus (SPFMV, genus potyvirus; Potyviridae and caused severe symptoms in co-infected sweetpotato plants. One SPCSV isolate enhanced accumulation of SPFMV, but no severe symptoms developed. A new whitefly-transmitted virus (KML33b encoding an RNase3 homolog (<56% identity to SPCSV RNase3 able to suppresses sense-mediated RNA silencing was detected in I. sinensis. CONCLUSIONS/SIGNIFICANCE: SPCSV isolates infecting wild species and sweetpotato in Uganda were genetically undifferentiated, suggesting inter-species transmission of SPCSV. Most isolates in Uganda contained p22, unlike SPCSV isolates characterized from other countries and continents. Enhanced accumulation of SPFMV and increased disease severity were found to be uncoupled phenotypic outcomes of RNase3-mediated viral synergism in

  2. Gene silencing of the tick protective antigens, Bm86, Bm91 and subolesin, in the one-host tick Boophilus microplus by RNA interference

    OpenAIRE

    Nijhof, A. M.; Taoufik, A.; de la Fuente, M.R.; Kocan, K M; de Vries, E; Jongejan, F

    2007-01-01

    The use of RNA interference (RNAi) to assess gene function has been demonstrated in several three-host tick species but adaptation of RNAi to the one-host tick, Boophilus microplus, has not been reported. We evaluated the application of RNAi in B. microplus and the effect of gene silencing on three tick-protective antigens: Bm86, Bm91 and subolesin. Gene-specific double-stranded (dsRNA) was injected into two tick stages, freshly molted unfed and engorged females, and specific gene silencing w...

  3. A Single RNaseIII Domain Protein from Entamoeba histolytica Has dsRNA Cleavage Activity and Can Help Mediate RNAi Gene Silencing in a Heterologous System.

    Directory of Open Access Journals (Sweden)

    Justine M Pompey

    Full Text Available Dicer enzymes process double-stranded RNA (dsRNA into small RNAs that target gene silencing through the RNA interference (RNAi pathway. Dicer enzymes are complex, multi-domain RNaseIII proteins, however structural minimalism of this protein has recently emerged in parasitic and fungal systems. The most minimal Dicer, Saccharomyces castellii Dicer1, has a single RNaseIII domain and two double stranded RNA binding domains. In the protozoan parasite Entamoeba histolytica 27nt small RNAs are abundant and mediate silencing, yet no canonical Dicer enzyme has been identified. Although EhRNaseIII does not exhibit robust dsRNA cleavage in vitro, it can process dsRNA in the RNAi-negative background of Saccharomyces cerevisiae, and in conjunction with S. castellii Argonaute1 can partially reconstitute the RNAi pathway. Thus, although EhRNaseIII lacks the domain architecture of canonical or minimal Dicer enzymes, it has dsRNA processing activity that contributes to gene silencing via RNAi. Our data advance the understanding of small RNA biogenesis in Entamoeba as well as broaden the spectrum of non-canonical Dicer enzymes that contribute to the RNAi pathway.

  4. Investigation of siRNA Nanoparticle Formation Using Mono-Cationic Detergents and Its Use in Gene Silencing in Human HeLa Cells

    International Nuclear Information System (INIS)

    The focus of recent research has been on the development of siRNA vectors to achieve an innovative gene therapy. Most of the conventional vectors are siRNA nanoparticles complexed with cationic polymers and liposomes, making it difficult to release siRNA. In this study, we report on the use of MCD, a quaternary ammonium salt detergent containing a long aliphatic chain (L-chain) as an siRNA complexation agent using human HeLa cells (a model cancer cell). We prepared siRNA nanoparticles using various MCDs, and measured the diameters and zeta-potentials of the particles. The use of an MCD with a long L-chain resulted in the formation of a positively charged nanoparticle. In contrast, a negatively charged nanoparticle was formed when a MCD with a short L-chain was used. We next evaluated the gene silencing efficiency of the nanoparticles using HeLa cells expressing the luciferase protein. The results showed that the siRNA/MCD nanoparticles showed a higher gene silencing efficiency than Lipofectamine 2000. We also found that the efficiency of gene silencing is a function of the length of the alkyl chain in MCD and zeta-potential of the siRNA/MCD nanoparticles. Such information provides another viewpoint for designing siRNA vectors

  5. A Single RNaseIII Domain Protein from Entamoeba histolytica Has dsRNA Cleavage Activity and Can Help Mediate RNAi Gene Silencing in a Heterologous System.

    Science.gov (United States)

    Pompey, Justine M; Foda, Bardees; Singh, Upinder

    2015-01-01

    Dicer enzymes process double-stranded RNA (dsRNA) into small RNAs that target gene silencing through the RNA interference (RNAi) pathway. Dicer enzymes are complex, multi-domain RNaseIII proteins, however structural minimalism of this protein has recently emerged in parasitic and fungal systems. The most minimal Dicer, Saccharomyces castellii Dicer1, has a single RNaseIII domain and two double stranded RNA binding domains. In the protozoan parasite Entamoeba histolytica 27nt small RNAs are abundant and mediate silencing, yet no canonical Dicer enzyme has been identified. Although EhRNaseIII does not exhibit robust dsRNA cleavage in vitro, it can process dsRNA in the RNAi-negative background of Saccharomyces cerevisiae, and in conjunction with S. castellii Argonaute1 can partially reconstitute the RNAi pathway. Thus, although EhRNaseIII lacks the domain architecture of canonical or minimal Dicer enzymes, it has dsRNA processing activity that contributes to gene silencing via RNAi. Our data advance the understanding of small RNA biogenesis in Entamoeba as well as broaden the spectrum of non-canonical Dicer enzymes that contribute to the RNAi pathway. PMID:26230096

  6. Expression of RNA interference triggers from an oncolytic herpes simplex virus results in specific silencing in tumour cells in vitro and tumours in vivo

    Directory of Open Access Journals (Sweden)

    Anesti Anna-Maria

    2010-09-01

    Full Text Available Abstract Background Delivery of small interfering RNA (siRNA to tumours remains a major obstacle for the development of RNA interference (RNAi-based therapeutics. Following the promising pre-clinical and clinical results with the oncolytic herpes simplex virus (HSV OncoVEXGM-CSF, we aimed to express RNAi triggers from oncolytic HSV, which although has the potential to improve treatment by silencing tumour-related genes, was not considered possible due to the highly oncolytic properties of HSV. Methods To evaluate RNAi-mediated silencing from an oncolytic HSV backbone, we developed novel replicating HSV vectors expressing short-hairpin RNA (shRNA or artificial microRNA (miRNA against the reporter genes green fluorescent protein (eGFP and β-galactosidase (lacZ. These vectors were tested in non-tumour cell lines in vitro and tumour cells that are moderately susceptible to HSV infection both in vitro and in mice xenografts in vivo. Silencing was assessed at the protein level by fluorescent microscopy, x-gal staining, enzyme activity assay, and western blotting. Results Our results demonstrate that it is possible to express shRNA and artificial miRNA from an oncolytic HSV backbone, which had not been previously investigated. Furthermore, oncolytic HSV-mediated delivery of RNAi triggers resulted in effective and specific silencing of targeted genes in tumour cells in vitro and tumours in vivo, with the viruses expressing artificial miRNA being comprehensibly more effective. Conclusions This preliminary data provide the first demonstration of oncolytic HSV-mediated expression of shRNA or artificial miRNA and silencing of targeted genes in tumour cells in vitro and in vivo. The vectors developed in this study are being adapted to silence tumour-related genes in an ongoing study that aims to improve the effectiveness of oncolytic HSV treatment in tumours that are moderately susceptible to HSV infection and thus, potentially improve response rates seen

  7. Global Effects on Gene Expression in Fission Yeast by Silencing and RNA Interference Machineries

    DEFF Research Database (Denmark)

    Hansen, Klavs R.; Burns, G.; Mata, J.;

    2005-01-01

    sequences such as long terminal repeats (LTRs). We analyzed the global effects of the Clr3 and Clr6 histone deacetylases, the Clr4 methyltransferase, the zinc finger protein Clr1, and the RNAi proteins Dicer, RdRP, and Argonaute on the transcriptome of Schizosaccharomyces pombe (fission yeast). The clr...... by histone deacetylation independent of RNAi. Our data indicate that the RNAi and Clr proteins show only a limited functional overlap and that the Clr proteins play more global roles in gene silencing....

  8. Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing.

    Science.gov (United States)

    Newton, Patrice; Latomanski, Eleanor A; Newton, Hayley J

    2016-07-06

    Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on β-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

  9. The ability of PVX p25 to form RL structures in plant cells is necessary for its function in movement, but not for its suppression of RNA silencing.

    Directory of Open Access Journals (Sweden)

    Fei Yan

    Full Text Available The p25 triple gene block protein of Potato virus X (PVX is multifunctional, participating in viral movement and acting as a suppressor of RNA silencing. The cell-to-cell movement of PVX is known to depend on the suppression function of p25. GFP-fused p25 accumulates in rod-like (RL structures with intense fluorescence in cells. By monitoring the location of fluorescence at different times, we have now shown that the RL structure is composed of filaments. P25 mutants without the conditional ability to recover movement function could not form RL structures while the mutants that had the ability did form the structure, suggesting that the ability of p25 to form RL structures is necessary for its function in cell-to-cell movement, but not for its suppressor function. Moreover, chemical inhibition of microfilaments in cells destroyed the formation of the complete RL structure. Additionally, TGBp2 and TGBp3 were recruited into the RL structure, suggesting a relationship between the TGBps in virus movement.

  10. Two virus-encoded RNA silencing suppressors, P14 of Beet necrotic yellow vein virus and S6 of Rice black streak dwarf virus

    Institute of Scientific and Technical Information of China (English)

    ZHANG Lingdi; WANG Zhaohui; WANG Xianbing; LI Dawei; HAN Chenggui; ZHAI Yafeng; YU Jialin

    2005-01-01

    Functional analysis for gene silencing suppressor of P14 gene of Beet necrotic yellow vein virus and S6 gene of Rice black streak dwarf virus was carried out by agro- infiltration with recombinant vectors of Potato virus X. The phenotype observation of green fluorescent protein (GFP) expression and Northern blot showed that the gene silencing of gfp transgenic Nicotiana benthamiana induced by homologous sequence was strongly suppressed by the immixture infiltration of either the P14 or the S6. In the suppressed plants, the gfp mRNA accumulation was higher than that in the non-suppressed controls and the symptoms caused by PVX infection became more severe, especially the gfp DNA methylation of plant genome was significantly inhabited when co-infiltrated with RBSDV S6 gene. These results suggested that these two virus genes were potentially to encode for proteins as RNA silencing suppressors.

  11. Graft-transmitted siRNA signal from the root induces visual manifestation of endogenous post-transcriptional gene silencing in the scion.

    Directory of Open Access Journals (Sweden)

    Atsushi Kasai

    Full Text Available In plants, post-transcriptional gene silencing (PTGS spreads systemically, being transmitted from the silenced stock to the scion expressing the corresponding transgene. It has been reported that a graft-transmitted siRNA signal can also induce PTGS of an endogenous gene, but this was done by top-grafting using silenced stock. In the present study involving grafting of Nicotiana benthamiana, we found that PTGS of an endogenous gene, glutamate-1-semialdehyde aminotransferase (GSA, which acts as a visible marker of RNAi via inhibition of chlorophyll synthesis, was manifested along the veins of newly developed leaves in the wild-type scion by the siRNA signal synthesized only in companion cells of the rootstock.

  12. MicroRNA silencing and the development of novel therapies for liver disease

    OpenAIRE

    Szabo, Gyongyi; Sarnow, Peter; Bala, Shashi

    2012-01-01

    In recent years microRNAs have emerged as crucial small non-coding RNA molecules with diverse roles in various diseases including diseases of the liver. In this review, we highlight the latest advances in the field of microRNA biology and their potential as emerging therapeutic targets in liver disease.

  13. Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection

    DEFF Research Database (Denmark)

    Lanford, Robert E; Hildebrandt-Eriksen, Elisabeth S; Petri, Andreas;

    2010-01-01

    The liver-expressed microRNA-122 (miR-122) is essential for hepatitis C virus (HCV) RNA accumulation in cultured liver cells, but its potential as a target for antiviral intervention has not been assessed. We found that treatment of chronically infected chimpanzees with a locked nucleic acid (LNA...

  14. Virus-induced gene silencing of the RPC5-like subunit of RNA polymerase III caused pleiotropic effects in Nicotiana benthamiana.

    Science.gov (United States)

    Nemchinov, Lev G; Boutanaev, Alexander M; Postnikova, Olga A

    2016-01-01

    In eukaryotic cells, RNA polymerase III is highly conserved and transcribes housekeeping genes such as ribosomal 5S rRNA, tRNA and other small RNAs. The RPC5-like subunit is one of the 17 subunits forming RNAPIII and its exact functional roles in the transcription are poorly understood. In this work, we report that virus-induced gene silencing of transcripts encoding a putative RPC5-like subunit of the RNA Polymerase III in a model species Nicotiana benthamiana had pleiotropic effects, including but not limited to severe dwarfing appearance, chlorosis, nearly complete reduction of internodes and abnormal leaf shape. Using transcriptomic analysis, we identified genes and pathways affected by RPC5 silencing and thus presumably related to the cellular roles of the subunit as well as to the downstream cascade of reactions in response to partial loss of RNA Polymerase III function. Our results suggest that silencing of the RPC5L in N. benthamiana disrupted not only functions commonly associated with the core RNA Polymerase III transcripts, but also more diverse cellular processes, including responses to stress. We believe this is the first demonstration that activity of the RPC5 subunit is critical for proper functionality of RNA Polymerase III and normal plant development. PMID:27282827

  15. Silencing of SOX12 by shRNA suppresses migration, invasion and proliferation of breast cancer cells

    Science.gov (United States)

    Ding, Hanzhi; Quan, Hong; Yan, Weiguo; Han, Jing

    2016-01-01

    Sex determining region Y-box protein 12 (SOX12) is essential for embryonic development and cell-fate determination. The role of SOX12 in tumorigenesis of breast cancer is not well-understood. Here, we found that SOX12 mRNA expression was up-regulated in human breast cancer tissues. To clarify the roles of SOX12 in breast cancer, we used lentiviral shRNAs to suppress its expression in two breast cancer cells with relatively higher expression of SOX12 (BT474 and MCF-7). Our findings strongly suggested that SOX12 was critical for cell migration and invasion of breast cancer cells. We found that silencing of SOX12 significantly decreased the mRNA and protein levels of MMP9 and Twist, while notably increased E-cadherin. Moreover, SOX12 knockdown significantly inhibited the proliferation of breast cancer cells in vitro and the growth of xenograft tumours in vivo. Flow cytometry analysis revealed that breast cancer cells with SOX12 knockdown showed cell cycle arrest and decreased mRNA and protein levels of proliferating cell nuclear antigen (PCNA), CDK2 and Cyclin D1. Taken together, SOX12 plays an important role in growth inhibition through cell-cycle arrest, as well as migration and invasion of breast cancer cells. PMID:27582508

  16. Melon RNA interference (RNAi) lines silenced for Cm-eIF4E show broad virus resistance.

    Science.gov (United States)

    Rodríguez-Hernández, Ana M; Gosalvez, Blanca; Sempere, Raquel N; Burgos, Lorenzo; Aranda, Miguel A; Truniger, Verónica

    2012-09-01

    Efficient and sustainable control of plant viruses may be achieved using genetically resistant crop varieties, although resistance genes are not always available for each pathogen; in this regard, the identification of new genes that are able to confer broad-spectrum and durable resistance is highly desirable. Recently, the cloning and characterization of recessive resistance genes from different plant species has pointed towards eukaryotic translation initiation factors (eIF) of the 4E family as factors required for the multiplication of many different viruses. Thus, we hypothesized that eIF4E may control the susceptibility of melon (Cucumis melo L.) to a broad range of viruses. To test this hypothesis, Cm-eIF4E knockdown melon plants were generated by the transformation of explants with a construct that was designed to induce the silencing of this gene, and the plants from T2 generations were genetically and phenotypically characterized. In transformed plants, Cm-eIF4E was specifically silenced, as identified by the decreased accumulation of Cm-eIF4E mRNA and the appearance of small interfering RNAs derived from the transgene, whereas the Cm-eIF(iso)4E mRNA levels remained unaffected. We challenged these transgenic melon plants with eight agronomically important melon-infecting viruses, and identified that they were resistant to Cucumber vein yellowing virus (CVYV), Melon necrotic spot virus (MNSV), Moroccan watermelon mosaic virus (MWMV) and Zucchini yellow mosaic virus (ZYMV), indicating that Cm-eIF4E controls melon susceptibility to these four viruses. Therefore, Cm-eIF4E is an efficient target for the identification of new resistance alleles able to confer broad-spectrum virus resistance in melon.

  17. P19 of Tomato Bushy Stunt Virus Suppresses RNA Silencing Induced by Short Hairpin RNA in Mammal Cells

    Institute of Scientific and Technical Information of China (English)

    Wei-xian CHEN; Juan CHEN; Zhen-zhen ZHANG; Ai-long HUANG

    2007-01-01

    To counteract the immune system in parasitic hosts, some viruses encode proteins to suppress the RNA interference (RNAi) effect. In this report, we established two RNAi systems to be easily observed with strong and obvious effect. The function of the P19 of tomato bushy stunt virus, which suppresses RNAi in mammal cells, was then studied using these two systems. Short hairpin RNAs targeting green fluorescence protein (pshRNA-GFP) and firefly luciferase (pshRNA-luc) were designed and inserted into a eukaryotic transcriptional vector pTZU6+1, respectively. The shRNA expressing vectors were co-transfected with plasmids containing the target gene with or without P19. The GFP expression level was assayed by fluorescence microscopy, Western blotting and RT-PCR. The luciferase expression level was analyzed by the dual-luciferase assay system. pshRNA designed in this study down-regulated the target gene specifically and efficiently, with a decrease of expression of both genes of about 70%, respectively. When P19 was introduced into the RNAi systems, the expression of both GFP and the luciferase were mostly recovered compared with the control groups. The RNAi systems of GFP and luciferase were constructed successfully, demonstrating that P19 of tomato bushy stunt virus has the ability to counteract the RNAi effect induced by shRNA in mammal cells.

  18. CTLA4 Silencing with siRNA Promotes Deviation of Th1/Th2 in Chronic Hepatitis B Patients

    Institute of Scientific and Technical Information of China (English)

    Yongsheng Yu; Hao Wu; Zhenghao Tang; Guoqing Zang

    2009-01-01

    To determine whether RNA interference (RNAi) could block cytotoxic T-lymphocyte antigen 4 (CTLA4) in human lymphocytes in vitro and promote IFN-γ and IL-2 secretions, three small interfering RNAs (siRNAs) were selected based on target specificity sequences of human CTLA4 and transfected into human lymphocytes of chronic HBV patients. As a result, the expression of human CTLA4 mRNA was efficiently suppressed by all the three siRNAs. Compared with negative control (siRNA-co), siRNA-1 inhibited the expression of CTLA4 most efficiently and was used in the further study. The expressions of IFN-γ and IL-2 were upregulated and the level of IL-4 was almost unchanged in lymphocytes transfected with siRNA-1 compared with the blank control. These results indicated that siRNA-1 led to IFN-γ and IL-2 secretions, which is a main response of Th1/Th2. In a conclusion, RNAi significantly suppressed the expression of human CTLA4 mRNA in human lymphocytes in vitro, and could induce Th1/Th2 response. It could be a new therapeutic strategy for chronic HBV infection. Cellular & Molecular Immunology.

  19. Specific degradation of 3' regions of GUS mRNA in posttranscriptionally silenced tobacco lines may be related to 5'-3' spreading of silencing

    DEFF Research Database (Denmark)

    Braunstein, Thomas Hartig; Moury, Benoit; Johannessen, Marina;

    2002-01-01

    Target regions for posttranscriptional silencing of transgenes often reside in the 3' region of the coding sequence, although there are exceptions. To resolve if the target region is determined by the gene undergoing silencing rather than by the structure of the transgene loci or the plant genetic...... background, we have performed detailed analyses of target regions in three spontaneously beta-glucuronidase (GUS) silencing tobacco lines of different origin. From quantitative cosuppression experiments, we show that the main target region in all three tobacco lines is found within the 3' half of the GUS...... coding region but upstream of the last 200 nt. The quantities of small (21-25 nt) RNAs homologous to 5' or 3' regions of the GUS coding sequence were found to correlate approximately with the target strength of the corresponding regions. These results suggest that transgene locus structure and plant...

  20. Carbon nanotube-mediated siRNA delivery for gene silencing in cancer cells

    Science.gov (United States)

    Hong, Tu; Guo, Honglian; Xu, Yaqiong

    2011-10-01

    Small interfering RNA (siRNA) is potentially a promising tool in influencing gene expression with a high degree of target specificity. However, its poor intracellular uptake, instability in vivo, and non-specific immune stimulations impeded its effect in clinical applications. In this study, carbon nanotubes (CNTs) functionalized with two types of phospholipid-polyethylene glycol (PEG) have shown capabilities to stabilize siRNA in cell culture medium during the transfection and efficiently deliver siRNA into neuroblastoma and breast cancer cells. Moreover, the intrinsic optical properties of CNTs have been investigated through absorption and fluorescence measurements. We have found that the directly-functionalized groups play an important role on the fluorescence imaging of functionalized CNTs. The unique fluorescence imaging and high delivery efficiency make CNTs a promising material to deliver drugs and evaluate the treatment effect simultaneously.

  1. Synthetic Polymer Hybridization with DNA and RNA Directs Nanoparticle Loading, Silencing Delivery, and Aptamer Function

    OpenAIRE

    Zhou, Zhun; Xia, Xin; Bong, Dennis

    2015-01-01

    We report herein discrete triplex hybridization of DNA and RNA with polyacrylates. Length-monodisperse triazine-derivatized polymers were prepared on gram-scale by reversible addition–fragmentation chain-transfer polymerization. Despite stereoregio backbone heterogeneity, the triazine polymers bind T/U-rich DNA or RNA with nanomolar affinity upon mixing in a 1:1 ratio, as judged by thermal melts, circular dichroism, gel-shift assays, and fluorescence quenching. We call these polyacrylates “bi...

  2. Resistance to Sri Lankan Cassava Mosaic Virus (SLCMV) in Genetically Engineered Cassava cv. KU50 through RNA Silencing

    KAUST Repository

    Ntui, Valentine Otang

    2015-04-22

    Cassava ranks fifth among the starch producing crops of the world, its annual bioethanol yield is higher than for any other crop. Cassava cultivar KU50, the most widely grown cultivar for non-food purposes is susceptible to Sri Lankan cassava mosaic virus (SLCMV). The objective of this work was to engineer resistance to SLCMV by RNA interference (RNAi) in order to increase biomass yield, an important aspect for bioethanol production. Here, we produced transgenic KU50 lines expressing dsRNA homologous to the region between the AV2 and AV1 of DNA A of SLCMV. High level expression of dsRNA of SLCMV did not induce any growth abnormality in the transgenic plants. Transgenic lines displayed high levels of resistance to SLCMV compared to the wild-type plants and no virus load could be detected in uninoculated new leaves of the infected resistant lines after PCR amplification and RT-PCR analysis. The agronomic performance of the transgenic lines was unimpaired after inoculation with the virus as the plants presented similar growth when compared to the mock inoculated control plants and revealed no apparent reduction in the amount and weight of tubers produced. We show that the resistance is correlated with post-transcriptional gene silencing because of the production of transgene specific siRNA. The results demonstrate that transgenic lines exhibited high levels of resistance to SLCMV. This resistance coupled with the desirable yield components in the transgenic lines makes them better candidates for exploitation in the production of biomass as well as bioethanol.

  3. Resistance to Sri Lankan cassava mosaic virus (SLCMV in genetically engineered cassava cv. KU50 through RNA silencing.

    Directory of Open Access Journals (Sweden)

    Valentine Otang Ntui

    Full Text Available Cassava ranks fifth among the starch producing crops of the world, its annual bioethanol yield is higher than for any other crop. Cassava cultivar KU50, the most widely grown cultivar for non-food purposes is susceptible to Sri Lankan cassava mosaic virus (SLCMV. The objective of this work was to engineer resistance to SLCMV by RNA interference (RNAi in order to increase biomass yield, an important aspect for bioethanol production. Here, we produced transgenic KU50 lines expressing dsRNA homologous to the region between the AV2 and AV1 of DNA A of SLCMV. High level expression of dsRNA of SLCMV did not induce any growth abnormality in the transgenic plants. Transgenic lines displayed high levels of resistance to SLCMV compared to the wild-type plants and no virus load could be detected in uninoculated new leaves of the infected resistant lines after PCR amplification and RT-PCR analysis. The agronomic performance of the transgenic lines was unimpaired after inoculation with the virus as the plants presented similar growth when compared to the mock inoculated control plants and revealed no apparent reduction in the amount and weight of tubers produced. We show that the resistance is correlated with post-transcriptional gene silencing because of the production of transgene specific siRNA. The results demonstrate that transgenic lines exhibited high levels of resistance to SLCMV. This resistance coupled with the desirable yield components in the transgenic lines makes them better candidates for exploitation in the production of biomass as well as bioethanol.

  4. Resistance to Sri Lankan cassava mosaic virus (SLCMV) in genetically engineered cassava cv. KU50 through RNA silencing.

    Science.gov (United States)

    Ntui, Valentine Otang; Kong, Kynet; Khan, Raham Sher; Igawa, Tomoko; Janavi, Gnanaguru Janaky; Rabindran, Ramalingam; Nakamura, Ikuo; Mii, Masahiro

    2015-01-01

    Cassava ranks fifth among the starch producing crops of the world, its annual bioethanol yield is higher than for any other crop. Cassava cultivar KU50, the most widely grown cultivar for non-food purposes is susceptible to Sri Lankan cassava mosaic virus (SLCMV). The objective of this work was to engineer resistance to SLCMV by RNA interference (RNAi) in order to increase biomass yield, an important aspect for bioethanol production. Here, we produced transgenic KU50 lines expressing dsRNA homologous to the region between the AV2 and AV1 of DNA A of SLCMV. High level expression of dsRNA of SLCMV did not induce any growth abnormality in the transgenic plants. Transgenic lines displayed high levels of resistance to SLCMV compared to the wild-type plants and no virus load could be detected in uninoculated new leaves of the infected resistant lines after PCR amplification and RT-PCR analysis. The agronomic performance of the transgenic lines was unimpaired after inoculation with the virus as the plants presented similar growth when compared to the mock inoculated control plants and revealed no apparent reduction in the amount and weight of tubers produced. We show that the resistance is correlated with post-transcriptional gene silencing because of the production of transgene specific siRNA. The results demonstrate that transgenic lines exhibited high levels of resistance to SLCMV. This resistance coupled with the desirable yield components in the transgenic lines makes them better candidates for exploitation in the production of biomass as well as bioethanol.

  5. Hydrophobic interactions between polymeric carrier and palmitic acid-conjugated siRNA improve PEGylated polyplex stability and enhance in vivo pharmacokinetics and tumor gene silencing.

    Science.gov (United States)

    Sarett, Samantha M; Werfel, Thomas A; Chandra, Irene; Jackson, Meredith A; Kavanaugh, Taylor E; Hattaway, Madison E; Giorgio, Todd D; Duvall, Craig L

    2016-08-01

    Formation of stable, long-circulating siRNA polyplexes is a significant challenge in translation of intravenously-delivered, polymeric RNAi cancer therapies. Here, we report that siRNA hydrophobization through conjugation to palmitic acid (siPA) improves stability, in vivo pharmacokinetics, and tumor gene silencing of PEGylated nanopolyplexes (siPA-NPs) with balanced cationic and hydrophobic content in the core relative to the analogous polyplexes formed with unmodified siRNA, si-NPs. Hydrophobized siPA loaded into the NPs at a lower charge ratio (N(+):P(-)) relative to unmodified siRNA, and siPA-NPs had superior resistance to siRNA cargo unpackaging in comparison to si-NPs upon exposure to the competing polyanion heparin and serum. In vitro, siPA-NPs increased uptake in MDA-MB-231 breast cancer cells (100% positive cells vs. 60% positive cells) but exhibited equivalent silencing of the model gene luciferase relative to si-NPs. In vivo in a murine model, the circulation half-life of intravenously-injected siPA-NPs was double that of si-NPs, resulting in a >2-fold increase in siRNA biodistribution to orthotopic MDA-MB-231 mammary tumors. The increased circulation half-life of siPA-NPs was dependent upon the hydrophobic interactions of the siRNA and the NP core component and not just siRNA hydrophobization, as siPA did not contribute to improved circulation time relative to unmodified siRNA when delivered using polyplexes with a fully cationic core. Intravenous delivery of siPA-NPs also achieved significant silencing of the model gene luciferase in vivo (∼40% at 24 h after one treatment and ∼60% at 48 h after two treatments) in the murine MDA-MB-231 tumor model, while si-NPs only produced a significant silencing effect after two treatments. These data suggest that stabilization of PEGylated siRNA polyplexes through a combination of hydrophobic and electrostatic interactions between siRNA cargo and the polymeric carrier improves in vivo pharmacokinetics and

  6. Silencing MicroRNA-155 Attenuates Cardiac Injury and Dysfunction in Viral Myocarditis via Promotion of M2 Phenotype Polarization of Macrophages.

    Science.gov (United States)

    Zhang, Yingying; Zhang, Mengying; Li, Xueqin; Tang, Zongsheng; Wang, Xiangmin; Zhong, Min; Suo, Qifeng; Zhang, Yao; Lv, Kun

    2016-01-01

    Macrophage infiltration is a hallmark feature of viral myocarditis. As studies have shown that microRNA-155 regulates the differentiation of macrophages, we aimed to investigate the role of microRNA-155 in VM. We report that silencing microRNA-155 protects mice from coxsackievirus B3 induced myocarditis. We found that microRNA-155 expression was upregulated and localized primarily in heart-infiltrating macrophages and CD4(+) T lymphocytes during acute myocarditis. In contrast with wildtype (WT) mice, microRNA-155(-/-) mice developed attenuated viral myocarditis, which was characterized by decreased cardiac inflammation and decreased intracardiac CD45(+) leukocytes. Hearts of microRNA-155(-/-) mice expressed decreased levels of the IFN-γ and increased levels of the cytokines IL-4 and IL-13. Although total CD4(+) and regulatory T cells were unchanged in miR-155(-/-) spleen proportionally, the activation of T cells and CD4(+) T cell proliferation in miR-155(-/-) mice were significantly decreased. Beyond the acute phase, microRNA-15(5-/-) mice had reduced mortality and improved cardiac function during 5 weeks of follow-up. Moreover, silencing microRNA-155 led to increased levels of alternatively-activated macrophages (M2) and decreased levels of classically-activated macrophages (M1) in the heart. Combined, our studies suggest that microRNA-155 confers susceptibility to viral myocarditis by affecting macrophage polarization, and thus may be a potential therapeutic target for viral myocarditis. PMID:26931072

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

    Institute of Scientific and Technical Information of China (English)

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

    2012-01-01

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

  8. Differential RNA silencing suppression activity of NS1 proteins from different influenza A virus strains

    NARCIS (Netherlands)

    W. de Vries; J. Haasnoot; R. Fouchier; P. de Haan; B. Berkhout

    2009-01-01

    The NS1 gene of influenza A virus encodes a multi-functional protein that plays an important role in counteracting cellular antiviral mechanisms such as the interferon (IFN), protein kinase R and retinoic acid-inducible gene product I pathways. In addition, NS1 has recently been shown to have RNA in

  9. Enhanced salt sensitivity following shRNA silencing of neuronal TRPV1 in rat spinal cord

    Institute of Scientific and Technical Information of China (English)

    Shuang-quan YU; Donna H WANG

    2011-01-01

    Aim: To investigate the effects of selective knockdown of TRPV1 channels in the lower thoracic and upper lumbar segments of spinal cord, dorsal root ganglia (DRG) and me senteric arteries on rat blood pressure responses to high salt intake.Methods: TRPV1 short-hairpin RNA (shRNA) was delivered using intrathecal injection (6 μg.kg1-d-1, for 3 d). Levels of TRPV1 and tyrosine hydroxylase expression were determined by Western blot analysis. Systolic blood pressure and mean arterial pressure (MAP) were examined using tail-cuff and direct arterial measurement, respectively.Results: In rats injected with control shRNA, high-salt diet (HS) caused higher systolic blood pressure compared with normal-salt diet(NS) (HS:149±4 mmHg; NS:126±2 mmHg, P<0.05). Intrathecal injection of TRPV1 shRNA significantly increased the systolic blood pressure in both HS rats and NS rats (HS:169±3 mmHg; NS:139±2 mmHg). The increases was greater in HS rats than in NS rats (HS:13.9%±1.8%; NS: 9.8±0.7, P<0.05). After TRPV1 shRNA treatment, TRPV1 expression in the dorsal horn and DRG of T8-L3 segments and in mesenteric arteries was knocked down to a greater extent in HS rats compared with NS rats. Blockade of α1-adrenoceptors abolished the TRPV1 shRNA-induced pressor effects. In rats injected with TRPV1 shRNA, level of tyrosine hydroxylase in mesenteric arteries was increased to a greater extent in HS rats compared with NS rats.Conclusion: Selective knockdown of TRPV1 expression in the lower thoracic and upper lumbar segments of spinal cord, DRG, and mesenteric arteries enhanced the prohypertensive effects of high salt intake, suggesting that TRPV1 channels in these sites protect against increased salt sensitivity, possibly via suppression of sympatho-excitatory responses.

  10. Viral RNA Silencing Suppressors (RSS): Novel Strategy of Viruses to Ablate the Host RNA Interference (RNAi) Defense System

    OpenAIRE

    Bivalkar-Mehla, Shalmali; Vakharia, Janaki; Mehla, Rajeev; Abreha, Measho; Kanwar, Jagat Rakesh; Tikoo, Akshay; Chauhan, Ashok

    2010-01-01

    Pathogenic viruses have developed a molecular defense arsenal for their survival by counteracting the host anti-viral system known as RNA interference (RNAi). Cellular RNAi, in addition to regulating gene expression through microRNAs, also serves as a barrier against invasive foreign nucleic acids. RNAi is conserved across the biological species, including plants, animals and invertebrates. Viruses in turn, have evolved mechanisms that can counteract this anti-viral defense of the host. Recen...

  11. APC/C-mediated degradation of dsRNA-binding protein 4 (DRB4 involved in RNA silencing.

    Directory of Open Access Journals (Sweden)

    Katia Marrocco

    Full Text Available BACKGROUND: Selective protein degradation via the ubiquitin-26S proteasome is a major mechanism underlying DNA replication and cell division in all Eukaryotes. In particular, the APC/C (Anaphase Promoting Complex or Cyclosome is a master ubiquitin protein ligase (E3 that targets regulatory proteins for degradation allowing sister chromatid separation and exit from mitosis. Interestingly, recent work also indicates that the APC/C remains active in differentiated animal and plant cells. However, its role in post-mitotic cells remains elusive and only a few substrates have been characterized. METHODOLOGY/PRINCIPAL FINDINGS: In order to identify novel APC/C substrates, we performed a yeast two-hybrid screen using as the bait Arabidopsis APC10/DOC1, one core subunit of the APC/C, which is required for substrate recruitment. This screen identified DRB4, a double-stranded RNA binding protein involved in the biogenesis of different classes of small RNA (sRNA. This protein interaction was further confirmed in vitro and in plant cells. Moreover, APC10 interacts with DRB4 through the second dsRNA binding motif (dsRBD2 of DRB4, which is also required for its homodimerization and binding to its Dicer partner DCL4. We further showed that DRB4 protein accumulates when the proteasome is inactivated and, most importantly, we found that DRB4 stability depends on APC/C activity. Hence, depletion of Arabidopsis APC/C activity by RNAi leads to a strong accumulation of endogenous DRB4, far beyond its normal level of accumulation. However, we could not detect any defects in sRNA production in lines where DRB4 was overexpressed. CONCLUSIONS/SIGNIFICANCE: Our work identified a first plant substrate of the APC/C, which is not a regulator of the cell cycle. Though we cannot exclude that APC/C-dependent degradation of DRB4 has some regulatory roles under specific growth conditions, our work rather points to a housekeeping function of APC/C in maintaining precise cellular

  12. A versatile building block: the structures and functions of negative-sense single-stranded RNA virus nucleocapsid proteins

    OpenAIRE

    Sun, Yuna; Guo, Yu; Lou, Zhiyong

    2012-01-01

    Nucleocapsid protein (NPs) of negative-sense single-stranded RNA (-ssRNA) viruses function in different stages of viral replication, transcription, and maturation. Structural investigations show that -ssRNA viruses that encode NPs preliminarily serve as structural building blocks that encapsidate and protect the viral genomic RNA and mediate the interaction between genomic RNA and RNA-dependent RNA polymerase. However, recent structural results have revealed other biological functions of -ssR...

  13. siRNA-induced silencing of hypoxia-inducible factor 3α (HIF3α) increases endurance capacity in rats.

    Science.gov (United States)

    Drozdovska, S; Gavenauskas, B; Drevytska, T; Nosar, V; Nagibin, V; Mankovska, I; Dosenko, V

    2016-06-01

    Molecular mechanisms of adaptation to exercise despite a large number of studies remain unclear. One of the crucial factors in this process is hypoxia inducible factor (HIF) that regulates transcription of many target genes encoding proteins that are implicated in molecular adaptation to hypoxia. Experiments were conducted on 24 adult male Fisher rats. Real-time PCR analysis was performed for quantitative evaluation of Hif3α, Igf1, Glut-4 and Pdk-1 in m. gastrocnemius, m. soleus, in lung and heart tissues. Mitochondrial respiratory function and electron microscopy were performed. Knockdown of Hif3α using siRNA increases time of swimming to exhaustion by 1.5 times. Level of mitochondrial NAD- and FAD-dependent oxidative pathways is decreased, however efficiency of phosphorylation is increased after Hif3α siRNA treatment. Expression of HIF target genes in muscles was not changed significantly, except for increasing of Pdk-1 expression in m. soleus by 2.1 times. More prominent changes were estimated in lung and heart: Igf1 gene expression was increased by 32.5 and 37.5 times correspondingly. Glut4 gene expression in lungs was increased from undetected level till 0.3 rel. units and by 84.2 times in heart. Level of Pdk1 gene expression was increased by 249.2 in lungs and by 35.1 times in hearts, correspondingly. Some destructive changes in muscle tissue were detected in animals with siRNA-inducing silencing of Hif3α. PMID:27274101

  14. Well-Defined Degradable Cationic Polylactide as Nanocarrier for the Delivery of siRNA to Silence Angiogenesis in Prostate Cancer

    OpenAIRE

    Chen, Chih-Kuang; Law, Wing-Cheung; Aalinkeel, Ravikumar; Nair, Bindukumar; Kopwitthaya, Atcha; Mahajan, Supriya D.; Reynolds, Jessica L.; Zou, Jiong; Schwartz, Stanley A; Paras N. Prasad; Cheng, Chong

    2012-01-01

    Well-defined tertiary amine-functionalized cationic polylactides (CPLAs) are synthesized by thiol-ene click functionalization of an allyl-functionalized polylactide, and utilized here for the delivery of interleukin-8 (IL-8) siRNA via CPLA-IL-8 siRNA nanoplexes. The CPLAs possess remarkable hydrolytic degradability, and their cytotoxicity is relatively low. The CPLA-IL-8 siRNA nanoplexes can be readily taken up by prostate cancer cells, resulting in significant IL-8 gene silencing. It is foun...

  15. Silencing Aurora-A with siRNA inhibits cell proliferation in human lung adenocarcinoma cells.

    Science.gov (United States)

    Zhong, Ning; Shi, Shunbin; Wang, Hongzhen; Wu, Guangzhou; Wang, Yunliang; Ma, Qiang; Wang, Hongwei; Liu, Yuanhua; Wang, Jinzhi

    2016-09-01

    Aurora kinase A (AURKA) is an oncogenic serine/threonine kinase, it plays important roles in tumorigenesis and chemoresistance. In this study, we investigated the expression of AURKA in lung adenocarcinoma tissues, the role of small interference RNA targeting AURKA on growth, cell cycle, and apoptosis of lung adenocarcinoma cell lines in vitro. The AURKA is highly expressed in lung adenocarcinoma tissues and human lung adenocarcinoma cell lines. Lentivirus-mediated short hairpin RNA (shRNA) was used to knock down AURKA expression in human lung adenocarcinoma cell lines H1299 and A549. The results indicated that depletion of AURKA could inhibit cell growth, cause cell cycle arrest and apoptosis. The potential mechanisms of AURKA inhibition induced cell cycle arrest and apoptosis are associated with downregulated RAF-1, CCND2, CCND3, CDK4, PAK4, EGFR and upregulated WEE1 expression. Furthermore, AURKA knockdown cooperated with vincristine (VCR) to repress A549 cell proliferation. Therefore, AURKA plays important roles in the proliferation of human lung adenocarcinoma cells, which suggests that AURKA could be a promising tool for lung adenocarcinoma therapy. PMID:27571708

  16. Expression of RNA-interference/antisense transgenes by the cognate promoters of target genes is a better gene-silencing strategy to study gene functions in rice.

    Science.gov (United States)

    Li, Jing; Jiang, Dagang; Zhou, Hai; Li, Feng; Yang, Jiawei; Hong, Laifa; Fu, Xiao; Li, Zhibin; Liu, Zhenlan; Li, Jianming; Zhuang, Chuxiong

    2011-03-03

    Antisense and RNA interference (RNAi)-mediated gene silencing systems are powerful reverse genetic methods for studying gene function. Most RNAi and antisense experiments used constitutive promoters to drive the expression of RNAi/antisense transgenes; however, several reports showed that constitutive promoters were not expressed in all cell types in cereal plants, suggesting that the constitutive promoter systems are not effective for silencing gene expression in certain tissues/organs. To develop an alternative method that complements the constitutive promoter systems, we constructed RNAi and/or antisense transgenes for four rice genes using a constitutive promoter or a cognate promoter of a selected rice target gene and generated many independent transgenic lines. Genetic, molecular, and phenotypic analyses of these RNAi/antisense transgenic rice plants, in comparison to previously-reported transgenic lines that silenced similar genes, revealed that expression of the cognate promoter-driven RNAi/antisense transgenes resulted in novel growth/developmental defects that were not observed in transgenic lines expressing constitutive promoter-driven gene-silencing transgenes of the same target genes. Our results strongly suggested that expression of RNAi/antisense transgenes by cognate promoters of target genes is a better gene-silencing approach to discovery gene function in rice.

  17. Expression of RNA-interference/antisense transgenes by the cognate promoters of target genes is a better gene-silencing strategy to study gene functions in rice.

    Directory of Open Access Journals (Sweden)

    Jing Li

    Full Text Available Antisense and RNA interference (RNAi-mediated gene silencing systems are powerful reverse genetic methods for studying gene function. Most RNAi and antisense experiments used constitutive promoters to drive the expression of RNAi/antisense transgenes; however, several reports showed that constitutive promoters were not expressed in all cell types in cereal plants, suggesting that the constitutive promoter systems are not effective for silencing gene expression in certain tissues/organs. To develop an alternative method that complements the constitutive promoter systems, we constructed RNAi and/or antisense transgenes for four rice genes using a constitutive promoter or a cognate promoter of a selected rice target gene and generated many independent transgenic lines. Genetic, molecular, and phenotypic analyses of these RNAi/antisense transgenic rice plants, in comparison to previously-reported transgenic lines that silenced similar genes, revealed that expression of the cognate promoter-driven RNAi/antisense transgenes resulted in novel growth/developmental defects that were not observed in transgenic lines expressing constitutive promoter-driven gene-silencing transgenes of the same target genes. Our results strongly suggested that expression of RNAi/antisense transgenes by cognate promoters of target genes is a better gene-silencing approach to discovery gene function in rice.

  18. Silencing of the metastasis-linked gene, AEG-1, using siRNA-loaded cholamine surface-modified gelatin nanoparticles in the breast carcinoma cell line MCF-7.

    Science.gov (United States)

    Abozeid, Salma M; Hathout, Rania M; Abou-Aisha, Khaled

    2016-09-01

    Cholamine surface-modified gelatin nanoparticles prepared by the double desolvation method using acetone as a dehydrating agent were selected and potentially evaluated as non viral vectors of siRNA targeting a metastatic gene AEG-1 in MCF-7 breast carcinoma cells. The ability of modified gelatin nanoparticle to complex and deliver siRNA for gene silencing was investigated. Hence, Particle size, surface charge (zeta potential) and morphology of siRNA/Gelatin nanoparticles (siGNPs) were characterized via dynamic light scattering (DLS), scanning electron microscopy (SEM) and transmission electron microscope (TEM). Moreover, the nanoparticles cytotoxicity, loading efficiency and interaction with MCF-7 human breast carcinoma cells were evaluated. Cationized GNPs of mean size range of 174nm and PDI of 0.101 were produced. The loading efficiency of siGNPs at a Nitrogen/Phosphate (N/P) ratio (w/w) of 200:1 was approximately 96%. Cellular uptake was evaluated after FITC conjugation where the particles produced high transfection efficiency. Finally, ELISA analysis of AEG-1/MTDH expression demonstrated the gene silencing effect of siGNPs, as more than 75% MTDH protein were inhibited. Our data indicate that cholamine modified GNPs pose a promising non-viral siRNA carrier for altering gene expression in MCF-7 breast cancer cells with many advantages such as relatively high gene transfection efficiency and efficient silencing ability. PMID:27285732

  19. Lentiviral vector-mediated gene transfer and RNA silencing technology in neuronal dysfunctions.

    Science.gov (United States)

    Dreyer, Jean-Luc

    2011-02-01

    Lentiviral-mediated gene transfer in vivo or in cultured mammalian neurons can be used to address a wide variety of biological questions, to design animals models for specific neurodegenerative pathologies, or to test potential therapeutic approaches in a variety of brain disorders. Lentiviruses can infect non-dividing cells, thereby allowing stable gene transfer in post-mitotic cells such as mature neurons. An important contribution has been the use of inducible vectors: the same animal can thus be used repeatedly in the doxycycline-on or -off state, providing a powerful mean for assessing the function of a gene candidate in a disorder within a specific neuronal circuit. Furthermore, lentivirus vectors provide a unique tool to integrate siRNA expression constructs with the aim to locally knockdown expression of a specific gene, enabling to assess the function of a gene in a very specific neuronal pathway. Lentiviral vector-mediated delivery of short hairpin RNA results in persistent knockdown of gene expression in the brain. Therefore, the use of lentiviruses for stable expression of siRNA in brain is a powerful aid to probe gene functions in vivo and for gene therapy of diseases of the central nervous system. In this chapter I review the applications of lentivirus-mediated gene transfer in the investigation of specific gene candidates involved in major brain disorders and neurodegenerative processes. Major applications have been in polyglutamine disorders, such as synucleinopathies and Parkinson's disease, or in investigating gene function in Huntington's disease, dystonia, or muscular dystrophy. Recently, lentivirus gene transfer has been an invaluable tool for evaluation of gene function in behavioral disorders such as drug addiction and attention-deficit hyperactivity disorder or in learning and cognition. PMID:20862616

  20. Novel mRNA-silencing bodies at the synapse: A never-ending story.

    Science.gov (United States)

    Thomas, María Gabriela; Boccaccio, Graciela Lidia

    2016-01-01

    Several cellular responses depend on translational regulation and in most cases, this involves the formation of cytoplasmic granules that contain repressed mRNAs. In neurons, numerous mRNAs travel along dendrites to be locally regulated upon synapse activity and we have recently shown that the exoribonuclease XRN1 forms dynamic aggregates at the post synapse that respond to specific stimuli.(1) These foci were termed SX-bodies and are distinct from stress granules (SGs), processing bodies (PBs) and other RNA granules previously described. Together with Smaug1-foci and FMRP-granules, the SX-bodies contribute to dynamically shape the transcriptome available for translation at the post-synapse.

  1. Experimental identification of microRNA-140 targets by silencing and overexpressing miR-140

    DEFF Research Database (Denmark)

    Nicolas, Francisco Esteban; Pais, Helio; Schwach, Frank;

    2008-01-01

    MicroRNAs (miRNAs) are short noncoding RNA molecules regulating the expression of mRNAs. Target identification of miRNAs is computationally difficult due to the relatively low homology between miRNAs and their targets. We present here an experimental approach to target identification where the ca...... on the presence of the seed sequence. Interestingly, none of these were predicted by the published target prediction methods we used. One of the potential target mRNAs, Cxcl12, was experimentally validated by Northern blot analysis and a luciferase reporter assay....

  2. Structure of the Cmr2 Subunit of the CRISPR-Cas RNA Silencing Complex

    Energy Technology Data Exchange (ETDEWEB)

    Cocozaki, Alexis I.; Ramia, Nancy F.; Shao, Yaming; Hale, Caryn R.; Terns, Rebecca M.; Terns, Michael P.; Li, Hong (FSU); (Georgia)

    2012-08-10

    Cmr2 is the largest and an essential subunit of a CRISPR RNA-Cas protein complex (the Cmr complex) that cleaves foreign RNA to protect prokaryotes from invading genetic elements. Cmr2 is thought to be the catalytic subunit of the effector complex because of its N-terminal HD nuclease domain. Here, however, we report that the HD domain of Cmr2 is not required for cleavage by the complex in vitro. The 2.3 {angstrom} crystal structure of Pyrococcus furiosus Cmr2 (lacking the HD domain) reveals two adenylyl cyclase-like and two {alpha}-helical domains. The adenylyl cyclase-like domains are arranged as in homodimeric adenylyl cyclases and bind ADP and divalent metals. However, mutagenesis studies show that the metal- and ADP-coordinating residues of Cmr2 are also not critical for cleavage by the complex. Our findings suggest that another component provides the catalytic function and that the essential role by Cmr2 does not require the identified ADP- or metal-binding or HD domains in vitro.

  3. Long noncoding RNA PVT1 promotes cervical cancer progression through epigenetically silencing miR-200b.

    Science.gov (United States)

    Zhang, Shaorong; Zhang, Guanli; Liu, Jingying

    2016-08-01

    Long noncoding RNA PVT1 has been reported to be dysregulated and play vital roles in a variety of cancers. However, the functions and molecular mechanisms of PVT1 in cervical cancer remain unclear. The objective of this study was to investigate the expression, clinical significance, biological roles, and underlying functional mechanisms of PVT1 in cervical cancer. Our results revealed that PVT1 is upregulated in cervical cancer tissues. Enhanced expression of PVT1 is associated with larger tumor size, advanced International Federation of Gynecology and Obstetrics stage, and poor prognosis of cervical cancer patients. Using gain-of-function and loss-of-function approaches, we demonstrated that overexpression of PVT1 promotes cervical cancer cells proliferation, cell cycle progression and migration, and depletion of PVT1 inhibits cervical cancer cell proliferation, cell cycle progression, and migration. Mechanistically, we verified that PVT1 binds to EZH2, recruits EZH2 to the miR-200b promoter, increases histone H3K27 trimethylation level on the miR-200b promoter, and inhibits miR-200b expression. Furthermore, the effects of PVT1 on cervical cell proliferation and migration depend upon silencing of miR-200b. Taken together, our findings confirmed that PVT1 functions as an oncogene in cervical cancer and indicated that PVT1 is not only an important prognostic marker, but also a potential therapy target for cervical cancer. PMID:27272214

  4. RNA interference silencing of chalcone synthase, the first step in the flavonoid biosynthesis pathway, leads to parthenocarpic tomato fruits.

    Science.gov (United States)

    Schijlen, Elio G W M; de Vos, C H Ric; Martens, Stefan; Jonker, Harry H; Rosin, Faye M; Molthoff, Jos W; Tikunov, Yury M; Angenent, Gerco C; van Tunen, Arjen J; Bovy, Arnaud G

    2007-07-01

    Parthenocarpy, the formation of seedless fruits in the absence of functional fertilization, is a desirable trait for several important crop plants, including tomato (Solanum lycopersicum). Seedless fruits can be of great value for consumers, the processing industry, and breeding companies. In this article, we propose a novel strategy to obtain parthenocarpic tomatoes by down-regulation of the flavonoid biosynthesis pathway using RNA interference (RNAi)-mediated suppression of chalcone synthase (CHS), the first gene in the flavonoid pathway. In CHS RNAi plants, total flavonoid levels, transcript levels of both Chs1 and Chs2, as well as CHS enzyme activity were reduced by up to a few percent of the corresponding wild-type values. Surprisingly, all strong Chs-silenced tomato lines developed parthenocarpic fruits. Although a relation between flavonoids and parthenocarpic fruit development has never been described, it is well known that flavonoids are essential for pollen development and pollen tube growth and, hence, play an essential role in plant reproduction. The observed parthenocarpic fruit development appeared to be pollination dependent, and Chs RNAi fruits displayed impaired pollen tube growth. Our results lead to novel insight in the mechanisms underlying parthenocarpic fruit development. The potential of this technology for applications in plant breeding and biotechnology will be discussed. PMID:17478633

  5. Short Hairpin RNA Silencing of PHD-2 Improves Neovascularization and Functional Outcomes in Diabetic Wounds and Ischemic Limbs.

    Directory of Open Access Journals (Sweden)

    Kevin J Paik

    Full Text Available The transcription factor hypoxia-inducible factor 1-alpha (HIF-1α is responsible for the downstream expression of over 60 genes that regulate cell survival and metabolism in hypoxic conditions as well as those that enhance angiogenesis to alleviate hypoxia. However, under normoxic conditions, HIF-1α is hydroxylated by prolyl hydroxylase 2, and subsequently degraded, with a biological half-life of less than five minutes. Here we investigated the therapeutic potential of inhibiting HIF-1α degradation through short hairpin RNA silencing of PHD-2 in the setting of diabetic wounds and limb ischemia. Treatment of diabetic mouse fibroblasts with shPHD-2 in vitro resulted in decreased levels of PHD-2 transcript demonstrated by qRT-PCR, higher levels of HIF-1α as measured by western blot, and higher expression of the downstream angiogenic genes SDF-1 and VEGFα, as measured by qRT-PCR. In vivo, shPHD-2 accelerated healing of full thickness excisional wounds in diabetic mice compared to shScr control, (14.33 ± 0.45 days vs. 19 ± 0.33 days and was associated with an increased vascular density. Delivery of shPHD-2 also resulted in improved perfusion of ischemic hind limbs compared to shScr, prevention of distal digit tip necrosis, and increased survival of muscle tissue. Knockdown of PHD-2 through shRNA treatment has the potential to stimulate angiogenesis through overexpression of HIF-1α and upregulation of pro-angiogenic genes downstream of HIF-1α, and may represent a viable, non-viral approach to gene therapy for ischemia related applications.

  6. Nanoparticle mediated P-glycoprotein silencing for improved drug delivery across the blood-brain barrier: a siRNA-chitosan approach.

    Directory of Open Access Journals (Sweden)

    Jostein Malmo

    Full Text Available The blood-brain barrier (BBB, composed of tightly organized endothelial cells, limits the availability of drugs to therapeutic targets in the central nervous system. The barrier is maintained by membrane bound efflux pumps efficiently transporting specific xenobiotics back into the blood. The efflux pump P-glycoprotein (P-gp, expressed at high levels in brain endothelial cells, has several drug substrates. Consequently, siRNA mediated silencing of the P-gp gene is one possible strategy how to improve the delivery of drugs to the brain. Herein, we investigated the potential of siRNA-chitosan nanoparticles in silencing P-gp in a BBB model. We show that the transfection of rat brain endothelial cells mediated effective knockdown of P-gp with subsequent decrease in P-gp substrate efflux. This resulted in increased cellular delivery and efficacy of the model drug doxorubicin.

  7. Chemical modification of chitosan with pH-sensitive molecules and specific ligands for efficient DNA transfection and siRNA silencing.

    Science.gov (United States)

    Singh, Bijay; Choi, Yun-Jaie; Park, In-Kyu; Akaike, Toshihiro; Cho, Chong-Su

    2014-01-01

    Successful gene therapy depends on the development of efficient and cell-specific gene delivery systems. Currently, animal viral vectors have been mostly used for in vivo and in clinical trials owing to their high transduction efficiency. However, they suffer from numerous limitations such as biosafety, immunogenicity, gene packaging capacity, complicated production and cell specificity. Therefore non-viral vectors are attractive alternatives to viral gene delivery systems due to their low toxicity, relatively easy production and greater diversity. Among non-viral vectors, chitosan and chitosan derivatives have been extensively utilized as gene carriers owing to their low immunogenicity, biocompatibility, biodegradability, low toxicity and ease of chemical modifications. However, low transfection efficiency of DNA (or low gene silencing of siRNA) and low cell specificity of chitosan should be overcome before clinical trials. The objective of this review is to summarize several parameters affecting the transfection efficiency of DNA (or gene silencing of siRNA) for the promising use of chitosan as gene carriers. Besides, chemical modifications of chitosan with pH-sensitive molecules and specific ligands so as to enhance the transfection efficiency of DNA (or gene silencing of siRNA) and cell specificity will be covered. PMID:24730283

  8. Heterologous expression of viral suppressors of RNA silencing complements virulence of the HC-Pro mutant of clover yellow vein virus in pea.

    Science.gov (United States)

    Atsumi, Go; Nakahara, Kenji S; Wada, Tomoko Sugikawa; Choi, Sun Hee; Masuta, Chikara; Uyeda, Ichiro

    2012-06-01

    Many plant viruses encode suppressors of RNA silencing, including the helper component-proteinase (HC-Pro) of potyviruses. Our previous studies showed that a D-to-Y mutation at amino acid position 193 in HC-Pro (HC-Pro-D193Y) drastically attenuated the virulence of clover yellow vein virus (ClYVV) in legume plants. Furthermore, RNA-silencing suppression (RSS) activity of HC-Pro-D193Y was significantly reduced in Nicotiana benthamiana. Here, we examine the effect of expression of heterologous suppressors of RNA silencing, i.e., tomato bushy stunt virus p19, cucumber mosaic virus 2b, and their mutants, on the virulence of the ClYVV point mutant with D193Y (Cl-D193Y) in pea. P19 and 2b fully and partially complemented Cl-D193Y multiplication and virulence, including lethal systemic HR in pea, respectively, but the P19 and 2b mutants with defects in their RSS activity did not. Our findings strongly suggest that the D193Y mutation exclusively affects RSS activity of HC-Pro and that RSS activity is necessary for ClYVV multiplication and virulence in pea. PMID:22398917

  9. Transgenic Sugarcane Resistant to Sorghum mosaic virus Based on Coat Protein Gene Silencing by RNA Interference

    Directory of Open Access Journals (Sweden)

    Jinlong Guo

    2015-01-01

    Full Text Available As one of the critical diseases of sugarcane, sugarcane mosaic disease can lead to serious decline in stalk yield and sucrose content. It is mainly caused by Potyvirus sugarcane mosaic virus (SCMV and/or Sorghum mosaic virus (SrMV, with additional differences in viral strains. RNA interference (RNAi is a novel strategy for producing viral resistant plants. In this study, based on multiple sequence alignment conducted on genomic sequences of different strains and isolates of SrMV, the conserved region of coat protein (CP genes was selected as the target gene and the interference sequence with size of 423 bp in length was obtained through PCR amplification. The RNAi vector pGII00-HACP with an expression cassette containing both hairpin interference sequence and cp4-epsps herbicide-tolerant gene was transferred to sugarcane cultivar ROC22 via Agrobacterium-mediated transformation. After herbicide screening, PCR molecular identification, and artificial inoculation challenge, anti-SrMV positive transgenic lines were successfully obtained. SrMV resistance rate of the transgenic lines with the interference sequence was 87.5% based on SrMV challenge by artificial inoculation. The genetically modified SrMV-resistant lines of cultivar ROC22 provide resistant germplasm for breeding lines and can also serve as resistant lines having the same genetic background for study of resistance mechanisms.

  10. RNA Interference-mediated Silencing of Phytochelatin Synthase Gene Reduce Cadmium Accumulation in Rice Seeds

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Phytochelatins (PCs) play an important role in heavy metal resistance and accumulation. To reduce the accumulation of cadmium (Cd) in rice seeds, the expression of phytochelatin synthase (PCS) gene OsPCS1 was suppressed by RNA interference (RNAi). A hairpin construct of a PCS fragment was designed in the pRNAi-OsPCS1 under the control of ZMM1, a seed-specific promoter from maize. The construct was introduced into rice (japonica) through Agrobacterium tumefaciens. The RNAi rice plantlets were selected and cultivated in pots exposured to 10 mg/kg Cd. The transcriptional level of OsPCS1 declined in seeds of some RNAi rice compared to the wild type. As a result Cd accumulation was reduced by about half in the seeds of RNAi rice. As expected, no apparent difference of growth appeared between RNAi and wild-type plants. The results suggest that this new approach can be used to control heavy metal accumulation in crops.

  11. Gliotoxin: inhibitor of poliovirus RNA synthesis that blocks the viral RNA polymerase 3Dpol.

    OpenAIRE

    Rodriguez, P L; Carrasco, L.

    1992-01-01

    The mode of action of gliotoxin against poliovirus has been analyzed in detail. This fungal metabolite inhibits the appearance of poliovirus proteins when present from the beginning of infection but has no effect on viral translation when added at late times. In agreement with previous findings, this toxin potently inhibited the incorporation of [3H]uridine into poliovirus RNA soon after its addition to the culture medium. Analysis of the synthesis of poliovirus plus- or minus-stranded RNA in...

  12. Silencing of the HER2/neu Gene by siRNA Inhibits Proliferation and Induces Apoptosis in HER2/neu-Overexpressing Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Timo Faltus

    2004-11-01

    Full Text Available In eukaryotes, double-stranded (ds RNA induces sequence-specific inhibition of gene expression referred to as RNA interference (RNAi. We exploited RNAi to define the role of HER2/neu in the neoplastic proliferation of human breast cancer cells. We transfected SK-BR-3, BT-474, MCF-7, and MDA-MB-468 breast cancer cells with short interfering RNA (siRNA targeted against human HER2/neu and analyzed the specific inhibition of HER2/neu expression by Northern and Western blots. Transfection with HER2/neu-specific siRNA resulted in a sequence-specific decrease in HER2/neu mRNA and protein levels. Moreover, transfection with HER2/neu siRNA caused cell cycle arrest at G0/G1 in the breast cancer cell lines SKBR-3 and BT-474, consistent with a powerful RNA silencing effect. siRNA treatment resulted in an antiproliferative and apoptotic response in cells overexpressing HER2/neu, but had no influence in cells with almost no expression of HER2/neu proteins like MDA-MB-468 cells. These data indicate that HER2/neu function is essential for the proliferation of HER2/neuoverexpressing breast cancer cells. Our observations suggest that siRNA targeted against human HER2/neu may be valuable tools as anti proliferative agents that display activity against neoplastic cells at very low doses.

  13. siRNA-mediated silencing of Cockayne Cyndrome group B gene potentiates radiation-induced apoptosis and antiproliferative effect in HeLa cells

    Institute of Scientific and Technical Information of China (English)

    LIU Feng; YU Zi-jian; SUI Jian-li; BAI Bei; ZHOU Ping-kun

    2006-01-01

    Background Cockayne syndrome (CS) is a rare human genetic disorder characterized by increased UV sensitivity, developmental abnormalities and premature aging. Cells isolated from individuals with CS have a defect in transcription-coupled DNA repair. Despite the repair defect, there is no any increased risk of spontaneous or UV-induced cancer for CS individuals. The strategy of RNA interfering was used here to explore the potential radiosensitizing and anticancer activity of targeting CS group B (CSB) gene.Methods The vectors encoding CSB-specific siRNAs were constructed by inserting duplex siRNA encoding oligonucleotides into the plasmid psilencer TM 3.1. The cell lines expressing the CSB-siRNA were generated from HeLa cells transfected with the above vectors. Colony-forming ability was used to assay cell survival. Cell cycle was analyzed by FACScan flow cytometry. The apoptosis was measured by detecting the accumulation of sub-G1 population as well as by fluorescence staining assay. Reverse transcriptase polymerase chain reaction (RT-PCR)was used to semi-quantify mRNA expression. Protein level was detected by Western blotting analysis.Results Two constructs encoding CSB-specific siRNA were generated, both of them resulted in remarkable suppression on CSB expression in HeLa cells, and led to an increased sensitivity to γ-ray and UV light.siRNA-mediated silencing of CSB decreased cell proliferation rate, increased spontaneous apoptosis as well as the occurrence of UV- or cisplatin-induced apoptosis by 2 to 3.5 fold. A significant S phase blockage and a remarkable reduction of G1 population were induced in control HeLa cells at 18 hours after being exposed to 10J/m2 of UV light. The S phase blockage was also observed in UV-irradiated CSB-siRNA transfected HeLa cells,but the extent of increased S phase population was lower than that in the UV-irradiated control cells. No or a relative weak reduction on G1 phase population was observed in UV-irradiated CSB-siRNA

  14. Small RNA Derived from the Virulence Modulating Region of the Potato spindle tuber viroid Silences callose synthase Genes of Tomato Plants.

    Science.gov (United States)

    Adkar-Purushothama, Charith Raj; Brosseau, Chantal; Giguère, Tamara; Sano, Teruo; Moffett, Peter; Perreault, Jean-Pierre

    2015-08-01

    The tomato (Solanum lycopersicum) callose synthase genes CalS11-like and CalS12-like encode proteins that are essential for the formation of callose, a major component of pollen mother cell walls; these enzymes also function in callose formation during pathogen infection. This article describes the targeting of these callose synthase mRNAs by a small RNA derived from the virulence modulating region of two Potato spindle tuber viroid variants. More specifically, viroid infection of tomato plants resulted in the suppression of the target mRNAs up to 1.5-fold, depending on the viroid variant used and the gene targeted. The targeting of these mRNAs by RNA silencing was validated by artificial microRNA experiments in a transient expression system and by RNA ligase-mediated rapid amplification of cDNA ends. Viroid mutants incapable of targeting callose synthase mRNAs failed to induce typical infection phenotypes, whereas a chimeric viroid obtained by swapping the virulence modulating regions of a mild and a severe variant of Potato spindle tuber viroid greatly affected the accumulation of viroids and the severity of disease symptoms. These data provide evidence of the silencing of multiple genes by a single small RNA derived from a viroid. PMID:26290537

  15. Synergistic effect of silencing the expression of tick protective antigens 4D8 and Rs86 in Rhipicephalus sanguineus by RNA interference.

    Science.gov (United States)

    de la Fuente, José; Almazán, Consuelo; Naranjo, Victoria; Blouin, Edmour F; Kocan, Katherine M

    2006-07-01

    Tick proteins have been shown to be useful for the development of vaccines which reduce tick infestations. Potential tick protective antigens have been identified and characterized, in part, by use of RNA interference (RNAi). RNAi allows for analysis of gene function by characterizing the impact of loss of gene expression on tick physiology. Herein, we used RNAi in Rhipicephalus sanguineus to evaluate gene functions of two tick protective antigens, 4D8 and Rs86, the homologue of Bm86, on tick infestation, feeding and oviposition. Silencing of 4D8 alone resulted in decreased tick attachment, survival, feeding and oviposition. Although the effect of Rs86 RNAi was less pronounced, silencing of this gene also reduced tick weight and oviposition. Most notably, simultaneous silencing of 4D8 and Rs86 by RNAi resulted in a synergistic effect in which tick survival, attachment, feeding, weight and oviposition were profoundly reduced. Microscopic evaluation of tick tissues revealed that guts from dual injected ticks were distended with epithelial cells sparsely distributed along the basement membrane. These results demonstrated the synergistic effect of the silencing expression of two tick protective genes. Inclusion of multiple tick protective antigens may, therefore, enhance the efficacy of tick vaccines. PMID:16518610

  16. Silencing of RhoA and RhoC expression by RNA interference suppresses human colorectal carcinoma growth in vivo

    Directory of Open Access Journals (Sweden)

    Wang Haibo

    2010-09-01

    Full Text Available Abstract Background RhoA and RhoC have been proved to be over-expressed in many solid cancers, including colorectal cancer. The reduction of RhoA and RhoC expression by RNA interference (RNAi resulted growth inhibition of cancer cells. The present study was to evaluate the effect of silencing of RhoA and RhoC expression by RNAi on growth of human colorectal carcinoma (CRC in tumor-bearing nude mice in vivo. Methods To establish HCT116 cell transplantable model, the nude mice were subcutaneously inoculated with 1.0 × 107 HCT116 cells and kept growing till the tumor xenografts reached 5-7 mm in diameter. Then the mice were randomly assigned to three groups(seven mice in each group: (1 normal saline(NS group, (2replication-defective recombinant adenovirus carrying the negative control shRNA (Ad-HK group and (3replication-defective recombinant adenovirus carrying the 4-tandem linked RhoA and RhoC shRNAs (Ad-RhoA-RhoC group. Ad-HK (4 × 108 pfu, 30 ul/mouse, Ad-RhoA-RhoC (4 × 108 pfu, 30 ul/mouse or PBS (30 ul/mouse was injected intratumorally four times once every other day. The weight and volumes of tumor xenografts were recorded. The levels of RhoA and RhoC mRNA transcripts and proteins in tumor xenografts were detected by reverse quantitative transcription polymerase chain reaction (QRT-PCR and immunohistochemical staining respectively. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL assay was used to detect the death of cells. Results The xenografts in mice could be seen at 5th day from the implantation of HCT116 cells and all had reached 5-7 mm in size at 9th day. After injection intratumorally, the growth speed of tumor xenografts in Ad-RhoA-RhoC group was significantly delayed compared with those in NS and Ad-HK group(P RhoA and RhoC reduced more in Ad-RhoA-RhoC group than those in NS and Ad-HK group. The relative RhoA and RhoC mRNA transcripts were decreased to 48% and 43% respectively (P RhoA and Rho

  17. A versatile building block: the structures and functions of negative-sense single-stranded RNA virus nucleocapsid proteins.

    Science.gov (United States)

    Sun, Yuna; Guo, Yu; Lou, Zhiyong

    2012-12-01

    Nucleocapsid protein (NPs) of negative-sense single-stranded RNA (-ssRNA) viruses function in different stages of viral replication, transcription, and maturation. Structural investigations show that -ssRNA viruses that encode NPs preliminarily serve as structural building blocks that encapsidate and protect the viral genomic RNA and mediate the interaction between genomic RNA and RNA-dependent RNA polymerase. However, recent structural results have revealed other biological functions of -ssRNA viruses that extend our understanding of the versatile roles of virally encoded NPs. PMID:23136065

  18. Enhancement of allele discrimination by introduction of nucleotide mismatches into siRNA in allele-specific gene silencing by RNAi.

    Directory of Open Access Journals (Sweden)

    Yusuke Ohnishi

    Full Text Available Allele-specific gene silencing by RNA interference (RNAi is therapeutically useful for specifically inhibiting the expression of disease-associated alleles without suppressing the expression of corresponding wild-type alleles. To realize such allele-specific RNAi (ASP-RNAi, the design and assessment of small interfering RNA (siRNA duplexes conferring ASP-RNAi is vital; however, it is also difficult. In a previous study, we developed an assay system to assess ASP-RNAi with mutant and wild-type reporter alleles encoding the Photinus and Renilla luciferase genes. In line with experiments using the system, we realized that it is necessary and important to enhance allele discrimination between mutant and corresponding wild-type alleles. Here, we describe the improvement of ASP-RNAi against mutant alleles carrying single nucleotide variations by introducing base substitutions into siRNA sequences, where original variations are present in the central position. Artificially mismatched siRNAs or short-hairpin RNAs (shRNAs against mutant alleles of the human Prion Protein (PRNP gene, which appear to be associated with susceptibility to prion diseases, were examined using this assessment system. The data indicates that introduction of a one-base mismatch into the siRNAs and shRNAs was able to enhance discrimination between the mutant and wild-type alleles. Interestingly, the introduced mismatches that conferred marked improvement in ASP-RNAi, appeared to be largely present in the guide siRNA elements, corresponding to the 'seed region' of microRNAs. Due to the essential role of the 'seed region' of microRNAs in their association with target RNAs, it is conceivable that disruption of the base-pairing interactions in the corresponding seed region, as well as the central position (involved in cleavage of target RNAs, of guide siRNA elements could influence allele discrimination. In addition, we also suggest that nucleotide mismatches at the 3'-ends of sense

  19. RNA Interference Silencing of Glycogen Synthase Kinase 3β Inhibites Tau Phosphorylation in Mice with Alzheimer Disease.

    Science.gov (United States)

    Bian, Hong; Bian, Wei; Lin, Xiaoying; Ma, Zhaoyin; Chen, Wen; Pu, Ying

    2016-09-01

    To explore the effect of glycogen synthase kinase 3β (GSK-3β) silencing on Tau-5 phosphorylation in mice suffering Alzheimer disease (AD). GSK-3β was firstly silenced in human neuroblastoma SH-SY5Y cells using special lentivirus (LV) and the content of Tau (A-12), p-Tau (Ser396) and p-Tau (PHF-6) proteins. GSK-3β was also silenced in APP/PS1 mouse model of AD mice, which were divided into three groups (n = 10): AD, vehicle, and LV group. Ten C57 mice were used as control. The memory ability of mice was tested by square water maze, and the morphological changes of hippocampus and neuron death were analyzed by haematoxylin-eosin staining. Moreover, the levels of Tau and phosphorylated Tau (p-Tau) were detected by western blotting and immunohistochemistry, respectively. The lentivirus-mediated GSK-3β silencing system was successfully developed and silencing GSK-3β at the cellular level reduced Tau phosphorylation obviously. Moreover, GSK-3β silence significantly improved the memory ability of AD mice in LV group compared with AD group (P < 0.05) according to the latency periods and error numbers. As for the hippocampus morphology and neuron death, no significant change was observed between LV group and normal control. Immunohistochemical detection and western blotting revealed that the levels of Tau and p-Tau were significantly down-regulated after GSK-3β silence. Silencing GSK-3β may have a positive effect on inhibiting the pathologic progression of AD through down-regulating the level of p-Tau. PMID:27255602

  20. Chitosan/siRNA functionalized titanium surface via a layer-by-layer approach for in vitro sustained gene silencing and osteogenic promotion

    Directory of Open Access Journals (Sweden)

    Song W

    2015-03-01

    Full Text Available Wen Song,1,* Xin Song,2,* Chuanxu Yang,2 Shan Gao,2 Lasse Hyldgaard Klausen,2 Yumei Zhang,1 Mingdong Dong,2 Jørgen Kjems21State Key Laboratory of Military Stomatology, Department of Prosthetic Dentistry, School of Stomatology, The Fourth Military Medical University, Xi’an, People’s Republic of China; 2Interdisciplinary Nanoscience Center (iNANO, Aarhus University, Aarhus, Denmark*These authors contributed equally to this workAbstract: Titanium surface modification is crucial to improving its bioactivity, mainly its bone binding ability in bone implant materials. In order to functionalize titanium with small interfering RNA (siRNA for sustained gene silencing in nearby cells, the layer-by-layer (LbL approach was applied using sodium hyaluronate and chitosan/siRNA (CS/siRNA nanoparticles as polyanion and polycation, respectively, to build up the multilayered film on smooth titanium surfaces. The CS/siRNA nanoparticle characterization was analyzed first. Dynamic contact angle, atomic force microscopy, and scanning electron microscopy were used to monitor the layer accumulation. siRNA loaded in the film was quantitated and the release profile of film in phosphate-buffered saline was studied. In vitro knockdown effect and cytotoxicity evaluation of the film were investigated using H1299 human lung carcinoma cells expressing green fluorescent protein (GFP. The transfection of human osteoblast-like cell MG63 and H1299 were performed and the osteogenic differentiation of MG63 on LbL film was analyzed. The CS/siRNA nanoparticles exhibited nice size distribution. During formation of the film, the surface wettability, topography, and roughness were alternately changed, indicating successful adsorption of the individual layers. The scanning electron microscope images clearly demonstrated the hybrid structure between CS/siRNA nanoparticles and sodium hyaluronate polymer. The cumulated load of siRNA increased linearly with the bilayer number and, more

  1. ShRNA-mediated gene silencing of MTA1 influenced on protein expression of ER alpha, MMP-9, CyclinD1 and invasiveness, proliferation in breast cancer cell lines MDA-MB-231 and MCF-7 in vitro

    Directory of Open Access Journals (Sweden)

    Zhang Ping

    2011-05-01

    Full Text Available Abstract Background MTA1(metastasis associated-1 is a tumor metastasis associated candidate gene and overexpression in many human tumors, including breast cancer. In this study, we investigated depressive effect on MTA1 by MTA1-specific short hairpin RNA(shRNA expression plasmids in human breast cancer cell lines MDA-MB-231 and MCF-7, and effect on protein levels of ER alpha, MMP-9, cyclinD1, and tumor cell invasion, proliferation. Methods ShRNA expression vectors targeting MTA1 was constructed and transfected into human breast cancer cell lines MDA-MB-231 and MCF-7. The transfection efficiency was evaluated by fluorescence microscopy, mRNA levels of MTA1 were detected by reverse transcription-polymerase chain reaction (RT-PCR, protein levels of ER alpha, MMP-9 and cyclinD1 were detected by Western blotting, respectively. Tumor cells invasive ability were evaluated by Boyden chamber assay, the cells proliferation were evaluated using cell growth curve and MTT analysis, the cell cycle analysis was performed using flow cytometry. Results Down-regulation of MTA1 by RNAi approach led to re-expression of ER alpha in ER-negative breast cancer cell lines MDA-MB-231, and reduced protein levels of MMP-9 and CyclinD1, as well as decreased tumor cell invasion and proliferation, more cells were blocked in G0/G1 stage(P 0.05. Conclusions ShRNA targeted against MTA1 could specifically mediate the MTA1 gene silencing and consequentially recover the protein expression of ER alpha, resulting in increase sensitivity of antiestrogens, as well as suppress the protein levels of MMP-9 and cyclinD1 in ER-negative human breast cancer cell lines MDA-MB-231. Silencing effect of MTA1 could efficiently inhibit the invasion and proliferation in MDA-MB-231 cells. The shRNA interference targeted against MTA1 may have potential therapeutic utility in human breast cancer.

  2. The RNA interference revolution

    Directory of Open Access Journals (Sweden)

    G. Lenz

    2005-12-01

    Full Text Available The discovery of double-stranded RNA-mediated gene silencing has rapidly led to its use as a method of choice for blocking a gene, and has turned it into one of the most discussed topics in cell biology. Although still in its infancy, the field of RNA interference has already produced a vast array of results, mainly in Caenorhabditis elegans, but recently also in mammalian systems. Micro-RNAs are short hairpins of RNA capable of blocking translation, which are transcribed from genomic DNA and are implicated in several aspects from development to cell signaling. The present review discusses the main methods used for gene silencing in cell culture and animal models, including the selection of target sequences, delivery methods and strategies for a successful silencing. Expected developments are briefly discussed, ranging from reverse genetics to therapeutics. Thus, the development of the new paradigm of RNA-mediated gene silencing has produced two important advances: knowledge of a basic cellular mechanism present in the majority of eukaryotic cells and access to a potent and specific new method for gene silencing.

  3. Effects of silencing RIP1 with siRNA on the biological behavior of the LoVo human colon cancer cell line.

    Science.gov (United States)

    You, Hong-Xia; Zhou, Yan-Hong; Tan, Shi-Yun; She, Tong-Hui

    2014-06-01

    The present study aimed to investigate the effects of silencing RIP1 by small interfering RNA (siRNA) on the biological behavior of the LoVo human colorectal carcinoma cell line and to provide evidence for the feasibility of colorectal cancer gene therapy. LoVo cells were divided into the RIP1 siRNA group, the blank control group and the negative control group. Chemically synthesized siRNA targeting RIP1 (RIP1 siRNA) was transfected into LoVo cells. Following transfection of the RIP1-targeted siRNA into the LoVo cells, the expression of the RIP1 gene was effectively inhibited. The results demonstrated that RIP1 effectively regulated the malignant biological behavior of the LoVo colon cancer cell line. Furthermore, the proliferation, motility and invasiveness of LoVo cells were inhibited by siRNA knockdown of RIP1. The results revealed that the RIP1 gene has an important role in the regulation of proliferation and apoptosis in colorectal carcinoma cells.

  4. Host-induced post-transcriptional hairpin RNA-mediated gene silencing of vital fungal genes confers efficient resistance against Fusarium wilt in banana.

    Science.gov (United States)

    Ghag, Siddhesh B; Shekhawat, Upendra K S; Ganapathi, Thumballi R

    2014-06-01

    Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is among the most destructive diseases of banana (Musa spp.). Because no credible control measures are available, development of resistant cultivars through genetic engineering is the only option. We investigated whether intron hairpin RNA (ihpRNA)-mediated expression of small interfering RNAs (siRNAs) targeted against vital fungal genes (velvet and Fusarium transcription factor 1) in transgenic banana could achieve effective resistance against Foc. Partial sequences of these two genes were assembled as ihpRNAs in suitable binary vectors (ihpRNA-VEL and ihpRNA-FTF1) and transformed into embryogenic cell suspensions of banana cv. Rasthali by Agrobacterium-mediated genetic transformation. Eleven transformed lines derived from ihpRNA-VEL and twelve lines derived from ihpRNA-FTF1 were found to be free of external and internal symptoms of Foc after 6-week-long greenhouse bioassays. The five selected transgenic lines for each construct continued to resist Foc at 8 months postinoculation. Presence of specific siRNAs derived from the two ihpRNAs in transgenic banana plants was confirmed by Northern blotting and Illumina sequencing of small RNAs derived from the transgenic banana plants. The present study represents an important effort in proving that host-induced post-transcriptional ihpRNA-mediated gene silencing of vital fungal genes can confer efficient resistance against debilitating pathogens in crop plants.

  5. Interference RNA (RNAi)-based silencing of endogenous thrombopoietin receptor (Mpl) in Dami cells resulted in decreased hNUDC-mediated megakaryocyte proliferation and differentiation

    International Nuclear Information System (INIS)

    Recently our laboratory reported evidence showing that hNUDC acts as an additional cytokine for thrombopoietin receptor (Mpl). Previously known as the human homolog of a fungal nuclear migration protein, hNUDC plays a critical role in megakaryocyte differentiation and maturation. Here we sought to further clarify the hNUDC-Mpl ligand-receptor relationship by utilizing interference RNA (RNAi) to knockdown Mpl expression in a megakaryocyte cell line. We created U6 promoter driven constructs to express short hairpin RNAs (shRNA) with affinity for different sites on Mpl mRNA. By including Mpl-EGFP fusion protein in these constructs, we were able to effectively screen the shRNA that was most efficient in inhibiting Mpl mRNA expression. This shRNA was subsequently transferred into a lentivirus vector and transduced into Dami cells, a cell line which constitutively expresses endogenous Mpl. This lentiviral vector was also designed to simultaneously express EGFP to monitor transfection efficiency. Our results show that lentivirus can be used to effectively deliver shRNAs into Dami cells and cause specific inhibition of Mpl protein expression after transduction. Furthermore, we show the functional effects of shRNA-mediated Mpl silencing by demonstrating reduced hNUDC stimulated megakaryocyte proliferation and differentiation. Thus, the use of a RNAi knockdown strategy has allowed us to pinpoint the connection of hNUDC with Mpl in the regulation of megakaryocyte maturation.

  6. Host-induced post-transcriptional hairpin RNA-mediated gene silencing of vital fungal genes confers efficient resistance against Fusarium wilt in banana.

    Science.gov (United States)

    Ghag, Siddhesh B; Shekhawat, Upendra K S; Ganapathi, Thumballi R

    2014-06-01

    Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is among the most destructive diseases of banana (Musa spp.). Because no credible control measures are available, development of resistant cultivars through genetic engineering is the only option. We investigated whether intron hairpin RNA (ihpRNA)-mediated expression of small interfering RNAs (siRNAs) targeted against vital fungal genes (velvet and Fusarium transcription factor 1) in transgenic banana could achieve effective resistance against Foc. Partial sequences of these two genes were assembled as ihpRNAs in suitable binary vectors (ihpRNA-VEL and ihpRNA-FTF1) and transformed into embryogenic cell suspensions of banana cv. Rasthali by Agrobacterium-mediated genetic transformation. Eleven transformed lines derived from ihpRNA-VEL and twelve lines derived from ihpRNA-FTF1 were found to be free of external and internal symptoms of Foc after 6-week-long greenhouse bioassays. The five selected transgenic lines for each construct continued to resist Foc at 8 months postinoculation. Presence of specific siRNAs derived from the two ihpRNAs in transgenic banana plants was confirmed by Northern blotting and Illumina sequencing of small RNAs derived from the transgenic banana plants. The present study represents an important effort in proving that host-induced post-transcriptional ihpRNA-mediated gene silencing of vital fungal genes can confer efficient resistance against debilitating pathogens in crop plants. PMID:24476152

  7. Cystathionine-γ-lyase gene silencing with siRNA in monocytes/ macrophages attenuates inflammation in cecal ligation and puncture-induced sepsis in the mouse

    Indian Academy of Sciences (India)

    A Badiei; ST Chambers; RR Gaddam; M Bhatia

    2016-03-01

    Hydrogen sulphide is an endogenous inflammatory mediator produced by cystathionine-γ-lyase (CSE) in macrophages. To determine the role of H2S and macrophages in sepsis, we used small interference RNA (siRNA) to target the CSE gene and investigated its effect in a mouse model of sepsis. Cecal ligation puncture (CLP)-induced sepsis is characterized by increased levels of myeloperoxidase (MPO) activity, morphological changes in liver and pro-inflammatory cytokines and chemokines in the liver and lung. SiRNA treatment attenuated inflammation in the liver and lungs of mice following CLP-induced sepsis. Liver MPO activity increased in CLP-induced sepsis and treatment with siRNA significantly reduced this. Similarly, lung MPO activity increased following induction of sepsis with CLP while siRNA treatment significantly reduced MPO activity. Liver and lung cytokine and chemokine levels in CLP-induced sepsis reduced following treatment with siRNA. These findings show a crucial pro-inflammatory role for H2S synthesized by CSE in macrophages in sepsis and suggest CSE gene silencing with siRNA as a potential therapeutic approach for this condition.

  8. Saturation of recognition elements blocks evolution of new tRNA identities

    Science.gov (United States)

    Saint-Léger, Adélaïde; Bello, Carla; Dans, Pablo D.; Torres, Adrian Gabriel; Novoa, Eva Maria; Camacho, Noelia; Orozco, Modesto; Kondrashov, Fyodor A.; Ribas de Pouplana, Lluís

    2016-01-01

    Understanding the principles that led to the current complexity of the genetic code is a central question in evolution. Expansion of the genetic code required the selection of new transfer RNAs (tRNAs) with specific recognition signals that allowed them to be matured, modified, aminoacylated, and processed by the ribosome without compromising the fidelity or efficiency of protein synthesis. We show that saturation of recognition signals blocks the emergence of new tRNA identities and that the rate of nucleotide substitutions in tRNAs is higher in species with fewer tRNA genes. We propose that the growth of the genetic code stalled because a limit was reached in the number of identity elements that can be effectively used in the tRNA structure. PMID:27386510

  9. P-glycoprotein silencing with siRNA delivered by DOPE-modified PEI overcomes doxorubicin resistance in breast cancer cells

    Science.gov (United States)

    Navarro, Gemma; Sawant, Rupa R; Biswas, Swati; Essex, Sean; Tros de Ilarduya, Conchita; Torchilin, Vladimir P

    2012-01-01

    Aims Multidrug resistance (MDR) mediated by overexpression of drug efflux transporters such as P-glycoprotein (P-gp), is a major problem, limiting successful chemotherapy of breast cancer. The use of siRNA to inhibit P-gp expression in MDR tumors is an attractive strategy to improve the effectiveness of anticancer drugs. Method We have synthesized a novel conjugate between a phospholipid (dioleoylphosphatidylethanolamine) and polyethylenimine (PEI) for siRNA delivery, for the purpose of silencing P-gp to overcome doxorubicin resistance in MCF-7 human breast cancer cells. Results The dioleoylphosphatidylethanolamine-PEI conjugate enhanced the transfection efficacy of low-molecular-weight PEI, which was otherwise totally ineffective. In addition, the polyethylene glycol/lipid coating of the new complexes gave rise to small micelle-like nanoparticles with improved biocompatibility properties. Both coated and noncoated formulations delivered P-gp-specific siRNA to MDR cells. Discussion The combination of doxorubicin and P-gp silencing formulations led to a twofold increase of doxorubicin uptake and a significant improvement of the therapeutic effect of doxorubicin in resistant cells. PMID:22191778

  10. Silencing Bag-1 gene via magnetic gold nanoparticle-delivered siRNA plasmid for colorectal cancer therapy in vivo and in vitro.

    Science.gov (United States)

    Huang, Wenbai; Liu, Zhan'ao; Zhou, Guanzhou; Ling, Jianmin; Tian, Ailing; Sun, Nianfeng

    2016-08-01

    Apoptosis disorder is generally regarded as an important mechanism of carcinogenesis. Inducement of tumor cell apoptosis can be an effectual way to treat cancer. Bcl-2-associated athanogene 1 (Bag-1) is a positive regulator of Bcl-2 which is an anti-apoptotic gene. Bag-1 is highly expressed in colorectal cancer, which plays a critical role in promoting metastasis, poor prognosis, especially in anti-apoptotic function, and is perhaps a valuable gene target for colorectal cancer therapy. Recently, we applied a novel non-viral gene carrier, magnetic gold nanoparticle, and mediated plasmid pGPH1/GFP/Neo-Bag-1-homo-825 silencing Bag-1 gene for treating colorectal cancer in vivo and in vitro. By mediating with magnetic gold nanoparticle, siRNA plasmid was successfully transfected into cell. In 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, magnetic gold nanoparticle had no significant cytotoxicity and by which delivered RNA plasmid inhibited cell viability significantly (P plasmid complex-transfected Balb c/nude tumor xenograft. In conclusion, Bag-1 is confirmed an anti-apoptosis gene that functioned in colorectal cancer, and the mechanism of Bag-1 gene causing colorectal cancer may be related to Wnt/β-catenin signaling pathway abnormality and suggested that magnetic gold nanoparticle-delivered siRNA plasmid silencing Bag-1 is an effective gene therapy method for colorectal cancer. PMID:26846101

  11. Silencing of microRNA-155 in mice during acute inflammatory response leads to derepression of c/ebp Beta and down-regulation of G-CSF

    OpenAIRE

    Worm, Jesper; Stenvang, Jan; Petri, Andreas; Frederiksen, Klaus Stensgaard; Obad, Susanna; Elmén, Joacim; Hedtjärn, Maj; Straarup, Ellen Marie; Hansen, Jens Bo; Kauppinen, Sakari

    2009-01-01

    microRNA-155 (miR-155) has been implicated as a central regulator of the immune system, but its function during acute inflammatory responses is still poorly understood. Here we show that exposure of cultured macrophages and mice to lipopolysaccharide (LPS) leads to up-regulation of miR-155 and that the transcription factor c/ebp Beta is a direct target of miR-155. Interestingly, expression profiling of LPS-stimulated macrophages combined with overexpression and silencing of miR-155 in murine ...

  12. RNA viruses and their silencing suppressors boost Abutilon mosaic virus, but not the Old World Tomato yellow leaf curl Sardinia virus.

    Science.gov (United States)

    Sardo, Luca; Wege, Christina; Kober, Sigrid; Kocher, Conny; Accotto, Gian Paolo; Noris, Emanuela

    2011-11-01

    Mixed viral infections can induce different changes in symptom development, genome accumulation and tissue tropism. These issues were investigated for two phloem-limited begomoviruses, Abutilon mosaic virus (AbMV) and Tomato yellow leaf curl Sardinia virus (TYLCSV) in Nicotiana benthamiana plants doubly infected by either the potyvirus Cowpea aphid-borne mosaic virus (CABMV) or the tombusvirus Artichoke mottled crinkle virus (AMCV). Both RNA viruses induced an increase of the amount of AbMV, led to its occasional egress from the phloem and induced symptom aggravation, while the amount and tissue tropism of TYLCSV were almost unaffected. In transgenic plants expressing the silencing suppressors of CABMV (HC-Pro) or AMCV (P19), AbMV was supported to a much lesser extent than in the mixed infections, with the effect of CABMV HC-Pro being superior to that of AMCV P19. Neither of the silencing suppressors influenced TYLCSV accumulation. These results demonstrate that begomoviruses differentially respond to the invasion of other viruses and to silencing suppression. PMID:21843560

  13. Silencing of microRNA-155 in mice during acute inflammatory response leads to derepression of c/ebp Beta and down-regulation of G-CSF.

    Science.gov (United States)

    Worm, Jesper; Stenvang, Jan; Petri, Andreas; Frederiksen, Klaus Stensgaard; Obad, Susanna; Elmén, Joacim; Hedtjärn, Maj; Straarup, Ellen Marie; Hansen, Jens Bo; Kauppinen, Sakari

    2009-09-01

    microRNA-155 (miR-155) has been implicated as a central regulator of the immune system, but its function during acute inflammatory responses is still poorly understood. Here we show that exposure of cultured macrophages and mice to lipopolysaccharide (LPS) leads to up-regulation of miR-155 and that the transcription factor c/ebp Beta is a direct target of miR-155. Interestingly, expression profiling of LPS-stimulated macrophages combined with overexpression and silencing of miR-155 in murine macrophages and human monocytic cells uncovered marked changes in the expression of granulocyte colony-stimulating factor (G-CSF), a central regulator of granulopoiesis during inflammatory responses. Consistent with these data, we show that silencing of miR-155 in LPS-treated mice by systemically administered LNA-antimiR results in derepression of the c/ebp Beta isoforms and down-regulation of G-CSF expression in mouse splenocytes. Finally, we report for the first time on miR-155 silencing in vivo in a mouse inflammation model, which underscores the potential of miR-155 antagonists in the development of novel therapeutics for treatment of chronic inflammatory diseases. PMID:19596814

  14. Double-stranded RNA uptake through topical application, mediates silencing of five CYP4 genes and suppresses insecticide resistance in Diaphorina citri.

    Science.gov (United States)

    Killiny, Nabil; Hajeri, Subhas; Tiwari, Siddharth; Gowda, Siddarame; Stelinski, Lukasz L

    2014-01-01

    Silencing of genes through RNA interference (RNAi) in insects has gained momentum during the past few years. RNAi has been used to cause insect mortality, inhibit insect growth, increase insecticide susceptibility, and prevent the development of insecticide resistance. We investigated the efficacy of topically applied dsRNA to induce RNAi for five Cytochrome P450 genes family 4 (CYP4) in Diaphorina citri. We previously reported that these CYP4 genes are associated with the development of insecticide resistance in D. citri. We targeted five CYP4 genes that share a consensus sequence with one dsRNA construct. Quantitative PCR confirmed suppressed expression of the five CYP4 genes as a result of dsRNA topically applied to the thoracic region of D. citri when compared to the expression levels in a control group. Western blot analysis indicated a reduced signal of cytochrome P450 proteins (45 kDa) in adult D. citri treated with the dsRNA. In addition, oxidase activity and insecticide resistance were reduced for D. citri treated with dsRNA that targeted specific CYP4 genes. Mortality was significantly higher in adults treated with dsRNA than in adults treated with water. Our results indicate that topically applied dsRNA can penetrate the cuticle of D. citri and induce RNAi. These results broaden the scope of RNAi as a mechanism to manage pests by targeting a broad range of genes. The results also support the application of RNAi as a viable tool to overcome insecticide resistance development in D. citri populations. However, further research is needed to develop grower-friendly delivery systems for the application of dsRNA under field conditions. Considering the high specificity of dsRNA, this tool can also be used for management of D. citri by targeting physiologically critical genes involved in growth and development. PMID:25330026

  15. Double-stranded RNA uptake through topical application, mediates silencing of five CYP4 genes and suppresses insecticide resistance in Diaphorina citri.

    Directory of Open Access Journals (Sweden)

    Nabil Killiny

    Full Text Available Silencing of genes through RNA interference (RNAi in insects has gained momentum during the past few years. RNAi has been used to cause insect mortality, inhibit insect growth, increase insecticide susceptibility, and prevent the development of insecticide resistance. We investigated the efficacy of topically applied dsRNA to induce RNAi for five Cytochrome P450 genes family 4 (CYP4 in Diaphorina citri. We previously reported that these CYP4 genes are associated with the development of insecticide resistance in D. citri. We targeted five CYP4 genes that share a consensus sequence with one dsRNA construct. Quantitative PCR confirmed suppressed expression of the five CYP4 genes as a result of dsRNA topically applied to the thoracic region of D. citri when compared to the expression levels in a control group. Western blot analysis indicated a reduced signal of cytochrome P450 proteins (45 kDa in adult D. citri treated with the dsRNA. In addition, oxidase activity and insecticide resistance were reduced for D. citri treated with dsRNA that targeted specific CYP4 genes. Mortality was significantly higher in adults treated with dsRNA than in adults treated with water. Our results indicate that topically applied dsRNA can penetrate the cuticle of D. citri and induce RNAi. These results broaden the scope of RNAi as a mechanism to manage pests by targeting a broad range of genes. The results also support the application of RNAi as a viable tool to overcome insecticide resistance development in D. citri populations. However, further research is needed to develop grower-friendly delivery systems for the application of dsRNA under field conditions. Considering the high specificity of dsRNA, this tool can also be used for management of D. citri by targeting physiologically critical genes involved in growth and development.

  16. Double-stranded RNA uptake through topical application, mediates silencing of five CYP4 genes and suppresses insecticide resistance in Diaphorina citri.

    Science.gov (United States)

    Killiny, Nabil; Hajeri, Subhas; Tiwari, Siddharth; Gowda, Siddarame; Stelinski, Lukasz L

    2014-01-01

    Silencing of genes through RNA interference (RNAi) in insects has gained momentum during the past few years. RNAi has been used to cause insect mortality, inhibit insect growth, increase insecticide susceptibility, and prevent the development of insecticide resistance. We investigated the efficacy of topically applied dsRNA to induce RNAi for five Cytochrome P450 genes family 4 (CYP4) in Diaphorina citri. We previously reported that these CYP4 genes are associated with the development of insecticide resistance in D. citri. We targeted five CYP4 genes that share a consensus sequence with one dsRNA construct. Quantitative PCR confirmed suppressed expression of the five CYP4 genes as a result of dsRNA topically applied to the thoracic region of D. citri when compared to the expression levels in a control group. Western blot analysis indicated a reduced signal of cytochrome P450 proteins (45 kDa) in adult D. citri treated with the dsRNA. In addition, oxidase activity and insecticide resistance were reduced for D. citri treated with dsRNA that targeted specific CYP4 genes. Mortality was significantly higher in adults treated with dsRNA than in adults treated with water. Our results indicate that topically applied dsRNA can penetrate the cuticle of D. citri and induce RNAi. These results broaden the scope of RNAi as a mechanism to manage pests by targeting a broad range of genes. The results also support the application of RNAi as a viable tool to overcome insecticide resistance development in D. citri populations. However, further research is needed to develop grower-friendly delivery systems for the application of dsRNA under field conditions. Considering the high specificity of dsRNA, this tool can also be used for management of D. citri by targeting physiologically critical genes involved in growth and development.

  17. Silencing of signal transducer and activator of transcription 3 expression by RNA interference suppresses growth of human hepatocellular carcinoma in tumor-bearing nude mice

    Institute of Scientific and Technical Information of China (English)

    Jing Li; Yun-Feng Piao; Zheng Jiang; Li Chen; Hai-Bo Sun

    2009-01-01

    AIM: To explore the effect of silencing of signal transducer and activator of transcription 3 (STAT3) expression by RNA interference (RNAi) on growth of human hepatocellular carcinoma (HCC) in tumorbearing nude mice in vivo.METHODS: To construct the recombinant plasmid of pSilencer 3.0-H1-STAT3-siRNA-GFP (pSH1-siRNASTAT3) and establish the tumor-bearing nude mouse model of the HCC cell line SMMC7721, we used intratumoral injection together with electroblotting to transfect the recombinant plasmid pSH1-siRNASTAT3 into the transplanted tumor. The weight of the nude mice and tumor volumes were recorded. STAT3 gene transcription was detected by semi-quantitative reverse transcription polymerase chain reaction (RTPCR).Level of protein expression and location of STAT3 were determined by Western blotting and immunohistochemical staining. STAT3-related genes such as survivin, c-myc, VEGF, p53 and caspase3 mRNA and protein expression were detected in tumor tissues at the same time. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was used to detect apoptosis of tumor cells.RESULTS: The weight of the treated nude mice increased, and the tumor volume decreased markedly compared with those of the mock-treated and negative control groups ( P < 0.01). The results of RT-PCR and Western blotting showed that mRNA and protein levels of STAT3 declined markedly in the treated group. The change in STAT3-related gene expression in tumor tissues at the mRNA and protein level also varied, the expression of survivin, VEGF and c-myc were obviously reduced, and expression of p53 and caspase3 increased ( P < 0.01). Most of the tumor tissue cells in the treated group developed apoptosis that was detected by TUNEL assay.CONCLUSION: Silencing of STAT3 expression by RNAi significantly inhibits expression of STAT3 mRNA and protein, and suppresses growth of human HCC in tumor-bearing nude mice. The mechanism may be related to down-regulation of survivin, VEGF

  18. The effect of silencing 20E biosynthesis relative genes by feeding bacterially expressed dsRNA on the larval development of Chilo suppressalis.

    Science.gov (United States)

    Zhu, Jian; Dong, Yong-Cheng; Li, Ping; Niu, Chang-Ying

    2016-01-01

    RNA interference (RNAi) is a robust tool to study gene functions as well as potential for insect pest control. Finding suitable target genes is the key step in the development of an efficient RNAi-mediated pest control technique. Based on the transcriptome of Chilo suppressalis, 24 unigenes which putatively associated with insect hormone biosynthesis were identified. Amongst these, four genes involved in ecdysteroidogenesis i.e., ptth, torso, spook and nm-g were evaluated as candidate targets for function study. The partial cDNA of these four genes were cloned and their bacterially expressed dsRNA were fed to the insects. Results revealed a significant reduction in mRNA abundance of target genes after 3 days. Furthermore, knocked down of these four genes resulted in abnormal phenotypes and high larval mortality. After 15 days, the survival rates of insects in dsspook, dsptth, dstorso, and dsnm-g groups were significantly reduced by 32%, 38%, 56%, and 67% respectively, compared with control. Moreover, about 80% of surviving larvae showed retarded development in dsRNA-treated groups. These results suggest that oral ingestion of bacterially expressed dsRNA in C. suppressalis could silence ptth, torso, spook and nm-g. Oral delivery of bacterially expressed dsRNA provides a simple and potential management scheme against C. suppressalis. PMID:27352880

  19. The effect of silencing 20E biosynthesis relative genes by feeding bacterially expressed dsRNA on the larval development of Chilo suppressalis

    Science.gov (United States)

    Zhu, Jian; Dong, Yong-Cheng; Li, Ping; Niu, Chang-Ying

    2016-01-01

    RNA interference (RNAi) is a robust tool to study gene functions as well as potential for insect pest control. Finding suitable target genes is the key step in the development of an efficient RNAi-mediated pest control technique. Based on the transcriptome of Chilo suppressalis, 24 unigenes which putatively associated with insect hormone biosynthesis were identified. Amongst these, four genes involved in ecdysteroidogenesis i.e., ptth, torso, spook and nm-g were evaluated as candidate targets for function study. The partial cDNA of these four genes were cloned and their bacterially expressed dsRNA were fed to the insects. Results revealed a significant reduction in mRNA abundance of target genes after 3 days. Furthermore, knocked down of these four genes resulted in abnormal phenotypes and high larval mortality. After 15 days, the survival rates of insects in dsspook, dsptth, dstorso, and dsnm-g groups were significantly reduced by 32%, 38%, 56%, and 67% respectively, compared with control. Moreover, about 80% of surviving larvae showed retarded development in dsRNA-treated groups. These results suggest that oral ingestion of bacterially expressed dsRNA in C. suppressalis could silence ptth, torso, spook and nm-g. Oral delivery of bacterially expressed dsRNA provides a simple and potential management scheme against C. suppressalis. PMID:27352880

  20. Efficient and nontoxic biological response carrier delivering TNF-α shRNA for gene silencing in a murine model of rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    Jialin Song

    2016-08-01

    Full Text Available Small interfering RNA (siRNA is an effective and specific method for silencing genes. However, an efficient and nontoxic carrier is needed to deliver the siRNA into the target cells. Tumor necrosis factor α (TNF-α plays a central role in the occurrence and progression of rheumatoid arthritis. In this study, we pre-synthetized a degradable cationic polymer (PDAPEI from 2,6-pyridinedicarboxaldehyde and low molecular weight polyethyleneimine (PEI, Mw=1.8 kDa as a gene vector for the delivery of TNF-α shRNA. The PDAPEI/pDNA complex showed a suitable particle size and stable zeta potential for transfection. In vitro study of the PDAPEI/pDNA complex revealed a lower cytotoxicity and higher transfection efficiency when transfecting TNF-α shRNA to macrophages by significantly down-regulating the expression of TNF-α. Moreover, the complex was extremely efficient in decreasing the severity of arthritis in mice with collagen-induced arthritis (CIA. PDAPEI delivered TNF-α shRNA has great potential in the treatment of rheumatoid arthritis.

  1. Expression of plasmid-based shRNA against the E1 and nsP1 genes effectively silenced Chikungunya virus replication.

    Directory of Open Access Journals (Sweden)

    Shirley Lam

    Full Text Available BACKGROUND: Chikungunya virus (CHIKV is a re-emerging alphavirus that causes chikungunya fever and persistent arthralgia in humans. Currently, there is no effective vaccine or antiviral against CHIKV infection. Therefore, this study evaluates whether RNA interference which targets at viral genomic level may be a novel antiviral strategy to inhibit the medically important CHIKV infection. METHODS: Plasmid-based small hairpin RNA (shRNA was investigated for its efficacy in inhibiting CHIKV replication. Three shRNAs designed against CHIKV Capsid, E1 and nsP1 genes were transfected to establish stable shRNA-expressing cell clones. Following infection of stable shRNA cells clones with CHIKV at M.O.I. 1, viral plaque assay, Western blotting and transmission electron microscopy were performed. The in vivo efficacy of shRNA against CHIKV replication was also evaluated in a suckling murine model of CHIKV infection. RESULTS: Cell clones expressing shRNAs against CHIKV E1 and nsP1 genes displayed significant inhibition of infectious CHIKV production, while shRNA Capsid demonstrated a modest inhibitory effect as compared to scrambled shRNA cell clones and non-transfected cell controls. Western blot analysis of CHIKV E2 protein expression and transmission electron microscopy of shRNA E1 and nsP1 cell clones collectively demonstrated similar inhibitory trends against CHIKV replication. shRNA E1 showed non cell-type specific anti-CHIKV effects and broad-spectrum silencing against different geographical strains of CHIKV. Furthermore, shRNA E1 clones did not exert any inhibition against Dengue virus and Sindbis virus replication, thus indicating the high specificity of shRNA against CHIKV replication. Moreover, no shRNA-resistant CHIKV mutant was generated after 50 passages of CHIKV in the stable cell clones. More importantly, strong and sustained anti-CHIKV protection was conferred in suckling mice pre-treated with shRNA E1. CONCLUSION: Taken together, these

  2. miRNA-Mediated KHSRP Silencing Rewires Distinct Post-transcriptional Programs during TGF-β-Induced Epithelial-to-Mesenchymal Transition.

    Science.gov (United States)

    Puppo, Margherita; Bucci, Gabriele; Rossi, Martina; Giovarelli, Matteo; Bordo, Domenico; Moshiri, Arfa; Gorlero, Franco; Gherzi, Roberto; Briata, Paola

    2016-07-26

    Epithelial-to-mesenchymal transition (EMT) confers several traits to cancer cells that are required for malignant progression. Here, we report that miR-27b-3p-mediated silencing of the single-strand RNA binding protein KHSRP is required for transforming growth factor β (TGF-β)-induced EMT in mammary gland cells. Sustained KHSRP expression limits TGF-β-dependent induction of EMT factors and cell migration, whereas its knockdown in untreated cells mimics TGF-β-induced EMT. Genome-wide sequencing analyses revealed that KHSRP controls (1) levels of mature miR-192-5p, a microRNA that targets a group of EMT factors, and (2) alternative splicing of a cohort of pre-mRNAs related to cell adhesion and motility including Cd44 and Fgfr2. KHSRP belongs to a ribonucleoprotein complex that includes hnRNPA1, and the two proteins cooperate in promoting epithelial-type exon usage of select pre-mRNAs. Thus, TGF-β-induced KHSRP silencing is central in a pathway leading to gene-expression changes that contribute to the cellular changes linked to EMT. PMID:27396342

  3. Silencing of hpv16 e6 and e7 oncogenic activities by small interference rna induces autophagy and apoptosis in human cervical cancer cells

    Directory of Open Access Journals (Sweden)

    Jonathan Salazar-León

    2011-08-01

    Full Text Available Cervical cancer is the second most common form of death by cancer in women worldwide and has special attention for the development of new treatment strategies. Human Papilloma Virus (HPV persistent infection is the main etiological agent of this neoplasia, and the main cellular transformation mechanism is by disruption of p53 and pRb function by interaction with HPV E6 and E7 oncoproteins. This generates alterations in cellular differentiation and cellular death inhibition. Thus, HPV E6 and E7 oncogenes represent suitable targets for the development of gene therapy strategies against cervical cancer. An attractive technology platform is developing for post-transcriptional selective silencing of gene expression, using small interference RNA. Therefore, in the present study, we used SiHa cells (HPV16+ transiently transfected with specific siRNA expression plasmids for HPV16 E6 and E7 oncogenes. In this model we detected repression of E6 and E7 oncogene and oncoprotein expression, an increase in p53 and hypophosphorylated pRb isoform protein expression, and autophagy and apoptosis morphology features. These findings suggest that selective silencing of HPV16 E6 and E7 oncogenes by siRNAs, has significant biological effects on the survival of human cancer cells and is a potential gene therapy strategy against cervical cancer.

  4. Correction of Mutant p63 in EEC Syndrome Using siRNA Mediated Allele-Specific Silencing Restores Defective Stem Cell Function.

    Science.gov (United States)

    Barbaro, Vanessa; Nasti, Annamaria A; Del Vecchio, Claudia; Ferrari, Stefano; Migliorati, Angelo; Raffa, Paolo; Lariccia, Vincenzo; Nespeca, Patrizia; Biasolo, Mariangela; Willoughby, Colin E; Ponzin, Diego; Palù, Giorgio; Parolin, Cristina; Di Iorio, Enzo

    2016-06-01

    Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) syndrome is a rare autosomal dominant disease caused by heterozygous mutations in the p63 gene and characterized by limb defects, orofacial clefting, ectodermal dysplasia, and ocular defects. Patients develop progressive total bilateral limbal stem cell deficiency, which eventually results in corneal blindness. Medical and surgical treatments are ineffective and of limited benefit. Oral mucosa epithelial stem cells (OMESCs) represent an alternative source of stem cells capable of regenerating the corneal epithelium and, combined with gene therapy, could provide an attractive therapeutic avenue. OMESCs from EEC patients carrying the most severe p63 mutations (p.R279H and p.R304Q) were characterized and the genetic defect of p.R279H silenced using allele-specific (AS) small interfering RNAs (siRNAs). Systematic screening of locked nucleic acid (LNA)-siRNAs against R279H-p63 allele in (i) stable WT-ΔNp63α-RFP and R279H-ΔNp63α-EGFP cell lines, (ii) transient doubly transfected cell lines, and (iii) p.R279H OMESCs, identified a number of potent siRNA inhibitors for the mutant allele, which had no effect on wild-type p63. In addition, siRNA treatment led to longer acquired life span of mutated stem cells compared to controls, less accelerated stem cell differentiation in vitro, reduced proliferation properties, and effective ability in correcting the epithelial hypoplasia, thus giving rise to full thickness stratified and differentiated epithelia. This study demonstrates the phenotypic correction of mutant stem cells (OMESCs) in EEC syndrome by means of siRNA mediated AS silencing with restoration of function. The application of siRNA, alone or in combination with cell-based therapies, offers a therapeutic strategy for corneal blindness in EEC syndrome. Stem Cells 2016;34:1588-1600. PMID:26891374

  5. Cytopathological evaluations combined RNA and protein analyses on defined cell regions using single frozen tissue block.

    Science.gov (United States)

    Li, Hong; Chen, Xiao Yan; Kong, Qing You; Liu, Jia

    2002-06-01

    The co-existence of multiple cell components in tissue samples is the main obstacle for precise molecular evaluation on defined cell types. Based on morphological examination, we developed an efficient approach for paralleled RNA and protein isolations from an identical histological region in frozen tissue section. The RNA and protein samples prepared were sufficient for RT-PCR and Western blot analyses, and the results obtained were well coincident each other as well as with the corresponding parameters revealed from immunohistochemical examinations. By this way, the sampling problem caused by cell-cross contamination can be largely avoided, committing the experimental data more specific to a defined cell type. These novel methods thus allow us to use single tissue block for a comprehensive study by integration of conventional cytological evaluations with nucleic acid and protein analyses.

  6. Cytopathological evaluations combined RNA and protein analyses on defined cell regions using single frozen tissue block

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The co-existence of multiple cell components in tissue samples is the main obstacle for precise molecular evaluation on defined cell types. Based on morphological examination, we developed an efficient approach for paralleled RNA and protein isolations from an identical histological region in frozen tissue section.The RNA and protein samples prepared were sufficient for RT-PCR and Western blot analyses, and the results obtained were well coincident each other as well as with the corresponding parameters revealed from immunohistochemical examinations. By this way, the sampling problem caused by cell-cross contamination can be largely avoided, committing the experimental data more specific to a defined cell type. These novel methods thus allow us to use single tissue block for a comprehensive study by integration of conventional cytological evaluations with nucleic acid and protein analyses.

  7. A lincRNA connected to cell mortality and epigenetically-silenced in most common human cancers.

    Science.gov (United States)

    Vrba, Lukas; Garbe, James C; Stampfer, Martha R; Futscher, Bernard W

    2015-01-01

    Immortality is an essential characteristic of human carcinoma cells. We recently developed an efficient, reproducible method that immortalizes human mammary epithelial cells (HMEC) in the absence of gross genomic changes by targeting 2 critical senescence barriers. Consistent transcriptomic changes associated with immortality were identified using microarray analysis of isogenic normal finite pre-stasis, abnormal finite post-stasis, and immortal HMECs from 4 individuals. A total of 277 genes consistently changed in cells that transitioned from post-stasis to immortal. Gene ontology analysis of affected genes revealed biological processes significantly altered in the immortalization process. These immortalization-associated changes showed striking similarity to the gene expression changes seen in The Cancer Genome Atlas (TCGA) clinical breast cancer data. The most dramatic change in gene expression seen during the immortalization step was the downregulation of an unnamed, incompletely annotated transcript that we called MORT, for mortality, since its expression was closely associated with the mortal, finite lifespan phenotype. We show here that MORT (ZNF667-AS1) is expressed in all normal finite lifespan human cells examined to date and is lost in immortalized HMEC. MORT gene silencing at the mortal/immortal boundary was due to DNA hypermethylation of its CpG island promoter. This epigenetic silencing is also seen in human breast cancer cell lines and in a majority of human breast tumor tissues. The functional importance of DNA hypermethylation in MORT gene silencing is supported by the ability of 5-aza-2'-deoxycytidine to reactivate MORT expression. Analysis of TCGA data revealed deregulation of MORT expression due to DNA hypermethylation in 15 out of the 17 most common human cancers. The epigenetic silencing of MORT in a large majority of the common human cancers suggests a potential fundamental role in cellular immortalization during human carcinogenesis. PMID

  8. High Throughput Sequencing of Entamoeba 27nt Small RNA Population Reveals Role in Permanent Gene Silencing But No Effect on Regulating Gene Expression Changes during Stage Conversion, Oxidative, or Heat Shock Stress.

    Science.gov (United States)

    Zhang, Hanbang; Ehrenkaufer, Gretchen M; Manna, Dipak; Hall, Neil; Singh, Upinder

    2015-01-01

    The human parasite Entamoeba histolytica has an active RNA interference (RNAi) pathway with an extensive repertoire of 27nt small RNAs that silence genes. However the role of this pathway in regulating amebic biology remains unknown. In this study, we address whether silencing via 27nt small RNAs may be a mechanism for controlling gene expression changes during conversion between the trophozoite and cyst stages of the parasite. We sequenced small RNA libraries generated from trophozoites, early cysts, mature cysts, and excysting cells and mapped them to the E. invadens genome. Our results show that, as in E. histolytica, small RNAs in E. invadens are largely ~27nt in length, have an unusual 5'-polyphosphate structure and mediate gene silencing. However, when comparing the libraries from each developmental time-point we found few changes in the composition of the small RNA populations. Furthermore, genes targeted by small RNAs were permanently silenced with no changes in transcript abundance during development. Thus, the E. invadens 27nt small RNA population does not mediate gene expression changes during development. In order to assess the generalizability of our observations, we examined whether small RNAs may be regulating gene expression changes during stress response in E. histolytica. Comparison of the 27nt small RNA populations from E. histolytica trophozoites from basal conditions, or after heat shock or exposure to oxidative stress showed few differences. Similar to data in E. invadens development, genes targeted by small RNAs were consistently silenced and did not change expression under tested stress conditions. Thus, the biological roles of the 27nt small RNA population in Entamoeba remain elusive. However, as the first characterization of the RNAi pathway in E. invadens these data serve as a useful resource for the study of Entamoeba development and open the door to the development of RNAi-based gene silencing tools in E. invadens.

  9. Silencing of Bcl-XL Expression in Human MGC-803 Gastric Cancer Cells by siRNA

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

    To investigate the inhibitory effect of the Bcl-XL small interfering RNA (siRNA) on Bcl-XL gene expression in the human gastric cancer cell line MGC-803, green fluorescent protein (GFP) siRNA was constructed and transfected into MGC-803 ceils, together with GFP expression vector pTrace SV40.GFP expression levels were observed using fluorescence microscopy. Bcl-XL siRNA and negative siRNA were then constructed and stably transfected into MGC-803 cells. RT-PCR and immunofluorescence were used to detect the expression of Bcl-XL. Spontaneous apoptosis was detected by acridine orange (AO) and flow cytometry. Results were as follows: (1) 48 h after GFP expression vector and GFP siRNA co-transfection, the expression level of GFP in the GFP siRNA group was much lower than the negative siRNA group,according to fluorescence microscopy results. The mRNA and protein levels of Bcl-XL in Bcl-XL siRNA stable transfectants were reduced to almost background level compared with negative siRNA transfectants or untreated cells. (2) Changes in nucleus morphology was observed by AO staining nucleic and flow cytometry analysis, which showed that stable Bcl-XL siRNA transfectants have an increased spontaneous apoptosis (21.17%± 1.26% vs. 1.19%±0.18% and 1.56%±0.15 % respectively, P<0.05 vs. negative siRNA or untreated control). siRNA targeting GFP or Bcl-XL genes can specifically suppress GFP or Bcl-XL expression in MGC-803 cells, and Bcl-XL siRNA can increase spontaneous apoptosis. Bcl-XL siRNA may be a beneficial agent against human gastric adenocarcinoma.

  10. Epigenetic silencing of miRNA-9 is associated with HES1 oncogenic activity and poor prognosis of medulloblastoma

    OpenAIRE

    Fiaschetti, G; Abela, L; Nonoguchi, N; Dubuc, A. M.; Remke, M; A. Boro; Grunder, E; Siler, U; Ohgaki, H.; M. D. Taylor; M. Baumgartner; Shalaby, T.; Grotzer, M. A.

    2013-01-01

    Background: microRNA-9 is a key regulator of neuronal development aberrantly expressed in brain malignancies, including medulloblastoma. The mechanisms by which microRNA-9 contributes to medulloblastoma pathogenesis remain unclear, and factors that regulate this process have not been delineated. Methods: Expression and methylation status of microRNA-9 in medulloblastoma cell lines and primary samples were analysed. The association of microRNA-9 expression with medulloblastoma patients' clinic...

  11. Proof-of-concept Studies for siRNA-mediated Gene Silencing for Coagulation Factors in Rat and Rabbit

    OpenAIRE

    Chen, Zhu; Luo, Bin; Cai, Tian-Quan; Thankappan, Anil; Xu, Yiming; Wu, Weizhen; DiMuzio, Jillian; Lifsted, Traci; DiPietro, Marty; Disa, Jyoti; Ng, Bruce; Leander, Karen; Clark, Seth; Hoos, Lizbeth; Zhou, Yuchen

    2015-01-01

    The present study aimed at establishing feasibility of delivering short interfering RNA (siRNA) to target the coagulation cascade in rat and rabbit, two commonly used species for studying thrombosis and hemostasis. siRNAs that produced over 90% mRNA knockdown of rat plasma prekallikrein and rabbit Factor X (FX) were identified from in vitro screens. An ionizable amino lipid based lipid nanoparticle (LNP) formulation for siRNA in vivo delivery was characterized as tolerable and exerting no app...

  12. Chitosan/siRNA functionalized titanium surface via a layer-by-layer approach for in vitro sustained gene silencing and osteogenic promotion.

    Science.gov (United States)

    Song, Wen; Song, Xin; Yang, Chuanxu; Gao, Shan; Klausen, Lasse Hyldgaard; Zhang, Yumei; Dong, Mingdong; Kjems, Jørgen

    2015-01-01

    Titanium surface modification is crucial to improving its bioactivity, mainly its bone binding ability in bone implant materials. In order to functionalize titanium with small interfering RNA (siRNA) for sustained gene silencing in nearby cells, the layer-by-layer (LbL) approach was applied using sodium hyaluronate and chitosan/siRNA (CS/siRNA) nanoparticles as polyanion and polycation, respectively, to build up the multilayered film on smooth titanium surfaces. The CS/siRNA nanoparticle characterization was analyzed first. Dynamic contact angle, atomic force microscopy, and scanning electron microscopy were used to monitor the layer accumulation. siRNA loaded in the film was quantitated and the release profile of film in phosphate-buffered saline was studied. In vitro knockdown effect and cytotoxicity evaluation of the film were investigated using H1299 human lung carcinoma cells expressing green fluorescent protein (GFP). The transfection of human osteoblast-like cell MG63 and H1299 were performed and the osteogenic differentiation of MG63 on LbL film was analyzed. The CS/siRNA nanoparticles exhibited nice size distribution. During formation of the film, the surface wettability, topography, and roughness were alternately changed, indicating successful adsorption of the individual layers. The scanning electron microscope images clearly demonstrated the hybrid structure between CS/siRNA nanoparticles and sodium hyaluronate polymer. The cumulated load of siRNA increased linearly with the bilayer number and, more importantly, a gradual release of the film allowed the siRNA to be maintained on the titanium surface over approximately 1 week. In vitro transfection revealed that the LbL film-associated siRNA could consistently suppress GFP expression in H1299 without showing significant cytotoxicity. The LbL film loading with osteogenic siRNA could dramatically increase the osteogenic differentiation in MG63. In conclusion, LbL technology can potentially modify titanium

  13. siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes.

    Science.gov (United States)

    Matsuda, Shigeo; Keiser, Kristofer; Nair, Jayaprakash K; Charisse, Klaus; Manoharan, Rajar M; Kretschmer, Philip; Peng, Chang G; V Kel'in, Alexander; Kandasamy, Pachamuthu; Willoughby, Jennifer L S; Liebow, Abigail; Querbes, William; Yucius, Kristina; Nguyen, Tuyen; Milstein, Stuart; Maier, Martin A; Rajeev, Kallanthottathil G; Manoharan, Muthiah

    2015-05-15

    Asialoglycoprotein receptor (ASGPR) mediated delivery of triantennary N-acetylgalactosamine (GalNAc) conjugated short interfering RNAs (siRNAs) to hepatocytes is a promising paradigm for RNAi therapeutics. Robust and durable gene silencing upon subcutaneous administration at therapeutically acceptable dose levels resulted in the advancement of GalNAc-conjugated oligonucleotide-based drugs into preclinical and clinical developments. To systematically evaluate the effect of display and positioning of the GalNAc moiety within the siRNA duplex on ASGPR binding and RNAi activity, nucleotides carrying monovalent GalNAc were designed. Evaluation of clustered and dispersed incorporation of GalNAc units to the sense (S) strand indicated that sugar proximity is critical for ASGPR recognition, and location of the clustered ligand impacts the intrinsic potency of the siRNA. An array of nucleosidic GalNAc monomers resembling a trivalent ligand at or near the 3' end of the S strand retained in vitro and in vivo siRNA activity, similar to the parent conjugate design. This work demonstrates the utility of simple, nucleotide-based, cost-effective siRNA-GalNAc conjugation strategies.

  14. The Drosophila HP1 homolog Rhino is required for transposon silencing and piRNA production by dual-strand clusters.

    Science.gov (United States)

    Klattenhoff, Carla; Xi, Hualin; Li, Chengjian; Lee, Soohyun; Xu, Jia; Khurana, Jaspreet S; Zhang, Fan; Schultz, Nadine; Koppetsch, Birgit S; Nowosielska, Anetta; Seitz, Herve; Zamore, Phillip D; Weng, Zhiping; Theurkauf, William E

    2009-09-18

    Piwi-interacting RNAs (piRNAs) silence transposons and maintain genome integrity during germline development. In Drosophila, transposon-rich heterochromatic clusters encode piRNAs either on both genomic strands (dual-strand clusters) or predominantly one genomic strand (uni-strand clusters). Primary piRNAs derived from these clusters are proposed to drive a ping-pong amplification cycle catalyzed by proteins that localize to the perinuclear nuage. We show that the HP1 homolog Rhino is required for nuage organization, transposon silencing, and ping-pong amplification of piRNAs. rhi mutations virtually eliminate piRNAs from the dual-strand clusters and block production of putative precursor RNAs from both strands of the major 42AB dual-strand cluster, but not of transcripts or piRNAs from the uni-strand clusters. Furthermore, Rhino protein associates with the 42AB dual-strand cluster,but does not bind to uni-strand cluster 2 or flamenco. Rhino thus appears to promote transcription of dual-strand clusters, leading to production of piRNAs that drive the ping-pong amplification cycle. PMID:19732946

  15. The response of trypanosomes and other eukaryotes to ER stress and the spliced leader RNA silencing (SLS) pathway in Trypanosoma brucei.

    Science.gov (United States)

    Michaeli, Shulamit

    2015-01-01

    The unfolded protein response (UPR) is induced when the quality control machinery of the cell is overloaded with unfolded proteins or when one of the functions of the endoplasmic reticulum (ER) is perturbed. Here, I describe UPR in yeast and mammals, and compare it to what we know about pathogenic fungi and the parasitic protozoans from the order kinetoplastida, focusing on the novel pathway the spliced leader silencing (SLS) in Trypanosoma brucei. Trypanosomes lack conventional transcription regulation, and thus, lack most of the UPR machinery present in other eukaryotes. Trypanosome genes are transcribed in polycistronic units that are processed by trans-splicing and polyadenylation. In trans-splicing, which is essential for processing of each mRNA, an exon known as the spliced leader (SL) is added to all mRNAs from a small RNA, the SL RNA. Under severe ER stress, T. brucei elicits the SLS pathway. In SLS, the transcription of the SL RNA gene is extinguished, and the entire transcription complex dissociates from the SL RNA promoter. Induction of SLS is mediated by an ER-associated kinase (PK3) that migrates to the nucleus, where it phosphorylates the TATA-binding protein (TRF4), leading shut-off of SL RNA transcription. As a result, trans-splicing is inhibited and the parasites activate a programmed cell death (PCD) pathway. Despite the ability to sense the ER stress, the different eukaryotes, especially unicellular parasites and pathogenic fungi, developed a variety of unique and different ways to sense and adjust to this stress in a manner different from their host.

  16. DICER/AGO-dependent epigenetic silencing of D4Z4 repeats enhanced by exogenous siRNA suggests mechanisms and therapies for FSHD.

    Science.gov (United States)

    Lim, Jong-Won; Snider, Lauren; Yao, Zizhen; Tawil, Rabi; Van Der Maarel, Silvère M; Rigo, Frank; Bennett, C Frank; Filippova, Galina N; Tapscott, Stephen J

    2015-09-01

    Facioscapulohumeral muscular dystrophy (FSHD) is caused by the aberrant expression of the DUX4 transcription factor in skeletal muscle. The DUX4 retrogene is encoded in the D4Z4 macrosatellite repeat array, and smaller array size or a mutation in the SMCHD1 gene results in inefficient epigenetic repression of DUX4 in skeletal muscle, causing FSHD1 and FSHD2, respectively. Previously we showed that the entire D4Z4 repeat is bi-directionally transcribed with the generation of small si- or miRNA-like fragments and suggested that these might suppress DUX4 expression through the endogenous RNAi pathway. Here we show that exogenous siRNA targeting the region upstream of the DUX4 transcription start site suppressed DUX4 mRNA expression and increased both H3K9 methylation and AGO2 recruitment. In contrast, similarly targeted MOE-gapmer antisense oligonucleotides that degrade RNA but do not engage the RNAi pathway did not repress DUX4 expression. In addition, knockdown of DICER or AGO2 using either siRNA or MOE-gapmer chemistries resulted in the induction of DUX4 expression in control muscle cells that normally do not express DUX4, indicating that the endogenous RNAi pathway is necessary to maintain repression of DUX4 in control muscle cells. Together these data demonstrate a role of the endogenous RNAi pathway in repeat-mediated epigenetic repression of the D4Z4 macrosatellite repeat, and show that enhancing the activity of this pathway by supplying exogenous siRNA oligonucleotides represents a potential therapeutic approach to silencing DUX4 in FSHD. PMID:26041815

  17. Post-transcriptional silencing of the SGE1 gene induced by a dsRNA hairpin in Fusarium oxysporum f. sp cubense, the causal agent of Panama disease.

    Science.gov (United States)

    Fernandes, J S; Angelo, P C S; Cruz, J C; Santos, J M M; Sousa, N R; Silva, G F

    2016-01-01

    Fusarium oxysporum f. sp cubense (Foc), the causal agent of Panama disease, is responsible for economic losses in banana crops worldwide. The identification of genes that effectively act on pathogenicity and/or virulence may contribute to the development of different strategies for disease control and the production of resistant plants. The objective of the current study was to analyze the importance of SGE1 gene expression in Foc virulence through post-transcriptional silencing using a double-stranded RNA hairpin. Thirteen transformants were selected based on different morphological characteristics, and sporulation in these transformants was significantly reduced by approximately 95% (P < 0.05) compared to that of the wild-type strain. The relative SGE1 expression levels in the transformant strains were reduced by 27 to 47% compared to those in the wild-type strain. A pathogenicity analysis revealed that the transformants were able to reach the rhizomes and pseudostems of the inoculated banana plants. However, the transformants induced initial disease symptoms in the banana plants approximately 10 days later than that by the wild-type Foc, and initial disease symptoms persisted even at 45 days after inoculation. These results indicate that the SGE1 gene is directly involved in the virulence of Foc. Therefore, SGE1 may be a potential candidate for host-induced gene silencing in banana plants. PMID:27173186

  18. Enhanced Gene Delivery and siRNA Silencing by Gold Nanoparticles Coated with Charge-Reversal Polyelectrolyte

    OpenAIRE

    Guo, Shutao; Huang, Yuanyu; Jiang, Qiao; Sun, Yun; Deng, Liandong; Liang, Zicai; Du, Quan; Xing, Jinfeng; Zhao, Yuliang; Wang, Paul C.; Dong, Anjie; Liang, Xing-Jie

    2010-01-01

    Charge-reversal functional gold nanoparticles first prepared by layer-by-layer technique were employed to deliver small interfering RNA (siRNA) and plasmid DNA into cancer cells. Polyacrylamide gel electrophoresis measurements of siRNA confirmed the occurrence of the charge-reversal property of functional gold nanoparticles. The expression efficiency of enhanced green fluorescent protein (EGFP) was improved by adjuvant transfection with charge-reversal functional gold nanoparticles, which als...

  19. Proof-of-concept Studies for siRNA-mediated Gene Silencing for Coagulation Factors in Rat and Rabbit

    Science.gov (United States)

    Chen, Zhu; Luo, Bin; Cai, Tian-Quan; Thankappan, Anil; Xu, Yiming; Wu, Weizhen; DiMuzio, Jillian; Lifsted, Traci; DiPietro, Marty; Disa, Jyoti; Ng, Bruce; Leander, Karen; Clark, Seth; Hoos, Lizbeth; Zhou, Yuchen; Jochnowitz, Nina; Jachec, Christine; Szczerba, Peter; Gindy, Marian E.; Strapps, Walter; Sepp-Lorenzino, Laura; Seiffert, Dietmar A.; Lubbers, Laura; Tadin-Strapps, Marija

    2015-01-01

    The present study aimed at establishing feasibility of delivering short interfering RNA (siRNA) to target the coagulation cascade in rat and rabbit, two commonly used species for studying thrombosis and hemostasis. siRNAs that produced over 90% mRNA knockdown of rat plasma prekallikrein and rabbit Factor X (FX) were identified from in vitro screens. An ionizable amino lipid based lipid nanoparticle (LNP) formulation for siRNA in vivo delivery was characterized as tolerable and exerting no appreciable effect on coagulability at day 7 postdosing in both species. Both prekallikrein siRNA-LNP and FX siRNA-LNP resulted in dose-dependent and selective knockdown of target gene mRNA in the liver with maximum reduction of over 90% on day 7 following a single dose of siRNA-LNP. Knockdown of plasma prekallikrein was associated with modest clot weight reduction in the rat arteriovenous shunt thrombosis model and no increase in the cuticle bleeding time. Knockdown of FX in the rabbit was accompanied with prolongation in ex vivo clotting times. Results fit the expectations with both targets and demonstrate for the first time, the feasibility of targeting coagulation factors in rat, and, more broadly, targeting a gene of interest in rabbit, via systemic delivery of ionizable LNP formulated siRNA. PMID:25625614

  20. RNA干扰对阿尔茨海默病家族性瑞典型APP突变的沉默作用%Induction of silencing effect of Alzheimer disease familial Swedish mutant APP by RNA interference

    Institute of Scientific and Technical Information of China (English)

    邱昕; 潘纪安; 陈宇; 张玮莹; 杨华静; 张苏明

    2005-01-01

    Objectives To observe the silencing effect of amyloid precursor protein (APP) carrying Swedish mutation of familial Alzheimer disease triggered by small interference RNA (siRNA). Methods We designed one oligonucleotide against Swedish mutant APP(APPswe) which was endogenously expressed by small interference RNA(siRNA) expressing plasmid . In order to observe the silence effect of siRNA on APP, COS-7 cells were transiently co-transfected with APPEGFP recombinant plasmid and siRNA expressing plasmid. The silencing effect of siRNA was determined by the assay of reporter gene-GFP expression levels. Results APPswe expression in COS-7 cells was knocked down by the application of siRNA. APPswesiRNA was more effective in silencing APPswe gene than wild type APP (APPwt) gene. Conclusion APPswe siRNA can effectively silence APPswe gene expression and such effect is allele-specific. Therefore, siRNA directed against APPswe may have therapeutic potential for the treatment of Alzheimer disease.%目的诱导小干涉RNA(small interference RNA,siRNA)对阿尔茨海默病淀粉样前体蛋白家族性瑞典型突变(APPswe)的沉默作用.方法设计一对针对APPswe的寡核苷酸,构建成内源性表达相应siRNA的质粒,与构建的APP-EGFP重组质粒瞬时共转染COS-7细胞,利用GFP报告基因来间接观察siRNA对APPswe的沉默作用.结果siRNA可有效地下调突变型APP,相比APPwt而言,针对突变型APP的siRNA对APPswe的沉默作用更强.结论针对APPswe的siRNA可有效地沉默APPswe基因,并有一定的等位基因特异性.因此,针对APPswe的siRNA将在阿尔茨海默病治疗研究领域扮演重要角色.

  1. Silence multiple

    DEFF Research Database (Denmark)

    Søndergaard, Katia Dupret

    -Network Theoretical approach to organization studies silence is conceptualised as both a means and an effect of innovative efforts. It is a way of ordering practices. Thus silencing is thought of as a central potential change agent both in composing a kind of specific organizational collectivity and in composing new...

  2. A Comparison of Target Gene Silencing using Synthetically Modified siRNA and shRNA That Express Recombinant Lentiviral Vectors.

    Science.gov (United States)

    Spirin, P V; Baskaran, D; Rubtsov, P M; Zenkova, M A; Vlassov, V V; Chernolovskaya, E L; Prassolov, V S

    2009-07-01

    RNA-interference is an effective natural mechanism of post-transcriptional modulation of gene expression. RNA-interference mechanism exist as in high eukaryotes both animals and plants as well in lower eukaryotes and viruses. RNA-interference is now used as a powerful tool in study of functional gene activity and many essential for fundamental biology results was obtained with this approach. Also it's widely believed that RNA-interference could be used in working out of new therapeutic medicine against malignant, infectious and hereditary diseases. One of the main problems of these developments is search of effective methods of siRNA transfer into the target cells. At present time for these purpose different sorts of transfect ions or viral transduction are used. At present article the results of comparison of inhibition of expression of oncogene AML-ET O by synthetic siRNA and by recombinant lentiviruses coding for corresponding shRNA are presented.

  3. Silencing by blasting: combination of laser pulse induced stress waves and magnetophoresis for siRNA delivery

    Science.gov (United States)

    Babincová, M.; Babincová, N.; Durdík, S.; Bergemann, C.; Sourivong, P.

    2016-06-01

    A new method is developed for efficient delivery of short interference RNA into cells using combination of magnetophoresis for pre-concentration of siRNA-magnetic nanoparticle complex on the surface of cells with subsequent nanosecond laser pulse generating stress waves in transfection chamber, which is able to permeabilize cell membrane for the facilitated delivery of siRNA into the cell interior. As has been shown using siRNA inducing cell apoptosis, combination of these two physical factors increased the efficiency of three different human carcinoma cells transfection to 93%, 89%, and 84%, for HeLa (cervical carcinoma), MCF-7 (breast carcinoma), and UCI-107 (ovarian carcinoma) cells, respectively. This new physical method of siRNA delivery may have therefore far reaching applications in biotechnology and functional genomics.

  4. Progress in molecular mechanism of RNA silencing in plants%植物RNA沉默的分子机制研究进展

    Institute of Scientific and Technical Information of China (English)

    王鹏; 赵显军; 朱国萍

    2008-01-01

    RNA沉默(RNA silencing)是真核生物中的一种抵抗外源遗传因子(病毒、转座子或转基因)及调控基因表达的防御机制.参与植物RNA沉默的酶及蛋白质主要包括6种RNA依赖的RNA聚合酶、4种Dicer-like(DCL)核酸内切酶和10种Argonautes蛋白.植物中4条RNA沉默途径分别由微小RNA(miRNAs)和3种小干扰RNA(siRNAs)介导,包括反式作用siRNAs(ta-siRNAs)、天然反义siRNAs(nat-siRNAs)和异染色质siRNAs(hc-siRNAs).在植物RNA沉默的系统性传播中,由DCL4或DCL2将dsRNAs裁剪为次级siRNAs,以放大RNA沉默信号和增强沉默效应.

  5. Dynamic Contrast Enhanced MRI Assessing the Antiangiogenic Effect of Silencing HIF-1α with Targeted Multifunctional ECO/siRNA Nanoparticles.

    Science.gov (United States)

    Malamas, Anthony S; Jin, Erlei; Gujrati, Maneesh; Lu, Zheng-Rong

    2016-07-01

    Stabilization of hypoxia inducible factor 1α (HIF-1α), a biomarker of hypoxia, in hypoxic tumors mediates a variety of downstream genes promoting tumor angiogenesis and cancer cell survival as well as invasion, and compromising therapeutic outcome. In this study, dynamic contrast enhanced MRI (DCE-MRI) with a biodegradable macromolecular MRI contrast agent was used to noninvasively assess the antiangiogenic effect of RGD-targeted multifunctional lipid ECO/siHIF-1α nanoparticles in a mouse HT29 colon cancer model. The RGD-targeted ECO/siHIF-1α nanoparticles resulted in over 50% reduction in tumor size after intravenous injection at a dose of 2.0 mg of siRNA/kg every 3 days for 3 weeks compared to a saline control. DCE-MRI revealed significant decline in vascularity and over a 70% reduction in the tumor blood flow, permeability-surface area product, and plasma volume fraction vascular parameters in the tumor treated with the targeted ECO/siHIF-1α nanoparticles. The treatment with targeted ECO/siRNA nanoparticles resulted in significant silencing of HIF-1α expression at the protein level, which also significantly suppressed the expression of VEGF, Glut-1, HKII, PDK-1, LDHA, and CAIX, which are all important players in tumor angiogenesis, glycolytic metabolism, and pH regulation. By possessing the ability to elicit a multifaceted effect on tumor biology, silencing HIF-1α with RGD-targeted ECO/siHIF-1α nanoparticles has great promise as a single therapy or in combination with traditional chemotherapy or radiation strategies to improve cancer treatment. PMID:27264671

  6. Transgenic tobacco plants expressing siRNA targeted against the Mungbean yellow mosaic virus transcriptional activator protein gene efficiently block the viral DNA accumulation.

    Science.gov (United States)

    Shanmugapriya, Gnanasekaran; Das, Sudhanshu Sekhar; Veluthambi, Karuppannan

    2015-06-01

    Mungbean yellow mosaic virus (MYMV) is a bipartite begomovirus that infects many pulse crops such as blackgram, mungbean, mothbean, Frenchbean, and soybean. We tested the efficacy of the transgenically expressed intron-spliced hairpin RNA gene of the transcriptional activator protein (hpTrAP) in reducing MYMV DNA accumulation. Tobacco plants transformed with the MYMV hpTrAP gene accumulated 21-22 nt siRNA. Leaf discs of the transgenic plants, agroinoculated with the partial dimers of MYMV, displayed pronounced reduction in MYMV DNA accumulation. Thus, silencing of the TrAP gene, a suppressor of gene silencing, emerged as an effective strategy to control MYMV. PMID:26436122

  7. Antitumor activity and prolonged survival by carbon-nanotube-mediated therapeutic siRNA silencing in a human lung xenograft model.

    Science.gov (United States)

    Podesta, Jennifer E; Al-Jamal, Khuloud T; Herrero, M Antonia; Tian, Bowen; Ali-Boucetta, Hanene; Hegde, Vikas; Bianco, Alberto; Prato, Maurizio; Kostarelos, Kostas

    2009-05-01

    Carbon nanotubes are novel nanomaterials that are thought to offer potential benefits to a variety of biomedical and clinical applications. In this study, the treatment of a human lung carcinoma model in vivo using siRNA sequences leading to cytotoxicity and cell death is carried out using either cationic liposomes (DOTAP:cholesterol) or amino-functionalized multi-walled carbon nanotubes (MWNT - NH(+)(3)). Validation for the most cytotoxic siRNA sequence using a panel of human carcinoma and murine cells reveals that the proprietary siTOX sequence is human specific and can lead to significant cytotoxic activities delivered both by liposome or MWNT - NH(+)(3) in vitro. A comparative study using both types of vector indicates that only MWNT - NH(+)(3):siRNA complexes administered intratumorally can elicit delayed tumor growth and increased survival of xenograft-bearing animals. siTOX delivery via the cationic MWNT - NH(+)(3) is biologically active in vivo by triggering an apoptotic cascade, leading to extensive necrosis of the human tumor mass. This suggests that carbon-nanotube-mediated delivery of siRNA by intratumoral administration leads to successful and statistically significant suppression of tumor volume, followed by a concomitant prolongation of survival of human lung tumor-bearing animals. The direct comparison between carbon nanotubes and liposomes demonstrates the potential advantages offered by carbon nanotubes for the intracellular delivery of therapeutic agents in vivo. The present work may act as the impetus for further studies to explore the therapeutic capacity of chemically functionalized carbon nanotubes to deliver siRNA directly into the cytoplasm of target cells and achieve effective therapeutic silencing in various disease indications where local delivery is feasible or desirable.

  8. Silencing of cystatin M in metastatic oral cancer cell line MDA-686Ln by siRNA increases cysteine proteinases and legumain activities, cell proliferation and in vitro invasion.

    NARCIS (Netherlands)

    Vigneswaran, N.; Wu, J.; Nagaraj, N.; James, R.; Zeeuwen, P.L.J.M.; Zacharias, W.

    2006-01-01

    Cystatins are inhibitors of lysosomal cysteine proteinases. Cystatin M demonstrates more diverse tissue distribution, target specificity and biological function than other cystatins from the same family. We utilized small interference RNAs (siRNA) to silence cystatin M gene expression in a metastati

  9. Enhancement of Gene Silencing Effect and Membrane Permeability by Peptide-Conjugated 27-Nucleotide Small Interfering RNA

    Directory of Open Access Journals (Sweden)

    Toshio Seyama

    2012-09-01

    Full Text Available Two different sizes of siRNAs, of which one type was 21-nucleotide (nt siRNA containing 2-nt dangling ends and the other type was 27-nt siRNA with blunt ends, were conjugated with a nuclear export signal peptide of HIV-1 Rev at the 5′-sense end. Processing by Dicer enzyme, cell membrane permeability, and RNAi efficiency of the peptide-conjugated siRNAs were examined. Dicer cleaved the peptide-conjugated 27-nt siRNA leading to the release of 21-nt siRNA, whereas the peptide-conjugated 21-nt siRNA was not cleaved. High membrane permeability and cytoplasmic localization was found in the conjugates. Moreover, the peptide-conjugated 27-nt siRNA showed increased potency of RNAi in comparison with the nonmodified 21-nt and 27-nt siRNAs, whereas the peptide-conjugated 21-nt siRNA showed decreased RNAi efficacy. This potent RNAi efficacy is probably owing to acceleration of RISC through recognition by Dicer, as well as to the improvement of cell membrane permeability and intracellular accumulation.

  10. Nanotechnology approach for drug addiction therapy: Gene silencing using delivery of gold nanorod-siRNA nanoplex in dopaminergic neurons

    OpenAIRE

    Bonoiu, Adela C.; Mahajan, Supriya D.; Ding, Hong; Roy, Indrajit; Yong, Ken-Tye; Kumar, Rajiv; Hu, Rui; Bergey, Earl J.; Schwartz, Stanley A.; Prasad, Paras N.

    2009-01-01

    Drug abuse is a worldwide health concern in which addiction involves activation of the dopaminergic signaling pathway in the brain. Here, we introduce a nanotechnology approach that utilizes gold nanorod-DARPP-32 siRNA complexes (nanoplexes) that target this dopaminergic signaling pathway in the brain. The shift in the localized longitudinal plasmon resonance peak of gold nanorods (GNRs) was used to show their interaction with siRNA. Plasmonic enhanced dark field imaging was used to visualize...

  11. The QKI-6 and QKI-7 RNA binding proteins block proliferation and promote Schwann cell myelination.

    Directory of Open Access Journals (Sweden)

    Daniel Larocque

    Full Text Available BACKGROUND: The quaking viable (qk(v mice have uncompacted myelin in their central and peripheral nervous system (CNS, PNS. The qk gene encodes 3 major alternatively spliced isoforms that contain unique sequence at their C-terminus dictating their cellular localization. QKI-5 is a nuclear isoform, whereas QKI-6 and QKI-7 are cytoplasmic isoforms. The qk(v mice harbor an enhancer/promoter deletion that prevents the expression of isoforms QKI-6 and QKI-7 in myelinating cells resulting in a dysmyelination phenotype. It was shown that QKI regulates the differentiation of oligodendrocytes, the myelinating cells of the CNS, however, little is known about the role of the QKI proteins, or RNA binding proteins in PNS myelination. METHODOLOGY/PRINCIPAL FINDINGS: To define the role of the QKI proteins in PNS myelination, we ectopically expressed QKI-6 and QKI-7 in primary rat Schwann cell/neuron from dorsal root ganglia cocultures. We show that the QKI isoforms blocked proliferation and promoted Schwann cell differentiation and myelination. In addition, these events were coordinated with elevated proteins levels of p27(KIP1 and myelin basic protein (MBP, markers of Schwann cell differentiation. QKI-6 and QKI-7 expressing co-cultures contained myelinated fibers that had directionality and contained significantly thicker myelin, as assessed by electron microscopy. Moreover, QKI-deficient Schwann cells had reduced levels of MBP, p27(KIP1 and Krox-20 mRNAs, as assessed by quantitative RT-PCR. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that the QKI-6 and QKI-7 RNA binding proteins are positive regulators of PNS myelination and show that the QKI RNA binding proteins play a key role in Schwann cell differentiation and myelination.

  12. MicroRNA-mediated Silencing of RhoC Inhibits Tumor Invasion and Increases Chemosensitivity to Paclitaxel in SKOV3 Cells in vitro

    Institute of Scientific and Technical Information of China (English)

    PAN Ying; DU Zhen-wu; LENG Wei-chun; ZHOU Jia-wen; WANG Ying-jian; SHENG Min-jia; WANG Jun-rong; ZHANG Gui-zhen

    2011-01-01

    RhoC is a member of the Ras-homologous family of genes which are implicated in tumorigenesis and tumor progression. Up-regulation of RhoC is associated with tumor progression in ovarian carcinoma and RhoC is significantly correlated with the invasive capability of ovarian cancer cell lines in vitro. We developed a system that blocks RhoC in the human ovarian cancer SKOV3 cells using specific MicroRNA(miRNA) interference. By transfecting SKOV3 cells with the plasmid vector to express specific MiRNA that targets human RhoC, we were able to establish a stable clone in which RhoC expression was significantly downregnlated. This resulted in the decreased invasive potential of SKOV3 cells as well as increased chemosensitivity to paclitaxel. RhoC involves in invasion and chemosensitivity of SKOV3, indicating that RhoC may be a promising therapeutic target for ovarian cancer.

  13. Blocking the Wnt/β-Catenin Pathway by Lentivirus-Mediated Short Hairpin RNA Targeting β-Catenin Gene Suppresses Silica-Induced Lung Fibrosis in Mice

    Directory of Open Access Journals (Sweden)

    Xin Wang

    2015-09-01

    Full Text Available Silicosis is a form of occupational lung disease caused by inhalation of crystalline silica dust. While the pathogenesis of silicosis is not clearly understood, the Wnt/β-catenin signaling pathway is thought to play a major role in lung fibrosis. To explore the role of Wnt/β-catenin pathway in silicosis, we blocked Wnt/β-catenin pathway both in silica-treated MLE-12 cells (a mouse pulmonary epithelial cell line and in a mouse silicosis model by using a lentiviral vector expressing a short hairpin RNA silencing β-catenin (Lv-shβ-catenin. In vitro, Lv-shβ-catenin significantly decreased the expression of β-catenin, MMP2 and MMP9, and secretion of TGF-β1. In vivo, intratracheal treatment with Lv-shβ-catenin significantly reduced expression of β-catenin in the lung and levels of TGF-β1 in bronchoalveolar lavage fluid, and notably attenuated pulmonary fibrosis as evidenced by hydroxyproline content and collagen I\\III synthesis in silica-administered mice. These results indicate that blockade of the Wnt/β-catenin pathway can prevent the development of silica-induced lung fibrosis. Thus Wnt/β-catenin pathway may be a target in prevention and treatment of silicosis.

  14. Arabidopsis RRP6L1 and RRP6L2 function in FLOWERING LOCUS C silencing via regulation of antisense RNA synthesis.

    Directory of Open Access Journals (Sweden)

    Jun-Hye Shin

    2014-09-01

    Full Text Available The exosome complex functions in RNA metabolism and transcriptional gene silencing. Here, we report that mutations of two Arabidopsis genes encoding nuclear exosome components AtRRP6L1 and AtRRP6L2, cause de-repression of the main flowering repressor FLOWERING LOCUS C (FLC and thus delay flowering in early-flowering Arabidopsis ecotypes. AtRRP6L mutations affect the expression of known FLC regulatory antisense (AS RNAs AS I and II, and cause an increase in Histone3 K4 trimethylation (H3K4me3 at FLC. AtRRP6L1 and AtRRP6L2 function redundantly in regulation of FLC and also act independently of the exosome core complex. Moreover, we discovered a novel, long non-coding, non-polyadenylated antisense transcript (ASL, for Antisense Long originating from the FLC locus in wild type plants. The AtRRP6L proteins function as the main regulators of ASL synthesis, as these mutants show little or no ASL transcript. Unlike ASI/II, ASL associates with H3K27me3 regions of FLC, suggesting that it could function in the maintenance of H3K27 trimethylation during vegetative growth. AtRRP6L mutations also affect H3K27me3 levels and nucleosome density at the FLC locus. Furthermore, AtRRP6L1 physically associates with the ASL transcript and directly interacts with the FLC locus. We propose that AtRRP6L proteins participate in the maintenance of H3K27me3 at FLC via regulating ASL. Furthermore, AtRRP6Ls might participate in multiple FLC silencing pathways by regulating diverse antisense RNAs derived from the FLC locus.

  15. Comparison of helper component-protease RNA silencing suppression activity, subcellular localization, and aggregation of three Korean isolates of Turnip mosaic virus.

    Science.gov (United States)

    Han, Jae-Yeong; Chung, Jinsoo; Kim, Jungkyu; Seo, Eun-Young; Kilcrease, James P; Bauchan, Gary R; Lim, Seungmo; Hammond, John; Lim, Hyoun-Sub

    2016-08-01

    In 2014, we performed a nationwide survey in Korean radish fields to investigate the distribution and variability of Turnip mosaic virus (TuMV). Brassica rapa ssp. pekinensis sap-inoculated with three isolates of TuMV from infected radish tissue showed different symptom severities, whereas symptoms in Raphanus sativus were similar for each isolate. The helper component-protease (HC-Pro) genes of each isolate were sequenced, and phylogenetic analysis showed that the three Korean isolates were clustered into the basal-BR group. The HC-Pro proteins of these isolates were tested for their RNA silencing suppressor (VSR) activity and subcellular localization in Nicotiana benthamiana. A VSR assay by co-agroinfiltration of HC-Pro with soluble-modified GFP (smGFP) showed that HC-Pro of isolate R007 and R041 showed stronger VSR activity than R065. The HC-Pros showed 98.25 % amino acid identity, and weak VSR isolate (R065) has a single variant residue in the C-terminal domain associated with protease activity and self-interaction compared to isolates with strong VSR activity. Formation of large subcellular aggregates of GFP:HC-Pro fusion proteins in N. benthamiana was only observed for HC-Pro from isolates with strong VSR activity, suggesting that R065 'weak' HC-Pro may have diminished self-association; substitution of the variant C-terminal residue largely reversed the HC-Pro aggregation and silencing suppressor characteristics. The lack of correlation between VSR efficiency and induction of systemic necrosis (SN) suggests that differences in viral accumulation due to HC-Pro are not responsible for SN. PMID:27059238

  16. Xist RNA is confined to the nuclear territory of the silenced X chromosome throughout the cell cycle

    NARCIS (Netherlands)

    Jonkers, Iris; Monkhorst, Kim; Rentmeester, Eveline; Grootegoed, J Anton; Grosveld, Frank; Gribnau, Joost

    2008-01-01

    In mammalian female cells, one X chromosome is inactivated to prevent a dose difference in the expression of X-encoded proteins between males and females. Xist RNA, required for X chromosome inactivation, is transcribed from the future inactivated X chromosome (Xi), where it spreads in cis, to initi

  17. Expression levels of the microRNA maturing microprocessor complex component DGCR8 and the RNA-induced silencing complex (RISC) components argonaute-1, argonaute-2, PACT, TARBP1, and TARBP2 in epithelial skin cancer.

    Science.gov (United States)

    Sand, Michael; Skrygan, Marina; Georgas, Dimitrios; Arenz, Christoph; Gambichler, Thilo; Sand, Daniel; Altmeyer, Peter; Bechara, Falk G

    2012-11-01

    The microprocessor complex mediates intranuclear biogenesis of precursor microRNAs from the primary microRNA transcript. Extranuclear, mature microRNAs are incorporated into the RNA-induced silencing complex (RISC) before interaction with complementary target mRNA leads to transcriptional repression or cleavage. In this study, we investigated the expression profiles of the microprocessor complex subunit DiGeorge syndrome critical region gene 8 (DGCR8) and the RISC components argonaute-1 (AGO1), argonaute-2 (AGO2), as well as double-stranded RNA-binding proteins PACT, TARBP1, and TARBP2 in epithelial skin cancer and its premalignant stage. Patients with premalignant actinic keratoses (AK, n = 6), basal cell carcinomas (BCC, n = 15), and squamous cell carcinomas (SCC, n = 7) were included in the study. Punch biopsies were harvested from the center of the tumors (lesional), from healthy skin sites (intraindividual controls), and from healthy skin sites in a healthy control group (n = 16; interindividual control). The DGCR8, AGO1, AGO2, PACT, TARBP1, and TARBP2 mRNA expression levels were detected by quantitative real-time reverse transcriptase polymerase chain reaction. The DGCR8, AGO1, AGO2, PACT, and TARBP1 expression levels were significantly higher in the AK, BCC, and SCC groups than the healthy controls (P  0.05). This study indicates that major components of the miRNA pathway, such as the microprocessor complex and RISC, are dysregulated in epithelial skin cancer.

  18. Nanolayered siRNA delivery platforms for local silencing of CTGF reduce cutaneous scar contraction in third-degree burns.

    Science.gov (United States)

    Castleberry, Steven A; Golberg, Alexander; Sharkh, Malak Abu; Khan, Saiqa; Almquist, Benjamin D; Austen, William G; Yarmush, Martin L; Hammond, Paula T

    2016-07-01

    Wound healing is an incredibly complex biological process that often results in thickened collagen-enriched healed tissue called scar. Cutaneous scars lack many functional structures of the skin such as hair follicles, sweat glands, and papillae. The absence of these structures contributes to a number of the long-term morbidities of wound healing, including loss of function for tissues, increased risk of re-injury, and aesthetic complications. Scar formation is a pervasive factor in our daily lives; however, in the case of serious traumatic injury, scars can create long-lasting complications due to contraction and poor tissue remodeling. Within this report we target the expression of connective tissue growth factor (CTGF), a key mediator of TGFβ pro-fibrotic response in cutaneous wound healing, with controlled local delivery of RNA interference. Through this work we describe both a thorough in vitro analysis of nanolayer coated sutures for the controlled delivery of siRNA and its application to improve scar outcomes in a third-degree burn induced scar model in rats. We demonstrate that the knockdown of CTGF significantly altered the local expression of αSMA, TIMP1, and Col1a1, which are known to play roles in scar formation. The knockdown of CTGF within the healing burn wounds resulted in improved tissue remodeling, reduced scar contraction, and the regeneration of papillary structures within the healing tissue. This work adds support to a number of previous reports that indicate CTGF as a potential therapeutic target for fibrosis. Additionally, we believe that the controlled local delivery of siRNA from ultrathin polymer coatings described within this work is a promising approach in RNA interference that could be applied in developing improved cancer therapies, regenerative medicine, and fundamental scientific research. PMID:27108403

  19. A possible role for miRNA silencing in disease phenotype variation in Swedish transthyretin V30M carriers

    Directory of Open Access Journals (Sweden)

    Olsson Malin

    2010-09-01

    Full Text Available Abstract Background Familial amyloidosis with polyneuropathy (FAP is an autosomal dominant disease caused by transthyretin (TTR mutations, of which V30M (TTR c.148G > A, p.Val50Met, "Val30Met" is the most common. Swedish V30M carriers display later age at onset and lower penetrance compared to other populations. Methods In the study, 130 Swedish V30M carriers (32 early, 30 late onset and 68 asymptomatic carriers and 50 controls, 23 French symptomatic V30M carriers and 29 controls and 18 Japanese symptomatic V30M carriers and 29 controls were included. We aimed to identify additional genetic factors in the TTR gene and its surrounding region that could have an impact on phenotype. Results We identified three SNPs (rs71383038, rs3794885 and rs62093482 with a significant difference in allele frequency between Swedish V30M carriers and controls. The two Swedish V30M homozygous patients present in the study also displayed homozygosity for the CA10 (rs71383038, A (rs3794885 and T (rs62093482 alleles in these SNPs. Hence, these alleles are present on the Swedish V30M haplotype. Of these, rs62093482 is located in the 3'UTR of TTR gene and thus more interesting since SNPs in the 3'UTR can affect gene expression levels by modifying microRNA (miRNA targeting activity. miRNA target predictions revealed four potential miRNAs with predicted targets unique for the polymorphic allele. Conclusions Our results are the first to show the presence of a 3'UTR polymorphism on the V30M haplotype in Swedish carriers, which can serve as a miRNA binding site potentially leading to down-regulated expression from the mutated TTR allele. This finding may be related to the low penetrance and high age at onset of the disease observed in the Swedish patient population.

  20. Reversible male sterility in eggplant (Solanum melongena L.) by artificial microRNA-mediated silencing of general transcription factor genes.

    Science.gov (United States)

    Toppino, Laura; Kooiker, Maarten; Lindner, Matias; Dreni, Ludovico; Rotino, Giuseppe L; Kater, Martin M

    2011-08-01

    Since decades, plant male sterility is considered a powerful tool for biological containment to minimize unwanted self-pollination for hybrid seed production. Furthermore, prevention of pollen dispersal also answers to concerns regarding transgene flow via pollen from Genetically Modified (GM) crops to traditional crop fields or wild relatives. We induced male sterility by suppressing endogenous general transcription factor genes, TAFs, using anther-specific promoters combined with artificial microRNA (amiRNA) technology (Schwab et al., 2006). The system was made reversible by the ethanol inducible expression of an amiRNA-insensitive form of the target gene. We provide proof of concept in eggplant, a cultivated crop belonging to the Solanaceae family that includes many important food crops. The transgenic eggplants that we generated are completely male sterile and fertility can be fully restored by short treatments with ethanol, confirming the efficiency but also the reliability of the system in view of open field cultivation. By combining this system with induced parthenocarpy (Rotino et al., 1997), we provide a novel example of complete transgene containment in eggplant, which enables biological mitigation measures for the benefit of coexistence or biosafety purposes for GM crop cultivation. PMID:20955179

  1. Thumb Site 2 Inhibitors of Hepatitis C Viral RNA-dependent RNA Polymerase Allosterically Block the Transition from Initiation to Elongation.

    Science.gov (United States)

    Li, Jiawen; Johnson, Kenneth A

    2016-05-01

    Replication of the hepatitis C viral genome is catalyzed by the NS5B (nonstructural protein 5B) RNA-dependent RNA polymerase, which is a major target of antiviral drugs currently in the clinic. Prior studies established that initiation of RNA replication could be facilitated by starting with a dinucleotide (pGG). Here we establish conditions for efficient initiation from GTP to form the dinucleotide and subsequent intermediates leading to highly processive elongation, and we examined the effects of four classes of nonnucleoside inhibitors on each step of the reaction. We show that palm site inhibitors block initiation starting from GTP but not when starting from pGG. In addition we show that nonnucleoside inhibitors binding to thumb site-2 (NNI2) lead to the accumulation of abortive intermediates three-five nucleotides in length. Our kinetic analysis shows that NNI2 do not significantly block initiation or elongation of RNA synthesis; rather, they block the transition from initiation to elongation, which is thought to proceed with significant structural rearrangement of the enzyme-RNA complex including displacement of the β-loop from the active site. Direct measurement in single turnover kinetic studies show that pyrophosphate release is faster than the chemistry step, which appears to be rate-limiting during processive synthesis. These results reveal important new details to define the steps involved in initiation and elongation during viral RNA replication, establish the allosteric mechanisms by which NNI2 inhibitors act, and point the way to the design of more effective allosteric inhibitors that exploit this new information. PMID:26851276

  2. Targeted silencing of anthrax toxin receptors protects against anthrax toxins.

    Science.gov (United States)

    Arévalo, Maria T; Navarro, Ashley; Arico, Chenoa D; Li, Junwei; Alkhatib, Omar; Chen, Shan; Diaz-Arévalo, Diana; Zeng, Mingtao

    2014-05-30

    Anthrax spores can be aerosolized and dispersed as a bioweapon. Current postexposure treatments are inadequate at later stages of infection, when high levels of anthrax toxins are present. Anthrax toxins enter cells via two identified anthrax toxin receptors: tumor endothelial marker 8 (TEM8) and capillary morphogenesis protein 2 (CMG2). We hypothesized that host cells would be protected from anthrax toxins if anthrax toxin receptor expression was effectively silenced using RNA interference (RNAi) technology. Thus, anthrax toxin receptors in mouse and human macrophages were silenced using targeted siRNAs or blocked with specific antibody prior to challenge with anthrax lethal toxin. Viability assays were used to assess protection in macrophages treated with specific siRNA or antibody as compared with untreated cells. Silencing CMG2 using targeted siRNAs provided almost complete protection against anthrax lethal toxin-induced cytotoxicity and death in murine and human macrophages. The same results were obtained by prebinding cells with specific antibody prior to treatment with anthrax lethal toxin. In addition, TEM8-targeted siRNAs also offered significant protection against lethal toxin in human macrophage-like cells. Furthermore, silencing CMG2, TEM8, or both receptors in combination was also protective against MEK2 cleavage by lethal toxin or adenylyl cyclase activity by edema toxin in human kidney cells. Thus, anthrax toxin receptor-targeted RNAi has the potential to be developed as a life-saving, postexposure therapy against anthrax.

  3. Silencing of ABCG2 by MicroRNA-3163 Inhibits Multidrug Resistance in Retinoblastoma Cancer Stem Cells.

    Science.gov (United States)

    Jia, Ming; Wei, Zhenhua; Liu, Peng; Zhao, Xiaoli

    2016-06-01

    To investigate the function and regulation mechanism of ATP-binding cassette, subfamily G, member 2 (ABCG2) in retinoblastoma cancer stem cells (RCSCs), a long-term culture of RCSCs from WERI-Rb1 cell line was successfully established based on the high expression level of ABCG2 on the surface of RCSCs. To further explore the molecular mechanism of ABCG2 on RCSCs, a microRNA that specifically targets ABCG2 was predicted. Subsequently, miR-3163 was selected and confirmed as the ABCG2-regulating microRNA. Overexpression of miR-3163 led to a significant decrease in ABCG2 expression. Additionally, ABCG2 loss-of-function induced anti-proliferation and apoptosis-promoting functions in RCSCs, and multidrug resistance to cisplatin, carboplatin, vincristine, doxorubicin, and etoposide was greatly improved in these cells. Our data suggest that miR-3163 has a significant impact on ABCG2 expression and can influence proliferation, apoptosis, and drug resistance in RCSCs. This work may provide new therapeutic targets for retinoblastoma. PMID:27247490

  4. Silencing stromal interaction molecule 1 by RNA interference inhibits the proliferation and migration of endothelial progenitor cells

    International Nuclear Information System (INIS)

    Research highlights: → STIM1 and TRPC1 are expressed in EPCs. → Knockdown of STIM1 inhibits the proliferation, migration and SOCE of EPCs. → TRPC1-SOC cooperates with STIM1 to mediate the SOCE of EPCs. -- Abstract: Knockdown of stromal interaction molecule 1 (STIM1) significantly suppresses neointima hyperplasia after vascular injury. Endothelial progenitor cells (EPCs) are the major source of cells that respond to endothelium repair and contribute to re-endothelialization by reducing neointima formation after vascular injury. We hypothesized that the effect of STIM1 on neointima hyperplasia inhibition is mediated through its effect on the biological properties of EPCs. In this study, we investigated the effects of STIM1 on the proliferation and migration of EPCs and examined the effect of STIM1 knockdown using cultured rat bone marrow-derived EPCs. STIM1 was expressed in EPCs, and knockdown of STIM1 by adenoviral delivery of small interfering RNA (siRNA) significantly suppressed the proliferation and migration of EPCs. Furthermore, STIM1 knockdown decreased store-operated channel entry 48 h after transfection. Replenishment with recombinant human STIM1 reversed the effects of STIM1 knockdown. Our data suggest that the store-operated transient receptor potential canonical 1 channel is involved in regulating the biological properties of EPCs through STIM1. STIM1 is a potent regulator of cell proliferation and migration in rat EPCs and may play an important role in the biological properties of EPCs.

  5. Investigation of a miRNA-Induced Gene Silencing Technique in Petunia Reveals Alterations in miR173 Precursor Processing and the Accumulation of Secondary siRNAs from Endogenous Genes.

    Directory of Open Access Journals (Sweden)

    Yao Han

    Full Text Available MIGS (miRNA-induced gene silencing is a straightforward and efficient gene silencing technique in Arabidopsis. It works by exploiting miR173 to trigger the production of phasiRNAs (phased small interfering RNAs. MIGS can be used in plant species other than Arabidopsis by co-expression of miR173 and target gene fragments fused to an upstream miR173 target site. However, the efficiency and technical mechanisms have not been thoroughly investigated in other plants. In this work, two vectors, pMIGS-chs and pMIGS-pds, were constructed and transformed into petunia plants. The transgenic plants showed CHS (chalcone synthase and PDS (phytoene desaturase gene-silencing phenotypes respectively, indicating that MIGS functions in petunia. MIGS-chs plants were used to investigate the mechanisms of this technique in petunia. Results of 5'- RACE showed that the miR173 target site was cleaved at the expected position and that endogenous CHS genes were cut at multiple positions. Small RNA deep sequencing analysis showed that the processing of Arabidopsis miR173 precursors in MIGS-chs transgenic petunia plants did not occur in exactly the same way as in Arabidopsis, suggesting differences in the machinery of miRNA processing between plant species. Small RNAs in-phase with the miR173 cleavage register were produced immediately downstream from the cleavage site and out-of-phase small RNAs were accumulated at relatively high levels from processing cycle 5 onwards. Secondary siRNAs were generated from multiple sites of endogenous CHS-A and CHS-J genes, indicating that miR173 cleavage induced siRNAs have the same ability to initiate siRNA transitivity as the siRNAs functioning in co-suppression and hpRNA silencing. On account of the simplicity of vector construction and the transitive amplification of signals from endogenous transcripts, MIGS is a good alternative gene silencing method for plants, especially for silencing a cluster of homologous genes with redundant

  6. Phylogenetic Studies of the Three RNA Silencing Suppressor Genes of South American CTV Isolates Reveal the Circulation of a Novel Genetic Lineage

    Directory of Open Access Journals (Sweden)

    María José Benítez-Galeano

    2015-07-01

    Full Text Available Citrus Tristeza Virus (CTV is the most economically important virus of citrus worldwide. Genetic diversity and population structure of CTV isolates from all citrus growing areas from Uruguay were analyzed by RT-PCR and cloning of the three RNA silencing suppressor genes (p25, p20 and p23. Bayesian phylogenetic analysis revealed the circulation of three known genotypes (VT, T3, T36 in the country, and the presence of a new genetic lineage composed by isolates from around the world, mainly from South America. Nucleotide and amino acid identity values for this new genetic lineage were both higher than 97% for the three analyzed regions. Due to incongruent phylogenetic relationships, recombination analysis was performed using Genetic Algorithms for Recombination Detection (GARD and SimPlot software. Recombination events between previously described CTV isolates were detected. High intra-sample variation was found, confirming the co-existence of different genotypes into the same plant. This is the first report describing: (1 the genetic diversity of Uruguayan CTV isolates circulating in the country and (2 the circulation of a novel CTV genetic lineage, highly present in the South American region. This information may provide assistance to develop an effective cross-protection program.

  7. Phylogenetic Studies of the Three RNA Silencing Suppressor Genes of South American CTV Isolates Reveal the Circulation of a Novel Genetic Lineage.

    Science.gov (United States)

    Benítez-Galeano, María José; Rubio, Leticia; Bertalmío, Ana; Maeso, Diego; Rivas, Fernando; Colina, Rodney

    2015-07-01

    Citrus Tristeza Virus (CTV) is the most economically important virus of citrus worldwide. Genetic diversity and population structure of CTV isolates from all citrus growing areas from Uruguay were analyzed by RT-PCR and cloning of the three RNA silencing suppressor genes (p25, p20 and p23). Bayesian phylogenetic analysis revealed the circulation of three known genotypes (VT, T3, T36) in the country, and the presence of a new genetic lineage composed by isolates from around the world, mainly from South America. Nucleotide and amino acid identity values for this new genetic lineage were both higher than 97% for the three analyzed regions. Due to incongruent phylogenetic relationships, recombination analysis was performed using Genetic Algorithms for Recombination Detection (GARD) and SimPlot software. Recombination events between previously described CTV isolates were detected. High intra-sample variation was found, confirming the co-existence of different genotypes into the same plant. This is the first report describing: (1) the genetic diversity of Uruguayan CTV isolates circulating in the country and (2) the circulation of a novel CTV genetic lineage, highly present in the South American region. This information may provide assistance to develop an effective cross-protection program. PMID:26205407

  8. Nanoparticles containing siRNA to silence CD4 and CCR5 reduce expression of these receptors and inhibit HIV-1 infection in human female reproductive tract tissue explants

    Directory of Open Access Journals (Sweden)

    Susan K. Eszterhas

    2011-09-01

    Full Text Available Human Immunodeficiency Virus-type 1 (HIV- 1 binds to CD4 and CCR5 receptors on target cells in the human female reproductive tract. We sought to determine whether reducing levels of messenger RNA (mRNA transcripts that encode these receptors in female reproductive tract cells could protect mucosal tissue explants from HIV- 1 infection. Explants prepared from the endometrium, endocervix, and ectocervix of hysterectomy tissues from HIV-1 sero-negative women were exposed to nanoparticles containing CD4- and CCR5-specific short-interfering RNA (siRNA sequences. Explants were then exposed two days later to HIV-1, and HIV-1 reverse transcripts were measured five days post-infection. Explants treated with nanoparticles containing CD4- and CCR5-specific siRNA showed reduced levels of CD4 and CCR5 transcripts, and significantly lower levels of HIV-1 reverse transcripts compared to those treated with an irrelevant siRNA. In female reproductive tract explants and in peripheral blood cell cultures, siRNA transfection induced the secretion of IFN-alpha (IFN-α, a potent antiviral cytokine. In female mice, murine-specific Cd4-siRNA nanoparticles instilled within the uterus significantly reduced murine Cd4 transcripts by day 3. Our findings demonstrate that siRNA nanoparticles reduce expression of HIV-1 infectivity receptors in human female reproductive tract tissues and also inhibit HIV-1 infection. Murine studies demonstrate that nanoparticles can penetrate the reproductive tract tissues in vivo and silence gene expression. The induction of IFN-α after siRNA transfection can potentially contribute to the antiviral effect. These findings support the therapeutic development of nanoparticles to deliver siRNA molecules to silence host cell receptors in the female reproductive tract as a novel microbicide to inhibit mucosal HIV-1 transmission.

  9. Suppression of iASPP-dependent aggressiveness in cervical cancer through reversal of methylation silencing of microRNA-124

    Science.gov (United States)

    Dong, Peixin; Xiong, Ying; Watari, Hidemichi; Hanley, Sharon JB; Konno, Yosuke; Ihira, Kei; Suzuki, Fumihiko; Yamada, Takahiro; Kudo, Masataka; Yue, Junming; Sakuragi, Noriaki

    2016-01-01

    Derepression of wild-type p53 by suppressing its negative inhibitor iASPP (Inhibitor of apoptosis-stimulating protein of p53) represents a potential therapeutic option for cervical cancer (CC). Here, we reported a novel functional significance of iASPP upregulation in cervical tumorigenesis: iASPP acts as a key promoter of CC cell proliferation, epithelial-mesenchymal transition, invasion and cancer stemness, by interacting with p53 to suppress p53-mediated transcription of target genes and reducing p53-responsive microRNA-34a levels. Moreover, we demonstrate that miR-124, directly targeting iASPP, reduces expression of iASPP and attenuates CC cell growth and invasiveness. Low miR-124 expression is inversely correlated with increased expression of iASPP mRNA in CC tissues. In a cohort of 40 patients with CC, the low miR-124 expression was correlated with poor 5-year overall survival (P = 0.0002) and shorter disease-free survival 5-year (P = 0006). Treatment with the DNA methyltransferase inhibitor Zebularine increases miR-124 expression and retards CC cell growth and invasion with minimal toxicity to normal cells. Even at a non-toxic concentration, Zebularine was effective in suppressing CC cell invasion and migration. Altogether, the restoration of miR-124 reduces iASPP expression and leads to p53-dependent tumor suppression, suggesting a therapeutic strategy to treat iASPP-associated CC. PMID:27765948

  10. Specific degradation of 3[prime prime or minute] regions of GUS mRNA in posttranscriptionally silenced tobacco lines may be related to 5[prime prime or minute]-3[prime prime or minute] spreading of silencing

    DEFF Research Database (Denmark)

    Braunstein, T.H.; Moury, B.; Johannessen, M.M.;

    2002-01-01

    Target regions for posttranscriptional silencing of transgenes often reside in the 3' region of the coding sequence, although there are exceptions. To resolve if the target region is determined by the gene undergoing silencing rather than by the structure of the transgene loci or the plant genetic...... background, we have performed detailed analyses of target regions in three spontaneously beta-glucuronidase (GUS) silencing tobacco lines of different origin. From quantitative cosuppression experiments, we show that the main target region in all three tobacco lines is found within the 3' half of the GUS...... coding region but upstream of the last 200 nt. The quantities of small (21-25 nt) RNAs homologous to 5' or 3' regions of the GUS coding sequence were found to correlate approximately with the target strength of the corresponding regions. These results suggest that transgene locus structure and plant...

  11. Silencing of Entamoeba histolytica Glucosamine 6-Phosphate Isomerase by RNA Interference Inhibits the Formation of Cyst-Like Structures

    Directory of Open Access Journals (Sweden)

    Hugo Aguilar-Díaz

    2013-01-01

    Full Text Available Encystment is an essential process in the biological cycle of the human parasite Entamoeba histolytica. In the present study, we evaluated the participation of E. histolytica Gln6Pi in the formation of amoeba cyst-like structures by RNA interference assay. Amoeba trophozoites transfected with two Gln6Pi siRNAs reduced the expression of the enzyme in 85%, which was confirmed by western blot using an anti-Gln6Pi antibody. The E. histolytica Gln6Pi knockdown with the mix of both siRNAs resulted in the loss of its capacity to form cyst-like structures (CLSs and develop a chitin wall under hydrogen peroxide treatment, as evidenced by absence of both resistance to detergent treatment and calcofluor staining. Thus, only 5% of treated trophozoites were converted to CLS, from which only 15% were calcofluor stained. These results represent an advance in the understanding of chitin biosynthesis in E. histolytica and provide insight into the encystment process in this parasite, which could allow for the developing of new control strategies for this parasite.

  12. 小干扰RNA对人淀粉样前体蛋白基因的沉默作用%Silencing effect of amyloid precursor protein triggered by small interfering RNA

    Institute of Scientific and Technical Information of China (English)

    邱昕; 潘纪安; 陈宇; 张苏明; 郭德银

    2005-01-01

    [Objective] Much needs to be learned about human amyloid precursor protein (APP) gene functions because APP may be etiologically involved in Alzheimer disease(AD). However, physiological function of the APP remains to be elucidated. As a novel technique, RNA interference (RNAi) is quite appropriate to analyze APP gene function. [Methods] In order to observe silencing effect of small interference RNA (siRNA) directed against APP gene, a designed oligonucleotide was transcribed endogenously into small hairpin RNA (shRNA) and two enhanced green fluorescence protein (EGFP) carried two APP isoforms recombinant plasmids were constructed. It will demonstrate the silencing effect by cotransfection of siRNA expression plasmid and GFP-APP recombinant plasmids into COS-7 cells because EGFP will act as a reporter gene. [Results] The siRNA directed against APP could silence APP gene effectively. [Conclusion] Therefore, siRNA directed against APP will play a crucial role in the functional exploration of APP gene.%目的观察小干扰RNA(siRNA)对人淀粉样前体蛋白(APP)基因的沉默作用.方法该研究将设计好的一对寡核甘酸在细胞内源性地转录成小发夹RNA(shRNA),同时构建了携带两个APP亚型的增强型绿色荧光蛋白(EGFP)的GFP-APP重组质粒.利用EGFP作为报告基因,在COS-7细胞内通过共转染siRNA表达质粒和GFP-APP的重组质粒来观察沉默效应.结果针对APP的siRNA可有效地下调APP基因.结论针对APP的siRNA将不仅在研究APP基因功能和深入研究阿尔茨海默病(AD)的病理分子机制上,而且在运用siRNA这种新型的反义核酸药物治疗AD方面扮演重要角色.

  13. MicroRNA-155 silencing enhances inflammatory response and lipid uptake in oxidized low-density lipoprotein-stimulated human THP-1 macrophages.

    Science.gov (United States)

    Huang, Ri-sheng; Hu, Guan-qiong; Lin, Bin; Lin, Zhi-yi; Sun, Cheng-chao

    2010-12-01

    It has been proposed that the inflammatory response of monocytes/macrophages induced by oxidized low-density lipoprotein (oxLDL) is a key event in the pathogenesis of atherosclerosis. MicroRNA-155 (miR-155) is an important regulator of the immune system and has been shown to be involved in acute inflammatory response. However, the function of miR-155 in oxLDL-stimulated inflammation and atherosclerosis remains unclear. Here, we show that the exposure of human THP-1 macrophages to oxLDL led to a marked up-regulation of miR-155 in a dose-dependent manner. Silencing of endogenous miR-155 in THP-1 cells using locked nucleic acid-modified antisense oligonucleotides significantly enhanced oxLDL-induced lipid uptake, up-regulated the expression of scavenger receptors (lectinlike oxidized LDL receptor-1, cluster of differentiation 36 [CD36], and CD68), and promoted the release of several cytokines including interleukin (IL)-6, -8, and tumor necrosis factor α (TNF-α). Luciferase reporter assay showed that targeting miR-155 promoted nuclear factor-kappa B (NF-κB) nuclear translocation and potentiated the NF-κB-driven transcription activity. Moreover, miR-155 knockdown resulted in a marked increase in the protein amount of myeloid differentiation primary response gene 88 (MyD88), an important adapter protein used by Toll-like receptors to activate the NF-κB pathway. Our data demonstrate that miR-155 serves as a negative feedback regulator in oxLDL-stimulated THP-1 inflammatory responses and lipid uptake and thus might have potential therapeutic implications in atherosclerosis. PMID:21030878

  14. Temporal Gene Expression Analysis and RNA Silencing of Single and Multiple Members of Gene Family in the Lone Star Tick Amblyomma americanum

    Science.gov (United States)

    Karim, Shahid

    2016-01-01

    Saliva is an integral factor in the feeding success of veterinary and medically important ticks. Therefore, the characterization of the proteins present in tick saliva is an important area of tick research. Here, we confirmed previously generated sialotranscriptome data using quantitative real-time PCR. The information obtained in this in-depth study of gene expression was used to measure the effects of metalloprotease gene silencing on tick feeding. We analyzed the temporal expression of seven housekeeping genes and 44 differentially expressed salivary molecules selected from a previously published Amblyomma americanum sialotranscriptome. Separate reference genes were selected for the salivary glands and midgut from among the seven housekeeping genes, to normalize the transcriptional expression of differentially expressed genes. The salivary gland reference gene, ubiquitin, was used to normalize the expression of 44 salivary genes. Unsurprisingly, each gene family was expressed throughout the blood meal, but the expression of specific genes differed at each time point. To further clarify the complex nature of the many proteins found in the saliva, we disrupted the translation of several members of the metalloprotease family. Intriguingly, the nucleotide sequence similarity of the reprolysin metalloprotease gene family is so homologous that a single synthesized dsRNA sequence knocked down multiple members of the family. The use of multigene knockdown yielded a more significant picture of the role of metalloproteases in tick feeding success, and changes were observed in the female engorgement weight and larval hatching success. Interestingly, the depletion of metalloprotease transcripts also reduced the total number of bacteria present in the salivary glands. These data provide insight into the expression and functions of tick salivary proteins expressed while feeding on its host. PMID:26872360

  15. Inorganic phosphate blocks binding of pre-miRNA to Dicer-2 via its PAZ domain

    OpenAIRE

    Fukunaga, Ryuya; Colpan, Cansu; Han, Bo W.; Zamore, Phillip D.

    2014-01-01

    In Drosophila, Dicer-1 produces microRNAs (miRNAs) from pre-miRNAs, whereas Dicer-2 generates small interfering RNAs from long double-stranded RNA (dsRNA), a process that requires ATP hydrolysis. We previously showed that inorganic phosphate inhibits Dicer-2 cleavage of pre-miRNAs, but not long dsRNAs. Here, we report that phosphate-dependent substrate discrimination by Dicer-2 reflects dsRNA substrate length. Efficient processing by Dicer-2 of short dsRNA requires a 5′ terminal phosphate and...

  16. Identification of Pns6, a putative movement protein of RRSV, as a silencing suppressor

    Directory of Open Access Journals (Sweden)

    Lin Qiying

    2010-11-01

    Full Text Available Abstract RNA silencing is a potent antiviral response in plants. As a counterdefense, most plant and some animal viruses encode RNA silencing suppressors. In this study, we showed that Pns6, a putative movement protein of Rice ragged stunt virus (RRSV, exhibited silencing suppressor activity in coinfiltration assays with the reporter green fluorescent protein (GFP in transgenic Nicotiana benthamiana line 16c. Pns6 of RRSV suppressed local silencing induced by sense RNA but had no effect on that induced by dsRNA. Deletion of a region involved in RNA binding abolished the silencing suppressor activity of Pns6. Further, expression of Pns6 enhanced Potato virus × pathogenicity in N. benthamiana. Collectively, these results suggested that RRSV Pns6 functions as a virus suppressor of RNA silencing that targets an upstream step of the dsRNA formation in the RNA silencing pathway. This is the first silencing suppressor to be identified from the genus Oryzavirus.

  17. Antisense gene silencing

    DEFF Research Database (Denmark)

    Nielsen, Troels T; Nielsen, Jørgen E

    2013-01-01

    Since the first reports that double-stranded RNAs can efficiently silence gene expression in C. elegans, the technology of RNA interference (RNAi) has been intensively exploited as an experimental tool to study gene function. With the subsequent discovery that RNAi could also be applied...... to mammalian cells, the technology of RNAi expanded from being a valuable experimental tool to being an applicable method for gene-specific therapeutic regulation, and much effort has been put into further refinement of the technique. This review will focus on how RNAi has developed over the years and how...

  18. Ligand-responsive RNA switches: viral translation regulators, therapeutic targets, and tunable building blocks for nanotechnology

    OpenAIRE

    Boerneke, Mark A.

    2016-01-01

    Ligand-responsive RNA mechanical switches represent a new class of simple and small switching modules which regulate viral translation initiation by adopting well-defined ligand-free and bound conformational states without undergoing large secondary structure rearrangements, distinguishing them from metabolite-sensing riboswitches. Initially discovered in the internal ribosome entry site (IRES) of hepatitis C virus (HCV), RNA switch motifs have now been discovered in the genomes of diverse o...

  19. The inhibition of cell proliferation using silencing of N-cadherin gene by siRNA process in human melanoma cell lines.

    Science.gov (United States)

    Ciołczyk-Wierzbicka, D; Gil, D; Laidler, P

    2012-01-01

    Malignant melanoma is a disease with high mortality rate caused by rapid metastasis. Cell motility is physically and biochemically restricted by cadherin-mediated cell interactions and signalling pathways, and alterations in cadherin expression strongly correlate with E to N-cadherin switch as well as the metastasis and progression of tumours. Contrary to E-cadherin, N-cadherin plays an important role in stimulating processes of cell division, migration, differentiation and death. In this study we investigated the role of N-cadherin in proliferation and AKT, ERK, beta-catenin signalling pathway in human melanoma cells: WM793(VGP), WM115(VGP) from the primary tumor site, as well as Lu1205(lung) and WM266-4(skin) from metastatic sites. N-cadherin, pAKT(S473), β-catenin, pERK1/2(T202/Y204), cyclin D1, cyclin D3, cyclin-dependent kinases CDK4, CDK6, and p15, p16, p21, p27 inhibitors expression was determined by western blot analysis. The study on proliferation of cells was performed with the use of BrdU incorporation and crystal violet staining assays. Knock-out of N-cadherin gene expression by siRNA process reduced the expression of: pAKT(S473), pERK1/2(T202/Y204), betacatenin, cyclin D1, cyclin D3, cyclin-dependent kinases CDK4, CDK6 while increasing expression of cell cycle inhibitors p21 and p27, and significantly decreased cell proliferation (50-70%). The collected data indicate that N-cadherin mediates the effect of cell cycle in G1 phase by AKT, β-catenin, and ERK signalling pathway. These results suggest that increased expression of N-cadherin significantly contributes to the increased invasive potential of melanoma cells. Silencing of N-cadherin arrests cell growth at G1 phase and inhibits the entry into S-phase which is of great importance as to its possible future use in cancer treatment. PMID:22300088

  20. Silencing of microRNA-21 confers radio-sensitivity through inhibition of the PI3K/AKT pathway and enhancing autophagy in malignant glioma cell lines.

    Directory of Open Access Journals (Sweden)

    Ho-Shin Gwak

    Full Text Available Radiation is a core part of therapy for malignant glioma and is often provided following debulking surgery. However, resistance to radiation occurs in most patients, and the underlying molecular mechanisms of radio-resistance are not fully understood. Here, we demonstrated that microRNA 21 (miR-21, a well-known onco-microRNA in malignant glioma, is one of the major players in radio-resistance. Radio-resistance in different malignant glioma cell lines measured by cytotoxic cell survival assay was closely associated with miR-21 expression level. Blocking miR-21 with anti-miR-21 resulted in radio-sensitization of U373 and U87 cells, whereas overexpression of miR-21 lead to a decrease in radio-sensitivity of LN18 and LN428 cells. Anti-miR-21 sustained γ-H2AX DNA foci formation, which is an indicator of double-strand DNA damage, up to 24 hours and suppressed phospho-Akt (ser473 expression after exposure to γ-irradiation. In a cell cycle analysis, a significant increase in the G₂/M phase transition by anti-miR-21 was observed at 48 hours after irradiation. Interestingly, our results showed that anti-miR-21 increased factors associated with autophagosome formation and autophagy activity, which was measured by acid vesicular organelles, LC3 protein expression, and the percentage of GFP-LC3 positive cells. Furthermore, augmented autophagy by anti-miR-21 resulted in an increase in the apoptotic population after irradiation. Our results show that miR-21 is a pivotal molecule for circumventing radiation-induced cell death in malignant glioma cells through the regulation of autophagy and provide a novel phenomenon for the acquisition of radio-resistance.

  1. Silencing of microRNA-155 in mice during acute inflammatory response leads to derepression of c/ebp Beta and down-regulation of G-CSF

    DEFF Research Database (Denmark)

    Worm, Jesper; Stenvang, Jan; Petri, Andreas;

    2009-01-01

    -stimulating factor (G-CSF), a central regulator of granulopoiesis during inflammatory responses. Consistent with these data, we show that silencing of miR-155 in LPS-treated mice by systemically administered LNA-antimiR results in derepression of the c/ebp Beta isoforms and down-regulation of G-CSF expression...

  2. Transcriptional Silencing of Retroviral Vectors

    DEFF Research Database (Denmark)

    Lund, Anders Henrik; Duch, M.; Pedersen, F.S.

    1996-01-01

    . Extinction of long-term vector expression has been observed after implantation of transduced hematopoietic cells as well as fibroblasts, myoblasts and hepatocytes. Here we review the influence of vector structure, integration site and cell type on transcriptional silencing. While down-regulation of proviral...... transcription is known from a number of cellular and animal models, major insight has been gained from studies in the germ line and embryonal cells of the mouse. Key elements for the transfer and expression of retroviral vectors, such as the viral transcriptional enhancer and the binding site for the t......RNA primer for reverse transcription may have a major influence on transcriptional silencing. Alterations of these elements of the vector backbone as well as the use of internal promoter elements from housekeeping genes may contribute to reduce transcriptional silencing. The use of cell culture and animal...

  3. Rickettsia prowazekii Transports UMP and GMP, but Not CMP, as Building Blocks for RNA Synthesis

    OpenAIRE

    Winkler, Herbert H.; Daugherty, Robin; Hu, Fuquan

    1999-01-01

    Rickettsia prowazekii, the etiological agent of epidemic typhus, is an obligate intracellular bacterium and is apparently unable to synthesize ribonucleotides de novo. Here, we show that as an alternative, isolated, purified R. prowazekii organisms transported exogenous uridyl- and guanylribonucleotides and incorporated these labeled precursors into their RNA in a rifampin-sensitive manner. Transport systems for nucleotides, which we have shown previously and show here are present in ricketts...

  4. Rickettsia prowazekii transports UMP and GMP, but not CMP, as building blocks for RNA synthesis.

    Science.gov (United States)

    Winkler, H H; Daugherty, R; Hu, F

    1999-05-01

    Rickettsia prowazekii, the etiological agent of epidemic typhus, is an obligate intracellular bacterium and is apparently unable to synthesize ribonucleotides de novo. Here, we show that as an alternative, isolated, purified R. prowazekii organisms transported exogenous uridyl- and guanylribonucleotides and incorporated these labeled precursors into their RNA in a rifampin-sensitive manner. Transport systems for nucleotides, which we have shown previously and show here are present in rickettsiae, have never been reported in free-living bacteria, and the usual nucleobase and nucleoside transport systems are absent in rickettsiae. There was a clear preference for the monophosphate form of ribonucleotides as the transported substrate. In contrast, rickettsiae did not transport cytidylribonucleotides. The source of rickettsial CTP appears to be the transport of UMP followed by its phosphorylation and the amination of intrarickettsial UTP to CTP by CTP synthetase. A complete schema of nucleotide metabolism in rickettsiae is presented that is based on a combination of biochemical, physiological, and genetic information. PMID:10322027

  5. Polycomb complexes and silencing mechanisms

    DEFF Research Database (Denmark)

    Lund, Anders H; van Lohuizen, Maarten

    2004-01-01

    Advances in the past couple of years have brought important new knowledge on the mechanisms by which Polycomb-group proteins regulate gene expression and on the consequences of their actions. The discovery of histone methylation imprints specific for Polycomb and Trithorax complexes has provided...... mechanistic insight on how this ancient epigenetic memory system acts to repress and indicates that it may share mechanistic aspects with other silencing and genome-protective processes, such as RNA interference....

  6. Silencing the city?

    OpenAIRE

    Jean-Paul Thibaud; Pascal Amphoux

    2013-01-01

    The notion of silence must be handled very carefully. In addition, its use reveals the way we deal with the urban environment as well as social life. What does the notion of silence convey about the current state of the urban sonic environment? How can we clarify the various meanings and the stakes involved in silence? Three themes are developed in order to answer these questions: silence as a research topic presents three complementary perspectives (acoustic, sociocultural, technological); s...

  7. Dramaturgy and Silence.

    Science.gov (United States)

    Proehl, Geoffrey S.

    2003-01-01

    Notes that in rehearsals and performances, a jumble of silences are encountered. Discusses silence in the following situations: as frustration; as imposition; as invisibility; as power; as pleasure; as safety; as humility; as necessity; and as potential. Contends that when dramaturgs enter into conversation and break silence, they must carefully…

  8. Micro RNA-induced gene silencing and neurodegenerative diseases%微小RNA介导的基因沉默与神经退行性疾病

    Institute of Scientific and Technical Information of China (English)

    张丽姝; 姜树林

    2011-01-01

    @@ 微小RNA(microRNA,miRNA) 是一类内源性的22个核苷酸左右的非蛋白编码单链短序列RNA,介导同源序列依赖的基因沉默.目前,miRNA数据库miRBase(16.0版)已收录了人类miRNA 1048个.miRNA及其靶基因构建了细胞内全方位多层次的基因表达调控网络系统,参与众多生命活动的调节并与人类疾病密切相关.miRNA的功能失常在多种神经退行性疾病的发生发展中均具有重要作用.

  9. Silencing expression of the catalytic subunit of DNA-dependent protein kinase by small interfering RNA sensitizes human cells for radiation-induced chromosome damage, cell killing, and mutation

    Science.gov (United States)

    Peng, Yuanlin; Zhang, Qinming; Nagasawa, Hatsumi; Okayasu, Ryuichi; Liber, Howard L.; Bedford, Joel S.

    2002-01-01

    Targeted gene silencing in mammalian cells by RNA interference (RNAi) using small interfering RNAs (siRNAs) was recently described by Elbashir et al. (S. M. Elbashir et al., Nature (Lond.), 411: 494-498, 2001). We have used this methodology in several human cell strains to reduce expression of the Prkdc (DNA-PKcs) gene coding for the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) that is involved in the nonhomologous end joining of DNA double-strand breaks. We have also demonstrated a radiosensitization for several phenotypic endpoints of radiation damage. In low-passage normal human fibroblasts, siRNA knock-down of DNA-PKcs resulted in a reduced capacity for restitution of radiation-induced interphase chromosome breaks as measured by premature chromosome condensation, an increased yield of acentric chromosome fragments at the first postirradiation mitosis, and an increased radiosensitivity for cell killing. For three strains of related human lymphoblasts, DNA-PKcs-targeted siRNA transfection resulted in little or no increase in radiosensitivity with respect to cell killing, a 1.5-fold decrease in induced mutant yield in TK6- and p53-null NH32 cells, but about a 2-fold increase in induced mutant yield in p53-mutant WTK1 cells at both the hypoxanthine quanine phosphoribosyl transferase (hprt) and the thymidine kinase loci.

  10. The distribution of a germline methylation marker suggests a regional mechanism of LINE-1 silencing by the piRNA-PIWI system

    Directory of Open Access Journals (Sweden)

    Sigurdsson Martin I

    2012-04-01

    Full Text Available Abstract Background A defense system against transposon activity in the human germline based on PIWI proteins and piRNA has recently been discovered. It represses the activity of LINE-1 elements via DNA methylation by a largely unknown mechanism. Based on the dispersed distribution of clusters of piRNA genes in a strand-specific manner on all human chromosomes, we hypothesized that this system might work preferentially on local and proximal sequences. We tested this hypothesis with a methylation-associated SNP (mSNP marker which is based on the density of C-T transitions in CpG dinucleotides as a surrogate marker for germline methylation. Results We found significantly higher density of mSNPs flanking piRNA clusters in the human genome for flank sizes of 1-16 Mb. A dose-response relationship between number of piRNA genes and mSNP density was found for up to 16 Mb of flanking sequences. The chromosomal density of hypermethylated LINE-1 elements had a significant positive correlation with the chromosomal density of piRNA genes (r = 0.41, P = 0.05. Genome windows of 1-16 Mb containing piRNA clusters had significantly more hypermethylated LINE-1 elements than windows not containing piRNA clusters. Finally, the minimum distance to the next piRNA cluster was significantly shorter for hypermethylated LINE-1 compared to normally methylated elements (14.4 Mb vs 16.1 Mb. Conclusions Our observations support our hypothesis that the piRNA-PIWI system preferentially methylates sequences in close proximity to the piRNA clusters and perhaps physically adjacent sequences on other chromosomes. Furthermore they suggest that this proximity effect extends up to 16 Mb. This could be due to an unknown localization signal, transcription of piRNA genes near the nuclear membrane or the presence of an unknown RNA molecule that spreads across the chromosome and targets the methylation directed by the piRNA-PIWI complex. Our data suggest a region specific molecular

  11. Silver nanoparticles-quercetin conjugation to siRNA against drug-resistant Bacillus subtilis for effective gene silencing: in vitro and in vivo.

    Science.gov (United States)

    Sun, Dongdong; Zhang, Weiwei; Li, Nuan; Zhao, Zhiwei; Mou, Zhipeng; Yang, Endong; Wang, Weiyun

    2016-06-01

    Quercetin (Qe) exhibited extremely low water solubility, and thus, it was modified using silver nanoparticles (AgNPs). We fabricated AgNPs combined with Qe (AgNPs-Qe). The modification suggested that the synergistic properties of Qe enhanced the antibacterial activity of AgNPs. However, AgNPs-Qe exerted no effect on many kinds of drug-resistant bacteria, including Pseudomonas aeruginosa and Bacillus subtilis. RNA interference has considerable therapeutic potential because of its high specificity and potential capability to evade drug resistance. Therefore, we stabilized AgNPs-Qe with a layer of molecules (siRNA). The newly fabricated nanoparticles exerted improved effect on many kinds of bacteria, including the most prominent drug-resistant species B. subtilis. Agarose gel electrophoresis showed that the highest critical nitrogen-to-phosphorus (N/P) ratio occurred at a vector/siRNA with a w/w ratio of 7:1. Characterization experiment indicated that the diameter of siRNA/AgNPs-Qe was approximately 40 nm (40 ± 10 nm). Moreover, AgNPs-Qe were stabilized with a layer of siRNA that was approximately 10nm thick. Results of the in vitro study suggested that siRNA/AgNPs-Qe could destroy the cell wall and inhibit bacterial propagation. Meanwhile, the in vivo experiment on the animal bacteremia model, as well as the optical imaging of nude mice and their isolated organs, demonstrated that bacteria accumulated in the blood, heart, liver, spleen, lungs, and kidneys after the intravenous injection of B. subtilis. The bacteria in the blood and organs, as well as the inflamed cells in the tissues, gradually decreased after the mice received intravenous tail injection of siRNA/AgNPs-Qe for treatment. Both the in vitro and the in vivo studies exhibit that siRNA/AgNPs-Qe can be a potential nanoscale drug delivery system for B. subtilis targeting bacterimia. PMID:27040247

  12. Influence of Bxpel1 Gene Silencing by dsRNA Interference on the Development and Pathogenicity of the Pine Wood Nematode, Bursaphelenchus xylophilus.

    Science.gov (United States)

    Qiu, Xiu-Wen; Wu, Xiao-Qin; Huang, Lin; Ye, Jian-Ren

    2016-01-01

    As the causal agent of pine wilt disease (PWD), the pine wood nematode (PWN), Bursaphelenchus xylophilus, causes huge economic losses by devastating pine forests worldwide. The pectate lyase gene is essential for successful invasion of their host plants by plant-parasitic nematodes. To demonstrate the role of pectate lyase gene in the PWD process, RNA interference (RNAi) is used to analyze the function of the pectate lyase 1 gene in B. xylophilus (Bxpel1). The efficiency of RNAi was detected by real-time PCR. The result demonstrated that the quantity of B. xylophilus propagated with control solution treatment was 62 times greater than that soaking in double-stranded RNA (dsRNA) after B. xylophilus inoculation in Botrytis cinerea for the first generation (F1). The number of B. xylophilus soaking in control solution was doubled compared to that soaking in Bxpel1 dsRNA four days after inoculation in Pinus thunbergii. The quantity of B. xylophilus was reduced significantly (p control solution treatment. Bxpel1 dsRNAi reduced the migration speed and reproduction of B. xylophilus in pine trees. The pathogenicity to P. thunbergii seedling of B. xylophilus was weaker after soaking in dsRNA solution compared with that after soaking in the control solution. Our results suggest that Bxpel1 gene is a significant pathogenic factor in the PWD process and this basic information may facilitate a better understanding of the molecular mechanism of PWD. PMID:26797602

  13. Silencing the city?

    Directory of Open Access Journals (Sweden)

    Jean-Paul Thibaud

    2013-12-01

    Full Text Available The notion of silence must be handled very carefully. In addition, its use reveals the way we deal with the urban environment as well as social life. What does the notion of silence convey about the current state of the urban sonic environment? How can we clarify the various meanings and the stakes involved in silence? Three themes are developed in order to answer these questions: silence as a research topic presents three complementary perspectives (acoustic, sociocultural, technological; silence as a polysemous notion emphasises the ideas of keeping quiet, tranquillity and pause; silence as a design issue relies on basic properties and principles in order to orient the design of the urban sonic environment.

  14. Identifizierung und Charakterisierung von Silencing Suppressoren in Arabidopsis

    OpenAIRE

    Thran, Moritz

    2013-01-01

    Post-transkriptionelles gene silencing (PTGS) kann ein limitierender Faktor für die Expression von Transgenen sein. Transgene mRNAs können von der RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) erkannt werden, was die Synthese doppelsträngiger RNA (dsRNA) initiiert und die Akkumulation von small interfering RNAs (siRNAs) zur Folge hat. Diese vermitteln die sequenzspezifische endonukleolytische Spaltung einer Ziel-mRNA und verhindern somit deren Akkumulation. Für die Initiierung des Transgen Silencing ...

  15. Increased expression of long noncoding RNA TUG1 predicts a poor prognosis of gastric cancer and regulates cell proliferation by epigenetically silencing of p57

    OpenAIRE

    Zhang, E; He, X; Yin, D; Han, L.; Qiu, M; XU, T; Xia, R; Xu, L.; Yin, R; De, W

    2016-01-01

    Recent evidence highlights long noncoding RNAs (lncRNAs) as crucial regulators of cancer biology that contribute to tumorigenesis. LncRNA TUG1 was initially detected in a genomic screen for genes upregulated in response to taurine treatment in developing mouse retinal cells. Our previous study showed that TUG1 could affect cell proliferation through epigenetically regulating HOXB7 in human non-small cell lung cancer. However, the clinical significance and potential role of TUG1 in GC remains ...

  16. Silencing of the hTERT gene by shRNA inhibits colon cancer SW480 cell growth in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Ai-Qun Liu

    Full Text Available Human telomerase reverse transcriptase (hTERT is the key enzyme responsible for synthesizing and maintaining the telomeres on the ends of chromosomes, and it is essential for cell proliferation. This has made hTERT a focus of oncology research and an attractive target for anticancer drug development. In this study, we designed a small interfering RNA (siRNA targeting the catalytic subunit of hTERT and tested its effects on the growth of telomerase-positive human colon carcinoma SW480 cells in vitro, as well as on the tumorigenicity of these cells in nude mice. Transient and stable transfection of hTERT siRNA into colon cancer SW480 cells suppressed hTERT expression, reduced telomerase activity and inhibited cell growth and proliferation. Knocking down hTERT expression in SW480 tumors xenografted into nude mice significantly slowed tumor growth and promoted tumor cell apoptosis. Our results suggest that hTERT is involved in carcinogenesis of human colon carcinoma, and they highlight the therapeutic potential of a hTERT knock-down approach.

  17. Inhibition of proliferation of human non-Hodgkin's lymphoma Raji cells by small interference RNA silencing Pokemon gene%siRNA 沉默 Pokemon 基因抑制人非霍奇金淋巴瘤 Raji细胞的增殖

    Institute of Scientific and Technical Information of China (English)

    董珂; 蒲业迪; 刘琼; 代广霞; 李丽珍; 李颢; 王玲玲; 王鲁群

    2014-01-01

    Objective Small interference RNA ( siRNA) was used to silence Pokemon gene in human non-Hodgkin's lymphoma Raji cells, then to observe the change of proliferation of Raji cells and explore its possible molecular mecha-nisms.Methods Pokemon-targeted siRNA was constructed with lentivirus vector and transfected to Raji cells. RT-PCR and Western blotting were adopted to confirm the silence effect of Pokemon gene in Raji cells, which were then used to determine the mRNA and protein expressions of bcl-6 and mutant p53.Flow cytometry was used to detect the cell apoptosis of each group.Results Pokemon-siRNA constructed with lentivirus vector could efficiently silence the expression of Pokemon gene in Raji cell (P<0.05).After that, the mRNA and protein expressions of bcl-6 and mutant p53 were significantly decreased (P<0.05) and the cell apoptosis rate was markedly elevated compared with the controls.Conclusion The siRNA Pokemon gene silencing promotes human non-Hodgkin's lymphoma Raji cells apoptosis by lowering the expressions of bcl-6 and mutant p53 gene and protein and inhibiting the proliferation.Poke-mon gene is expected to become a new target for non-Hodgkin's lymphoma treatment.%目的:采用siRNA干扰技术沉默人非霍奇金淋巴瘤Raji细胞中Pokemon基因,观察Raji细胞增殖活性的变化,并探讨其可能的分子机制。方法构建靶向Pokemon基因的siRNA重组慢病毒载体并转染Raji细胞。采用实时定量PCR法和Western blotting法检测Raji细胞Pokemon基因的沉默效果,在Raji细胞中沉默Pokemon基因的表达后采用实时定量PCR法和Western blotting法检测bcl-6和突变型p53表达水平的变化;采用流式细胞术检测Pokemon基因沉默后Raji细胞凋亡的情况。结果利用Pokemon靶向siRNA重组慢病毒载体感染Raji细胞有效沉默Raji细胞Pokemon基因表达(P<0.05)。沉默Pokemon基因后,bcl-6和突变型p53基因和蛋白表达均显著降低(P<0.05)

  18. siRNA抑制人乳腺癌KLK6基因表达的实验研究%Research of silencing kallikrein 6 gene with siRNA in human breast cancer

    Institute of Scientific and Technical Information of China (English)

    季丙元; 关晶

    2011-01-01

    目的 探讨siRNA沉默KLK6基因表达对乳腺癌细胞生长的抑制作用及其机制,为乳腺癌的基因诊断和治疗提供理论依据.方法 针对KLK6 mRNA 序列设计合成siRNA,构建2个重组质粒PGCsilencerTMH1/GFP/Neo/KLK6和PGCsilencerTMH1/GFP/Neo/Non;将重组质粒导入乳腺癌MCF-7细胞株,G418筛选获得稳定转染的细胞株;实验分为脂质体对照、阴性质粒转染对照及KLK6 siRNA 重组质粒转染组,实时荧光定量PCR 法检测KLK6 mRNA 的表达的变化,用四甲基偶氮唑盐(MTT)法测量细胞生长情况.结果 测序证实表达载体构建成功,在稳定转染重组质粒PGCsilencerTMH1/GFP/Neo/KLK6的乳腺癌MCF-7细胞株中,KLK6 mRNA抑制率(76%)明显高于阴性质粒对照组(2.7%),MCF-7细胞增殖活性显著低于阴性对照组和脂质体对照组.结论 重组质粒PGCsilencerTMH1/GFP/Neo/KLK6可抑制乳腺癌细胞中KLK6基因的表达,并抑制乳腺癌细胞的生长.%Objective To investigate the inhibitory effect of silencing KLK6 gene with siRNA on the growth of breast cancer cells and its mechanism,and to provide evidence in diagnosis and treatment for breast cancer.Methods Specific siRNA for KLK6 mRNA was designed and synthesized.Two recombinant plasmids PGCsilencer TM H1/GFP/Neo/KLK6 and PGCsilencer tm H1/GFP/Neo/Non were constructed.The two plasmids were transfected into MCF-7 cells and the positive cell clones were obtained by G418 selection.The breast cancer cell line MCF-7 were divided into three groups :liposome-treated control group,negative plasmid-transfected control group and KLK6-siRNA transfected group.The expression of KLK6 was determined by real time quantitative PCR,and the proliferation was also observed by MTT assay.Results The recombinant plasmid was successfully constructed.The results of real time quantitative PCR indicated that the inhibitory rates of mRNA in PGCsilencer TM H1/GFP/Neo/KLK6 transfected group (76%) was higher than that in negative plasmid

  19. A crucial role for GW182 and the DCP1:DCP2 decapping complex in miRNA-mediated gene silencing

    OpenAIRE

    Rehwinkel, Jan; BEHM-ANSMANT, ISABELLE; Gatfield, David; Izaurralde, Elisa

    2005-01-01

    In eukaryotic cells degradation of bulk mRNA in the 5′ to 3′ direction requires the consecutive action of the decapping complex (consisting of DCP1 and DCP2) and the 5′ to 3′ exonuclease XRN1. These enzymes are found in discrete cytoplasmic foci known as P-bodies or GW-bodies (because of the accumulation of the GW182 antigen). Proteins acting in other post-transcriptional processes have also been localized to P-bodies. These include SMG5, SMG7, and UPF1, which function in nonsense-mediated mR...

  20. MicroRNA-26a is Strongly Down-regulated in Melanoma and Induces Cell Death through Repression of Silencer of Death Domains (SODD)

    OpenAIRE

    Reuland, Steven N.; Smith, Shilo; Bemis, Lynne; Goldstein, Nathaniel B.; Almeida, Adam; Katie A. Partyka; Marquez, Victor E.; Zhang, Qinghong; David A Norris; Yiqun G Shellman

    2012-01-01

    Melanoma is an aggressive cancer that metastasizes rapidly, and is refractory to conventional chemotherapies. Identifying miRNAs that are responsible for this pathogenesis is therefore a promising means of developing new therapies. We identified miR-26a through microarray and qRT-PCR experiments as an miRNA that is strongly down-regulated in melanoma cell lines as compared to primary melanocytes. Treatment of cell lines with miR-26a mimic caused significant and rapid cell death compared to a ...

  1. Long non-coding RNA TUG1 is up-regulated in hepatocellular carcinoma and promotes cell growth and apoptosis by epigenetically silencing of KLF2

    OpenAIRE

    Huang, Ming-De; Chen, Wen-ming; Qi, Fu-zhen; Sun, Ming; Xu, Tong-peng; Ma, Pei; Shu, Yong-qian

    2015-01-01

    Background Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide, and the biology of this cancer remains poorly understood. Recent evidence indicates that long non-coding RNAs (lncRNAs) are found to be dysregulated in a variety of cancers, including HCC. Taurine Up-regulated Gene 1 (TUG1), a 7.1-kb lncRNA, recruiting and binding to polycomb repressive complex 2 (PRC2), is found to be disregulated in non-small cell lung carcinoma (NSCLC) and esophageal s...

  2. siRNA沉默c-FLIP对K562/ADR耐药性的影响%Effect of siRNA-mediated silencing of c-FLIP on adriamycin-resistance of K562/ADR cells

    Institute of Scientific and Technical Information of China (English)

    宋敏; 王建宁; 孟庆齐; 包红雨; 杨杰

    2015-01-01

    目的:探讨c-FLIP siRNA干扰c-FLIP mRNA水平对阿霉素耐药细胞K562/ADR的耐药性的影响及作用机制。方法应用siRNA干扰的方法抑制K562及K562/ADR细胞c-FLIP的表达,通过荧光定量PCR的方法检测c-FLIP mRNA表达及对多药耐药基因MDR1 mRNA水平的影响。MTT法检测c-FLIP干扰与否对K562及K562/ADR细胞增殖的影响,Annexin V/7-ADD双染研究c-FLIP干扰与否对K562及K562/ADR细胞凋亡的影响。结果与阴性siRNA转染组相比较,c-FLIP siRNA转染下调K562细胞中c-FLIP的mRNA后,K562细胞增殖受到一定程度的抑制(P<0.05),但是并未显著诱导细胞凋亡,c-FLIP干扰与否K562细胞48 h增殖率分别为(69.14±1.82)%和(60.69±2.23)%,凋亡率分别为(1.7±0.3)%和(1.8±0.2)%。与阴性siRNA转染组相比较, c-FLIP siRNA转染抑制K562/ADR细胞中c-FLIP的mRNA后,K562/ADR细胞增殖显著被抑制(P<0.05),并显著诱导了细胞凋亡增加(P<0.05),c-FLIP干扰与否K562/ADR细胞48 h增殖率分别为(-6.07±0.71)%和(-37.45±3.53)%,凋亡率分别为(5.2±0.4)%和(9.2±0.4)%。并且c-FLIP siRNA下调c-FLIP mRNA水平后,K562/ADR细胞中的多药耐药基因MDR1的mRNA表达水平也被显著下调(P<0.05)。结论 c-FLIP siRNA下调K562/ADR细胞中c-FLIP的mRNA水平抑制了多药耐药基因MDR1的表达,从而抑制了K562/ADR细胞对阿霉素的耐药性。%Objective To investigate the effect of silenced c-FLIP mRNA level by small interfering RNA (siRNA) on adriamycin-resistance of K562/ADR cells. Methods c-FLIP siRNA and negative siRNA were transfect-ed into K562 and K562/ADR cell lines respectively, and mRNA expression of c-FLIP and multi-drug resistance gene 1 (MDR1) were detected by quantitative PCR. Cell proliferation rate was detected by MTT assay, and cell apoptosis rate was assayed by Annexin V/7-ADD double-staining method. Results Compared with negative siRNA transfection group, siRNA transfection significantly decreased c-FLIP mRNA

  3. The rice yellow mottle virus P1 protein exhibits dual functions to suppress and activate gene silencing

    OpenAIRE

    Lacombe, Séverine; Bangratz, Martine; Vignols, Florence; Brugidou, Christophe

    2010-01-01

    In plants RNA silencing is a host defense mechanism against viral infection, in which double-strand RNA is processed into 21-24-nt short interfering RNA (siRNA). Silencing spreads from cell to cell and systemically through a sequence-specific signal to limit the propagation of the virus. To counteract this defense mechanism, viruses encode suppressors of silencing. The P1 protein encoded by the rice yellow mottle virus (RYMV) displays suppression activity with variable efficiency, according t...

  4. Expression analysis and localization of wt1, ad4bp/sf-1 and gata4 in the testis of catfish, Clarias batrachus: Impact of wt1-esiRNA silencing.

    Science.gov (United States)

    Murugananthkumar, Raju; Senthilkumaran, Balasubramanian

    2016-08-15

    In teleosts, a comprehensive role or interaction of wt1, ad4bp/sf-1 and gata4 genes in relation to gonadal development and/or recrudescence was never attempted. Present study aimed to identify the involvement of these genes during testicular development of catfish, Clarias batrachus. Dominant expression of wt1 and gata4 was observed in developing and adult testis, while ad4bp/sf-1 showed steady expression. Localization of these genes in adult testis revealed their presence in spermatogonia, spermatocytes and interstitial/Leydig cells. Significant high expression during pre-spawning and spawning phases, and upregulated levels of these genes after hCG induction authenticated gonadotropic regulation. Transient silencing of wt1-esiRNA displayed decrease in wt1 expression, which further downregulated the expression of ad4bp/sf-1 and gata4, and certain steroidogenic enzyme genes related to androgen production. These results suggest that wt1 might target ad4bp/sf-1 and gata4 expression, and also have regulatory influence either indirectly or directly on the steroidogenic enzyme genes of catfish. PMID:27173028

  5. Expression analysis and localization of wt1, ad4bp/sf-1 and gata4 in the testis of catfish, Clarias batrachus: Impact of wt1-esiRNA silencing.

    Science.gov (United States)

    Murugananthkumar, Raju; Senthilkumaran, Balasubramanian

    2016-08-15

    In teleosts, a comprehensive role or interaction of wt1, ad4bp/sf-1 and gata4 genes in relation to gonadal development and/or recrudescence was never attempted. Present study aimed to identify the involvement of these genes during testicular development of catfish, Clarias batrachus. Dominant expression of wt1 and gata4 was observed in developing and adult testis, while ad4bp/sf-1 showed steady expression. Localization of these genes in adult testis revealed their presence in spermatogonia, spermatocytes and interstitial/Leydig cells. Significant high expression during pre-spawning and spawning phases, and upregulated levels of these genes after hCG induction authenticated gonadotropic regulation. Transient silencing of wt1-esiRNA displayed decrease in wt1 expression, which further downregulated the expression of ad4bp/sf-1 and gata4, and certain steroidogenic enzyme genes related to androgen production. These results suggest that wt1 might target ad4bp/sf-1 and gata4 expression, and also have regulatory influence either indirectly or directly on the steroidogenic enzyme genes of catfish.

  6. Methylated DNA binding domain protein 2 (MBD2 coordinately silences gene expression through activation of the microRNA hsa-mir-496 promoter in breast cancer cell line.

    Directory of Open Access Journals (Sweden)

    Sebastian Alvarado

    Full Text Available Methylated DNA binding protein 2 (MBD2 binds methylated promoters and suppresses transcription in cis through recruitment of a chromatin modification repressor complex. We show here a new mechanism of action for MBD2: suppression of gene expression indirectly through activation of microRNA hsa-mir-496. Overexpression of MBD2 in breast epithelial cell line MCF-10A results in induced expression and demethylation of hsa-mir-496 while depletion of MBD2 in a human breast cancer cell lines MCF-7 and MDA-MB231 results in suppression of hsa-mir-496. Activation of hsa-mir-496 by MBD2 is associated with silencing of several of its target genes while depletion of MBD2 leads to induction of hsa-mir-496 target genes. Depletion of hsa-mir-496 by locked nucleic acid (LNA antisense oligonucleotide leads to activation of these target genes in MBD2 overexpressing cells supporting that hsa-mir-496 is mediating in part the effects of MBD2 on gene expression. We demonstrate that MBD2 binds the promoter of hsa-mir-496 in MCF-10A, MCF-7 and MDA-MB-231 cells and that it activates an in vitro methylated hsa-mir-496 promoter driving a CG-less luciferase reporter in a transient transfection assay. The activation of hsa-mir-496 is associated with reduced methylation of the promoter. Taken together these results describe a novel cascade for gene regulation by DNA methylation whereby activation of a methylated microRNA by MBD2 that is associated with loss of methylation triggers repression of downstream targets.

  7. AQP4 expression of the cerebral ischemic tissue following its mRNA silence in rat%基因沉默对缺血脑组织水通道蛋白4表达的影响

    Institute of Scientific and Technical Information of China (English)

    鲁宏; 胡惠; 何占平; 涂蓉; 舒庆杰

    2012-01-01

    To investigate the effect of RNA interference targeting AQP4 on its expression in the cerebral ischemic tissues. Methods: Total 108 Wistar rats were divided into three groups randomly: ischemic groups (MCAO), control group (shRNA-AQP4 + MCAO) and interference groups (siRNA-AQP4 + MCAO). Each group was subdivided into six sub groups according to the different interval of time points: 0. 25 h, 0. 5 h, 1 h, 2 h, 4 h and 6 h (n = 6 for each group). The right middle cerebral artery of rats was unilaterally occluded (MCAO) in ischemic groups. The reagent including nonsense short hairpin (shRNA) liposomes and short interference (siRNA)-AQP4 liposomes were injected to the right basal ganglia of the rats of MCAO served as a control group and an interference group, respectively. The animals were sacrificed and per fused with the mixture solution consisting formalin. The right basal ganglia tissues were examined with pathology, immuno histochemistry, real time fluorescence quantitative reverse transcript polymerase chain reaction (RT-PCR) and Western blot ting. Results: There was no significant change in AQP4 expression between the control group and the ischemic group. The expression of AQP4 increased rapidly within 2 h, and then increased slowly during 4-6 h. Whereas, there was no significant change in AQP4 expression between the control group and the interference group at 0. 25 h and 6 h after MCAO. But there was significant change in AQP4 expression between the control group and the interference group during 0. 5-4 h. The AQP4 expression could be inhibited efficiently by RNA silencing technique during 0. 5-4 h, but it became unconspicuous during 4-6 h. The corresponding histological changes within 4 h were intracellular edema in the ischemic group and the control group, but this pathological change was obviously mitigated in the interference group during the same period of time. With the pro gress of the time (4-6 h), The vasogenic brain edema was mostly observed in

  8. Effects of Nogo-A Silencing on TNF-α and IL-6 Secretion and TH Downregulation in Lipopolysaccharide-Stimulated PC12 Cells

    Directory of Open Access Journals (Sweden)

    Jianbin Zhong

    2015-01-01

    Full Text Available Parkinson’s disease (PD is a common degenerative disease that lacks efficient treatment. Myelin-associated neurite outgrowth inhibitor A (Nogo-A is relevant with inhibition of nerve regeneration and may play vital role in pathogenesis of PD. The study aimed to establish the shRNA expression plasmids of Nogo-A gene and explore the regulatory effects of Nogo-A silencing on the expression of inflammation factor tumor necrosis factor-alpha (TNF-alpha and interleukin-6 (IL-6 as well as tyrosine hydroxylase (TH in lipopolysaccharide- (LPS- stimulated rat PC12 cells. The results showed that both mRNA and protein levels of Nogo-A in pGenesil-nogoA-shRNA group were downregulated. The viabilities of PC12 cells decreased with increase of LPS concentrations. LPS significantly increased the supernatant TNF-alpha and IL-6 concentrations and reduced TH protein expression in PC12 cells, while silencing Nogo-A could block these effects. These results suggested that LPS can activate PC12 cells to secrete inflammatory cytokines and lower the TH expression, which can be regulated by Nogo-A gene silencing. Nogo-A silencing might provide new ideas for PD treatment in the future.

  9. ss-siRNAs allele selectively inhibit ataxin-3 expression: multiple mechanisms for an alternative gene silencing strategy.

    Science.gov (United States)

    Liu, Jing; Yu, Dongbo; Aiba, Yuichiro; Pendergraff, Hannah; Swayze, Eric E; Lima, Walt F; Hu, Jiaxin; Prakash, Thazha P; Corey, David R

    2013-11-01

    Single-stranded silencing RNAs (ss-siRNAs) provide an alternative approach to gene silencing. ss-siRNAs combine the simplicity and favorable biodistribution of antisense oligonucleotides with robust silencing through RNA interference (RNAi). Previous studies reported potent and allele-selective inhibition of human huntingtin expression by ss-siRNAs that target the expanded CAG repeats within the mutant allele. Mutant ataxin-3, the genetic cause of Machado-Joseph Disease, also contains an expanded CAG repeat. We demonstrate here that ss-siRNAs are allele-selective inhibitors of ataxin-3 expression and then redesign ss-siRNAs to optimize their selectivity. We find that both RNAi-related and non-RNAi-related mechanisms affect gene expression by either blocking translation or affecting alternative splicing. These results have four broad implications: (i) ss-siRNAs will not always behave similarly to analogous RNA duplexes; (ii) the sequences surrounding CAG repeats affect allele-selectivity of anti-CAG oligonucleotides; (iii) ss-siRNAs can function through multiple mechanisms and; and (iv) it is possible to use chemical modification to optimize ss-siRNA properties and improve their potential for drug discovery.

  10. 烟草丛顶病毒编码的沉默抑制子初步鉴定%Preliminary Identification of RNA Silencing Suppressors Encoded by Tobacco Bushy Top Virus

    Institute of Scientific and Technical Information of China (English)

    张润; 王连春; 陈海如; 李凡

    2011-01-01

    Tobacco bushy top virus ( TBTV) is a distinct species of the genus Umbravirus, and can cause severe damage to tobacco Plants. The genomic RNA of TBTV contains four putative open reading frames (ORFs) , and the nearly overlapping ORF3 and 0RF4 may play important role in the pathoge-nicity of TBTV. The TBTV 0RF3 and ORF4 were cloned into the vectors of pGR107 to construct the recombinant express vector of pGR107-ORF3 and pGR107-ORF4, respectively. pGR107-ORF3 or pGR107-ORF4 was co-inoculated with pGREEN208 into the GFP-transformed Nicotiana benthamiana plants. Obvious green fluorescence patterns were observed in the infiltrated areas under long wave ultraviolet light at 2 to 5 days post-inoculation (dpi). The green fluorescence areas extended to non-infiltrated zone at 12 dpi on the pGR107-ORF4 inoculated leaves, while the infiltrated zone recovered red and no more green was observed on the pGR107-ORF3 inoculated plants. The results suggested thatTBTV 0RF4 encoded protein may partially suppress the RNA silencing induced by the GFP-trans-formed N. Benthamiana line 16c to the foreign GFP gene, whereas TBTV 0RF3 encoded product may not a gene silencing suppressor. Real-time quantitative PCR analysis results revealed that the GFP gene transcripts of the plants infiltrated with pGR107-ORF4 at 15 dpi were higher than that of pGR107 inoculated control, and less lower than that of non-inoculated line 16c. The results further confirmed that TBTV 0RF4 product suppressed the RNA silencing triggered by the GFP transgene, and the foreign GFP gene could express normally in the 16c.' The TBTV ORF4 encoded protein is a gene silencing suppressor was thus proposed in this study.%烟草丛顶病毒(Tobacco bushy top virus,TBTV)为幽影病毒属成员,能在烟草上造成严重危害.TBTV基因组编码4个ORF,其中ORF3和ORF4几乎完全重叠,可能在TBTV的致病性中发挥重要作用.分别将TBTV的ORF3和ORF4与载体pGR107连接,构建植物表达载体pGR107-ORF3,pGR107

  11. MAPKAP Kinase 2 Blocks Tristetraprolin-directed mRNA Decay by Inhibiting CAF1 Deadenylase Recruitment

    OpenAIRE

    Marchese, Francesco P.; Aubareda, Anna; Tudor, Corina; Saklatvala, Jeremy; Clark, Andrew R; Dean, Jonathan L. E.

    2010-01-01

    Tristetraprolin (TTP) directs its target AU-rich element (ARE)-containing mRNAs for degradation by promoting removal of the poly(A) tail. The p38 MAPK pathway regulates mRNA stability via the downstream kinase MAPK-activated protein kinase 2 (MAPKAP kinase 2 or MK2), which phosphorylates and prevents the mRNA-destabilizing function of TTP. We show that deadenylation of endogenous ARE-containing tumor necrosis factor mRNA is inhibited by p38 MAPK. To investigate whether phosphorylation of TTP ...

  12. 慢病毒载体介导RNA干扰体外抑制人胰腺癌细胞VIM基因的表达%Construction of a lentiviral vector for RNA interference of human VIM gene and its silencing effect in pancreatic cancer cells

    Institute of Scientific and Technical Information of China (English)

    Jianxin Jiang; Ming Shen; Renyi Qin; Rui Tian; Jing Li; Min Wang

    2009-01-01

    Objective: To construct a lentiviral expression vector for RNA interference (RNAi) of human VIM gene; and assess its gene silencing effect in pancreatic cancer cell line Panc-1. Methods: Three pairs of human VIM gene short hairpin RNA (shRNA) sequences were designed using a software available on-line and one pair came from document. After synthesis and annealing, four double-stranded oligonucleotides (dsOligo) were cloned into the pGCL-GFP/U6 plasmid, which were subsequently confirmed by polymerase chain reaction (PCR) and DNA sequencing analysis. Real-time PCR and Western blotting were used to screen the effective pGCL-GFP-shRNA plasmid in 293T cells, then the most effective one was packed into the recombinant lentivirus Lv-VIM-shRNA with lentiviral packing materials pHelper 1.0 and pHelper 2.0 in 293T cells.The titer of lentivirus was determined by hole-by-dilution titer assay. The silencing effect of Lv-VIM-shRNA in Panc-1 cells were validated by real-time PCR and Western-blotting. Results: An effective Lv-VIM-shRNA was successfully constructed.The titer of lentivirus was determined on 2×109TU/mL. The expressions of VIM mRNA and vimentin were down-regluated in the Panc-1 cells infected with Lv-VIM-shRNA. Conclusion: An effective Lv-VIM-shRNA could inhibit the expression of VIM gene in Panc-1 cells in vitro, which provides a tool for investigating the role of VIM gene in the signaling pathway involved in tumorigenesis and progression of pancreatic cancer and searching new therapeutic targets.

  13. Rapid evolution and gene expression: a rapidly evolving Mendelian trait that silences field crickets has widespread effects on mRNA and protein expression.

    Science.gov (United States)

    Pascoal, S; Liu, X; Ly, T; Fang, Y; Rockliffe, N; Paterson, S; Shirran, S L; Botting, C H; Bailey, N W

    2016-06-01

    A major advance in modern evolutionary biology is the ability to start linking phenotypic evolution in the wild with genomic changes that underlie that evolution. We capitalized on a rapidly evolving Hawaiian population of crickets (Teleogryllus oceanicus) to test hypotheses about the genomic consequences of a recent Mendelian mutation of large effect which disrupts the development of sound-producing structures on male forewings. The resulting silent phenotype, flatwing, persists because of natural selection imposed by an acoustically orienting parasitoid, but it interferes with mate attraction. We examined gene expression differences in developing wing buds of wild-type and flatwing male crickets using RNA-seq and quantitative proteomics. Most differentially expressed (DE) transcripts were down-regulated in flatwing males (625 up vs. 1716 down), whereas up- and down-regulated proteins were equally represented (30 up and 34 down). Differences between morphs were clearly not restricted to a single pathway, and we recovered annotations associated with a broad array of functions that would not be predicted a priori. Using a candidate gene detection test based on homology, we identified 30% of putative Drosophila wing development genes in the cricket transcriptome, but only 10% were DE. In addition to wing-related annotations, endocrine pathways and several biological processes such as reproduction, immunity and locomotion were DE in the mutant crickets at both biological levels. Our results illuminate the breadth of genetic pathways that are potentially affected in the early stages of adaptation. PMID:26999731

  14. Crystallization and preliminary X-ray diffraction analysis of the Cmr2–Cmr3 subcomplex in the CRISPR–Cas RNA-silencing effector complex

    International Nuclear Information System (INIS)

    The Cmr2–Cmr3 subcomplex from P. furiosus was co-crystallized with 3′-AMP. X-ray diffraction data for the crystals were collected to 2.6 Å resolution using a synchrotron-radiation source. Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci, found in prokaryotes, are transcribed to produce CRISPR RNAs (crRNAs). The Cmr proteins (Cmr1–6) and crRNA form a ribonucleoprotein complex that degrades target RNAs derived from invading genetic elements. Cmr2dHD, a Cmr2 variant lacking the N-terminal putative HD nuclease domain, and Cmr3 were co-expressed in Escherichia coli cells and co-purified as a complex. The Cmr2dHD–Cmr3 complex was co-crystallized with 3′-AMP by the vapour-diffusion method. The crystals diffracted to 2.6 Å resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the orthorhombic space group I222, with unit-cell parameters a = 103.9, b = 136.7, c = 192.0 Å. The asymmetric unit of the crystals is expected to contain one Cmr2dHD–Cmr3 complex with a Matthews coefficient of 3.0 Å3 Da−1 and a solvent content of 59%

  15. Primers to block the amplification of symbiotic apostome ciliate 18S rRNA gene in a PCR-based copepod diet study

    Science.gov (United States)

    Yi, Xiaoyan; Zhang, Huan; Liu, Guangxing

    2014-05-01

    Pelagic copepods play an important role in the marine food web. However, a full understanding of the ecological status of this zooplankton group depends on the careful study of their natural diets. In previous PCR-based copepod diet studies, we found many apostome ciliates that live symbiotically under the exoskeleton of the copepods, and their sequences were often over-represented in the 18S rRNA gene (18S rDNA) libraries. As a first step to address this issue, we designed three apostome ciliate 18S rDNA blocking primers, and tested their blocking efficiency against apostome ciliate 18s rDNA under various PCR conditions. Using a semi-quantitative PCR method, we optimized the conditions to efficiently amplify the 18S rDNA of the prey while simultaneously excluding the symbiotic apostome ciliates. This technique will facilitate PCR-based diet studies of copepods and other zooplankton in their natural environments.

  16. Ombuds’ corner: Employee silence

    CERN Multimedia

    Vincent Vuillemin

    2013-01-01

    Although around a hundred cases a year are reported to the Ombuds, several issues may still not be disclosed due to employee silence*. The deliberate withholding of concerns, escalating misunderstandings or genuine conflicts can impede the global process of learning and development of a better respectful organizational workplace environment, and prevent the detection and correction of acts violating the CERN Code of Conduct.   For the employee him/herself, such silence can lead to feelings of anger, resentment, helplessness and humiliation. These feelings will inevitably contaminate personal and interpersonal relations, and poison creativity and effectiveness. Employee silence can be explained by many factors; sometimes it is connected to organizational forces. In their published paper*, authors Michael Knoll and Rolf van Dick found four forms of employee silence. People may stay silent if they feel that their opinion is neither welcomed nor valued by their management. They have gi...

  17. The trp RNA-binding attenuation protein of Bacillus subtilis regulates translation of the tryptophan transport gene trpP (yhaG) by blocking ribosome binding.

    Science.gov (United States)

    Yakhnin, Helen; Zhang, Hong; Yakhnin, Alexander V; Babitzke, Paul

    2004-01-01

    Expression of the Bacillus subtilis tryptophan biosynthetic genes (trpEDCFBA and pabA [trpG]) is regulated in response to tryptophan by TRAP, the trp RNA-binding attenuation protein. TRAP-mediated regulation of the tryptophan biosynthetic genes includes a transcription attenuation and two distinct translation control mechanisms. TRAP also regulates translation of trpP (yhaG), a single-gene operon that encodes a putative tryptophan transporter. Its translation initiation region contains triplet repeats typical of TRAP-regulated mRNAs. We found that regulation of trpP and pabA is unaltered in a rho mutant strain. Results from filter binding and gel mobility shift assays demonstrated that TRAP binds specifically to a segment of the trpP transcript that includes the untranslated leader and translation initiation region. While the affinities of TRAP for the trpP and pabA transcripts are similar, TRAP-mediated translation control of trpP is much more extensive than for pabA. RNA footprinting revealed that the trpP TRAP binding site consists of nine triplet repeats (five GAG, three UAG, and one AAG) that surround and overlap the trpP Shine-Dalgarno (S-D) sequence and translation start codon. Results from toeprint and RNA-directed cell-free translation experiments indicated that tryptophan-activated TRAP inhibits TrpP synthesis by preventing binding of a 30S ribosomal subunit. Taken together, our results establish that TRAP regulates translation of trpP by blocking ribosome binding. Thus, TRAP coordinately regulates tryptophan synthesis and transport by three distinct mechanisms: attenuation transcription of the trpEDCFBA operon, promoting formation of the trpE S-D blocking hairpin, and blocking ribosome binding to the pabA and trpP transcripts. PMID:14702295

  18. Zn(2+ inhibits coronavirus and arterivirus RNA polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture.

    Directory of Open Access Journals (Sweden)

    Aartjan J W te Velthuis

    Full Text Available Increasing the intracellular Zn(2+ concentration with zinc-ionophores like pyrithione (PT can efficiently impair the replication of a variety of RNA viruses, including poliovirus and influenza virus. For some viruses this effect has been attributed to interference with viral polyprotein processing. In this study we demonstrate that the combination of Zn(2+ and PT at low concentrations (2 µM Zn(2+ and 2 µM PT inhibits the replication of SARS-coronavirus (SARS-CoV and equine arteritis virus (EAV in cell culture. The RNA synthesis of these two distantly related nidoviruses is catalyzed by an RNA-dependent RNA polymerase (RdRp, which is the core enzyme of their multiprotein replication and transcription complex (RTC. Using an activity assay for RTCs isolated from cells infected with SARS-CoV or EAV--thus eliminating the need for PT to transport Zn(2+ across the plasma membrane--we show that Zn(2+ efficiently inhibits the RNA-synthesizing activity of the RTCs of both viruses. Enzymatic studies using recombinant RdRps (SARS-CoV nsp12 and EAV nsp9 purified from E. coli subsequently revealed that Zn(2+ directly inhibited the in vitro activity of both nidovirus polymerases. More specifically, Zn(2+ was found to block the initiation step of EAV RNA synthesis, whereas in the case of the SARS-CoV RdRp elongation was inhibited and template binding reduced. By chelating Zn(2+ with MgEDTA, the inhibitory effect of the divalent cation could be reversed, which provides a novel experimental tool for in vitro studies of the molecular details of nidovirus replication and transcription.

  19. Deletion of fucose residues in plant N-glycans by repression of the GDP-mannose 4,6-dehydratase gene using virus-induced gene silencing and RNA interference.

    Science.gov (United States)

    Matsuo, Kouki; Matsumura, Takeshi

    2011-02-01

    Production of pharmaceutical glycoproteins in plants has many advantages in terms of safety and reduced costs. However, plant-produced glycoproteins have N-glycans with plant-specific sugar residues (core β-1,2-xylose and α-1,3-fucose) and a Lewis a (Le(a) ) epitope, i.e., Galβ(1-3)[Fucα(1-4)]GlcNAc. Because these sugar residues and glycan structures seemed to be immunogenic, several attempts have been made to delete them by repressing their respective glycosyltransferase genes. However, until date, such deletions have not been successful in completely eliminating the fucose residues. In this study, we simultaneously reduced the plant-specific core α-1,3-fucose and α-1,4-fucose residues in the Le(a) epitopes by repressing the Guanosine 5'-diphosphate (GDP)-D-mannose 4,6-dehydratase (GMD) gene, which is associated with GDP-L-fucose biosynthesis, in Nicotiana benthamiana plants. Repression of GMD was achieved using virus-induced gene silencing (VIGS) and RNA interference (RNAi). The proportion of fucose-free N-glycans found in total soluble protein from GMD gene-repressed plants increased by 80% and 95% following VIGS and RNAi, respectively, compared to wild-type plants. A small amount of putative galactose substitution in N-glycans from the NbGMD gene-repressed plants was observed, similar to what has been previously reported GMD-knockout Arabidopsis mutant. On the other hand, the recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) with fucose-deleted N-glycans was successfully produced in NbGMD-RNAi transgenic N. benthamiana plants. Thus, repression of the GMD gene is thus very useful for deleting immunogenic total fucose residues and facilitating the production of pharmaceutical glycoproteins in plants.

  20. Efficient and Tumor Targeted siRNA Delivery by Polyethylenimine-graft-polycaprolactone-block-poly(ethylene glycol)-folate (PEI-PCL-PEG-Fol).

    Science.gov (United States)

    Liu, Li; Zheng, Mengyao; Librizzi, Damiano; Renette, Thomas; Merkel, Olivia M; Kissel, Thomas

    2016-01-01

    Efficient delivery of functional nucleic acids into specific cells or tissues is still a challenge for gene therapy and largely depends on targeted delivery strategies. The folate receptor (FR) is known to be overexpressed extracellularly on a variety of human cancers and is therefore an outstanding gate for tumor-targeted Trojan horse-like delivery of therapeutics. In this study, an amphiphilic and biodegradable ternary copolymer conjugated with folate as ligand, polyethylenimine-graft-polycaprolactone-block-poly(ethylene glycol)-folate (PEI-PCL-PEG-Fol) was synthesized and evaluated for targeted siRNA delivery via folate-FR recognition. The amphiphilic character of similar polymers was shown previously to support endosomal release of endocytosed nanocarriers and to promote formation of long circulating micelles. The obtained PEI-PCL-PEG-Fol exhibited less cytotoxicity in comparison with the corresponding ternary copolymer without folate (PEI-PCL-PEG) and with unmodified PEI25kDa. Stable micelle-like polyplexes with hydrodynamic diameters about 100 nm were found to have a zeta potential of +8.6 mV, which was lower than that of micelleplexes without folate-conjugation (+13-16 mV). Nonetheless, increased cellular uptake and in vitro gene knockdown of PEI-PCL-PEG-Fol/siRNA micelleplexes were observed in SKOV-3 cells, an FR overexpressing cell line, in comparison with the nonfolate-conjugated ones. Moreover, PEI-PCL-PEG-Fol/siRNA micelleplexes exhibited excellent stability in vivo during the analysis of 120 min and a longer circulation half life than hyPEI25kDa/siRNA polyplexes. Most interestingly, the targeted delivery system yielded 17% deposition of the i.v. injected siRNA per gram in the tumor after 24 h due to the effective folate targeting and the prolonged circulation. PMID:26641134

  1. The C. elegans CSR-1 Argonaute pathway counteracts epigenetic silencing to promote germline gene expression

    OpenAIRE

    Seth, Meetu; Shirayama, Masaki; Gu, Weifeng; Ishidate, Takao; Conte, Darryl; Mello, Craig C

    2013-01-01

    Organisms can develop adaptive sequence-specific immunity by re-expressing pathogen-specific small RNAs that guide gene silencing. For example, the C. elegans PIWI-Argonaute/piRNA pathway recruits RNA-dependent RNA polymerase RdRP to foreign sequences to amplify a trans-generational small RNA-induced epigenetic silencing signal (termed RNAe). Here we provide evidence that in addition to an adaptive memory of silenced sequences, C. elegans can also develop an opposing adaptive memory of expres...

  2. Breaking the Silence

    Institute of Scientific and Technical Information of China (English)

    1998-01-01

    Many women who suffer from vaginitis have kept silent about their illness because they think it is shameful to have such a disease. The International Women’s Health Coalition (IWHC) has publicized the problem, referring to it as a "culture of silence"inherited from traditional thinking. The coalition has made attempts to improve women’s health conditions by changing people’s misconceptions about the disease. In 1997, under a grant from the American Ford Foundation, the Sichuan Provincial Women’s Federation carried out a study on women’s repro-ductive health, aimed at "breaking the silence."

  3. Memories Persist in Silence

    Directory of Open Access Journals (Sweden)

    Sandra Patricia Arenas Grisales

    2012-08-01

    Full Text Available This article exposes the hypothesis that memory artifacts, created to commemorate the victims of armed conflict in Colombia, are an expression of the underground memories and a way of political action in the midst of war. We analyze three cases of creations of memory artifacts in Medellín, Colombia, as forms of suffering, perceiving and resisting the power of armed groups in Medellín. The silence, inherent in these objects, should not be treated as an absence of language, but as another form of expression of memory. Silence is a tactic used to overcome losses and reset everyday life in contexts of protracted violence.

  4. Illuminating employees’ organizational silence

    Directory of Open Access Journals (Sweden)

    Jaber Moghaddampour

    2013-08-01

    Full Text Available Undoubtedly, human capital is the main source of knowledge generation, which leads to a competitive advantage and sustainability of organizations. When managers pay more attention to such capital, they will be able to lead their organization, more effectively. In such route, the managers should look for their employees’ opinions on policies/plans of the organization and learn how to run the organization and organizational challenges. They should use their employees’ knowledge to improve the quality of decisions, they should encourage them to share their ideas and protect them from organizational silence. Thus, human capitals will be obviously considered as strategic capitals of an organization. Concerning the importance of organizational sound, the status of organizational silence is studied in 13 selected organizations in Qom Province – as one the biggest Iranian provinces. The findings indicate that organizational silence in Qom selected organizations is not in an ideal status and the current level should be mitigated. Likewise, findings show that there is no significant difference between organizational silence in Qom selected organizations in terms of demographic variables.

  5. The Silence of Michelangelo

    DEFF Research Database (Denmark)

    Foote, Jonathan

    2016-01-01

    of contemplation with Ovid's well-known myth of Pygmalion. Similarly, while sojourning in the Carrara quarries in 1516, he ignored repeated requests from the pope, friends and family to send a model for the facade of San Lorenzo. The silence from papal politics notwithstanding, it was in fact the hammering...

  6. FXR silencing in human colon cancer by DNA methylation and KRAS signaling.

    Science.gov (United States)

    Bailey, Ann M; Zhan, Le; Maru, Dipen; Shureiqi, Imad; Pickering, Curtis R; Kiriakova, Galina; Izzo, Julie; He, Nan; Wei, Caimiao; Baladandayuthapani, Veerabhadran; Liang, Han; Kopetz, Scott; Powis, Garth; Guo, Grace L

    2014-01-01

    Farnesoid X receptor (FXR) is a bile acid nuclear receptor described through mouse knockout studies as a tumor suppressor for the development of colon adenocarcinomas. This study investigates the regulation of FXR in the development of human colon cancer. We used immunohistochemistry of FXR in normal tissue (n = 238), polyps (n = 32), and adenocarcinomas, staged I-IV (n = 43, 39, 68, and 9), of the colon; RT-quantitative PCR, reverse-phase protein array, and Western blot analysis in 15 colon cancer cell lines; NR1H4 promoter methylation and mRNA expression in colon cancer samples from The Cancer Genome Atlas; DNA methyltransferase inhibition; methyl-DNA immunoprecipitation (MeDIP); bisulfite sequencing; and V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) knockdown assessment to investigate FXR regulation in colon cancer development. Immunohistochemistry and quantitative RT-PCR revealed that expression and function of FXR was reduced in precancerous lesions and silenced in a majority of stage I-IV tumors. FXR expression negatively correlated with phosphatidylinositol-4, 5-bisphosphate 3 kinase signaling and the epithelial-to-mesenchymal transition. The NR1H4 promoter is methylated in ~12% colon cancer The Cancer Genome Atlas samples, and methylation patterns segregate with tumor subtypes. Inhibition of DNA methylation and KRAS silencing both increased FXR expression. FXR expression is decreased early in human colon cancer progression, and both DNA methylation and KRAS signaling may be contributing factors to FXR silencing. FXR potentially suppresses epithelial-to-mesenchymal transition and other oncogenic signaling cascades, and restoration of FXR activity, by blocking silencing mechanisms or increasing residual FXR activity, represents promising therapeutic options for the treatment of colon cancer.

  7. Agrobacterium mediated transient gene silencing (AMTS in Stevia rebaudiana: insights into steviol glycoside biosynthesis pathway.

    Directory of Open Access Journals (Sweden)

    Praveen Guleria

    Full Text Available BACKGROUND: Steviol glycoside biosynthesis pathway has emerged as bifurcation from ent-kaurenoic acid, substrate of methyl erythritol phosphate pathway that also leads to gibberellin biosynthesis. However, the genetic regulation of steviol glycoside biosynthesis has not been studied. So, in present study RNA interference (RNAi based Agrobacterium mediated transient gene silencing (AMTS approach was followed. SrKA13H and three SrUGTs (SrUGT85C2, SrUGT74G1 and SrUGT76G1 genes encoding ent-kaurenoic acid-13 hydroxylase and three UDP glycosyltransferases of steviol glycoside biosynthesis pathway were silenced in Stevia rebaudiana to understand its molecular mechanism and association with gibberellins. METHODOLOGY/PRINCIPAL FINDINGS: RNAi mediated AMTS of SrKA13H and three SrUGTs has significantly reduced the expression of targeted endogenous genes as well as total steviol glycoside accumulation. While gibberellins (GA3 content was significantly enhanced on AMTS of SrUGT85C2 and SrKA13H. Silencing of SrKA13H and SrUGT85C2 was found to block the metabolite flux of steviol glycoside pathway and shifted it towards GA3 biosynthesis. Further, molecular docking of three SrUGT proteins has documented highest affinity of SrUGT76G1 for the substrates of alternate pathways synthesizing steviol glycosides. This could be a plausible reason for maximum reduction in steviol glycoside content on silencing of SrUGT76G1 than other genes. CONCLUSIONS: SrKA13H and SrUGT85C2 were identified as regulatory genes influencing carbon flux between steviol glycoside and gibberellin biosynthesis. This study has also documented the existence of alternate steviol glycoside biosynthesis route.

  8. Efficient Silencing of Endogenous MicroRNAs Using Artificial MicroRNAs in Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    Andrew L. Eamens; Claire Agius; Neil A. Smith; Peter M. Waterhouse; Ming-Bo Wang

    2011-01-01

    We report here that the expression of endogenous microRNAs (miRNAs) can be efficiently silenced in Arabidopsis thaliana (Arabidopsis) using artificial miRNA (amiRNA) technology. We demonstrate that an amiRNA designed to target a mature miRNA directs silencing against all miRNA family members, whereas an amiRNA designed to target the stem-loop region of a miRNA precursor transcript directs silencing against only the individual family member targeted.Furthermore, our results indicate that amiRNAs targeting both the mature miRNA and stem-loop sequence direct RNA silencing through cleavage of the miRNA precursor transcript, which presumably occurs in the nucleus of a plant cell during the initial stages of miRNA biogenesis. This suggests that small RNA (sRNA)-guided RNA cleavage in plants occurs not only in the cytoplasm, but also in the nucleus. Many plant miRNA gene families have been identified via sequencing and bioinformatic analysis, but, to date, only a small tranche of these have been functionally characterized due to a lack of effective forward or reverse genetic tools. Our findings therefore provide a new and powerful reverse-genetic tool for the analysis of miRNA function in plants.

  9. miR-150, a microRNA expressed in mature B and T cells, blocks early B cell development when expressed prematurely.

    Science.gov (United States)

    Zhou, Beiyan; Wang, Stephanie; Mayr, Christine; Bartel, David P; Lodish, Harvey F

    2007-04-24

    MicroRNAs (miRNAs) are a family of approximately 22-nt noncoding RNAs that can posttranscriptionally regulate gene expression. Several miRNAs are specifically expressed in hematopoietic cells. Here we show that one such miRNA, miR-150, is mainly expressed in the lymph nodes and spleen and is highly up-regulated during the development of mature T and B cells; expression of miR-150 is sharply up-regulated at the immature B cell stage. Overexpression of miR-150 in hematopoietic stem cells, followed by bone marrow transplantation, had little effect on the formation of either mature CD8- and CD4-positive T cells or granulocytes or macrophages, but the formation of mature B cells was greatly impaired. Furthermore, premature expression of miR-150 blocked the transition from the pro-B to the pre-B stage. Our results indicate that miR-150 most likely down-regulates mRNAs that are important for pre- and pro-B cell formation or function, and its ectopic expression in these cells blocks further development of B cells.

  10. A modular plasmid assembly kit for multigene expression, gene silencing and silencing rescue in plants.

    Directory of Open Access Journals (Sweden)

    Andreas Binder

    Full Text Available The Golden Gate (GG modular assembly approach offers a standardized, inexpensive and reliable way to ligate multiple DNA fragments in a pre-defined order in a single-tube reaction. We developed a GG based toolkit for the flexible construction of binary plasmids for transgene expression in plants. Starting from a common set of modules, such as promoters, protein tags and transcribed regions of interest, synthetic genes are assembled, which can be further combined to multigene constructs. As an example, we created T-DNA constructs encoding multiple fluorescent proteins targeted to distinct cellular compartments (nucleus, cytosol, plastids and demonstrated simultaneous expression of all genes in Nicotiana benthamiana, Lotus japonicus and Arabidopsis thaliana. We assembled an RNA interference (RNAi module for the construction of intron-spliced hairpin RNA constructs and demonstrated silencing of GFP in N. benthamiana. By combination of the silencing construct together with a codon adapted rescue construct into one vector, our system facilitates genetic complementation and thus confirmation of the causative gene responsible for a given RNAi phenotype. As proof of principle, we silenced a destabilized GFP gene (dGFP and restored GFP fluorescence by expression of a recoded version of dGFP, which was not targeted by the silencing construct.

  11. A Modular Plasmid Assembly Kit for Multigene Expression, Gene Silencing and Silencing Rescue in Plants

    Science.gov (United States)

    Binder, Andreas; Lambert, Jayne; Morbitzer, Robert; Popp, Claudia; Ott, Thomas; Lahaye, Thomas; Parniske, Martin

    2014-01-01

    The Golden Gate (GG) modular assembly approach offers a standardized, inexpensive and reliable way to ligate multiple DNA fragments in a pre-defined order in a single-tube reaction. We developed a GG based toolkit for the flexible construction of binary plasmids for transgene expression in plants. Starting from a common set of modules, such as promoters, protein tags and transcribed regions of interest, synthetic genes are assembled, which can be further combined to multigene constructs. As an example, we created T-DNA constructs encoding multiple fluorescent proteins targeted to distinct cellular compartments (nucleus, cytosol, plastids) and demonstrated simultaneous expression of all genes in Nicotiana benthamiana, Lotus japonicus and Arabidopsis thaliana. We assembled an RNA interference (RNAi) module for the construction of intron-spliced hairpin RNA constructs and demonstrated silencing of GFP in N. benthamiana. By combination of the silencing construct together with a codon adapted rescue construct into one vector, our system facilitates genetic complementation and thus confirmation of the causative gene responsible for a given RNAi phenotype. As proof of principle, we silenced a destabilized GFP gene (dGFP) and restored GFP fluorescence by expression of a recoded version of dGFP, which was not targeted by the silencing construct. PMID:24551083

  12. Low energy (0-12 eV) electron interaction with gas phase building blocks of DNA/RNA

    International Nuclear Information System (INIS)

    We review recent results on dissociative electron attachment (DEA) to gas phase D-ribose, tetraacetyl-D-ribose (TAR) and dibutylphosphate (DBP), which serve as model compounds for the DNA or RNA backbone. New results are presented on negative ion formation in D-ribose probed by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry. The two methods reveal that the transient D-ribose anion R- decomposes in the same way like the deprotonated D-ribose molecule [R-H]-, i.e. by abstraction of different numbers of water and formaldehyde units. In DEA the TNI R- is generated at very low energies close to 0 eV most likely through a vibrational feshbach resonance. The fragmentation pattern and the characteristic resonances of D-ribose are preserved in TAR, where a furanose is bound to four acetyl groups. The presence of an acetyl group leads additionally to fragmentation through a shape resonance. Shape resonances were also observed in DBP, followed by C-O and P-O bond breaking.

  13. Silencing Status Epilepticus-Induced BDNF Expression with Herpes Simplex Virus Type-1 Based Amplicon Vectors.

    Directory of Open Access Journals (Sweden)

    Chiara Falcicchia

    Full Text Available Brain-derived neurotrophic factor (BDNF has been found to produce pro- but also anti-epileptic effects. Thus, its validity as a therapeutic target must be verified using advanced tools designed to block or to enhance its signal. The aim of this study was to develop tools to silence the BDNF signal. We generated Herpes simplex virus type 1 (HSV-1 derived amplicon vectors, i.e. viral particles containing a genome of 152 kb constituted of concatameric repetitions of an expression cassette, enabling the expression of the gene of interest in multiple copies. HSV-1 based amplicon vectors are non-pathogenic and have been successfully employed in the past for gene delivery into the brain of living animals. Therefore, amplicon vectors should represent a logical choice for expressing a silencing cassette, which, in multiple copies, is expected to lead to an efficient knock-down of the target gene expression. Here, we employed two amplicon-based BDNF silencing strategies. The first, antisense, has been chosen to target and degrade the cytoplasmic mRNA pool of BDNF, whereas the second, based on the convergent transcription technology, has been chosen to repress transcription at the BDNF gene. Both these amplicon vectors proved to be effective in down-regulating BDNF expression in vitro, in BDNF-expressing mesoangioblast cells. However, only the antisense strategy was effective in vivo, after inoculation in the hippocampus in a model of status epilepticus in which BDNF mRNA levels are strongly increased. Interestingly, the knocking down of BDNF levels induced with BDNF-antisense was sufficient to produce significant behavioral effects, in spite of the fact that it was produced only in a part of a single hippocampus. In conclusion, this study demonstrates a reliable effect of amplicon vectors in knocking down gene expression in vitro and in vivo. Therefore, this approach may find broad applications in neurobiological studies.

  14. Silencing Status Epilepticus-Induced BDNF Expression with Herpes Simplex Virus Type-1 Based Amplicon Vectors.

    Science.gov (United States)

    Falcicchia, Chiara; Trempat, Pascal; Binaschi, Anna; Perrier-Biollay, Coline; Roncon, Paolo; Soukupova, Marie; Berthommé, Hervé; Simonato, Michele

    2016-01-01

    Brain-derived neurotrophic factor (BDNF) has been found to produce pro- but also anti-epileptic effects. Thus, its validity as a therapeutic target must be verified using advanced tools designed to block or to enhance its signal. The aim of this study was to develop tools to silence the BDNF signal. We generated Herpes simplex virus type 1 (HSV-1) derived amplicon vectors, i.e. viral particles containing a genome of 152 kb constituted of concatameric repetitions of an expression cassette, enabling the expression of the gene of interest in multiple copies. HSV-1 based amplicon vectors are non-pathogenic and have been successfully employed in the past for gene delivery into the brain of living animals. Therefore, amplicon vectors should represent a logical choice for expressing a silencing cassette, which, in multiple copies, is expected to lead to an efficient knock-down of the target gene expression. Here, we employed two amplicon-based BDNF silencing strategies. The first, antisense, has been chosen to target and degrade the cytoplasmic mRNA pool of BDNF, whereas the second, based on the convergent transcription technology, has been chosen to repress transcription at the BDNF gene. Both these amplicon vectors proved to be effective in down-regulating BDNF expression in vitro, in BDNF-expressing mesoangioblast cells. However, only the antisense strategy was effective in vivo, after inoculation in the hippocampus in a model of status epilepticus in which BDNF mRNA levels are strongly increased. Interestingly, the knocking down of BDNF levels induced with BDNF-antisense was sufficient to produce significant behavioral effects, in spite of the fact that it was produced only in a part of a single hippocampus. In conclusion, this study demonstrates a reliable effect of amplicon vectors in knocking down gene expression in vitro and in vivo. Therefore, this approach may find broad applications in neurobiological studies.

  15. 人胰腺癌 PANC-1细胞裸鼠移植瘤模型建立及其用于观察载 siRNA 纳米微粒体内效应的研究%Establishment of human pancreatic tumor xenograft mouse model for evaluating tumor-homing and gene-silencing effects of siRNA-loading nanoparticles

    Institute of Scientific and Technical Information of China (English)

    曾林涓; 李景果; 张秋波; 钱辰琛; 林忠; 陈茵婷; 黄开红

    2014-01-01

    AIM:To establish an effective and rapid method to develop transplanted subcutaneous pancreatic carcinoma by inducing PANC-1 cells into nude mice, and then use this mouse model to evaluate the tumor-homing and gene-silencing effects of siRNA-loading nanoparticles in vivo.METHODS:Different numbers of PANC-1 cells in 100 μL or 300 μL PBS were inoculated subcutaneously into the right flank of BALB /c (nu/nu) mice.When the tumor volume reached 100 mm 3 , siRNACY 5.5 nanoparticles were injected through the mouse tail vein to perform in vivo imaging assay.Be-sides, the mice were randomly divided into 3 treatment groups treated with PBS, scrambled control RNA nanoparticles and siKras nanoparticles, respectively.The protein expression of Kras was detected by Western blotting and immunohistochemi-cal staining.RESULTS:After inoculated with 1 ×10 7 PANC-1 cells in 300 μL PBS, all mice developed tumors within 2 weeks.The in vivo results showed that siRNA-loading nanoparticles accumulated in the tumor tissues and exerted gene si-lencing effect.CONCLUSION:In the present study, an effective and rapid method was established for PANC-1 cells to induce transplanted subcutaneous pancreatic carcinoma in nude mice within 2 weeks, which is suitable for in vivo imaging and treatment evaluations as a reproducible and reliable way for the further experiments .%目的:探讨建立人胰腺癌PANC-1裸鼠移植瘤模型的最佳实验方法,并应用该模型进行载基因纳米微粒体内效应的研究。方法:将不同数量PANC-1细胞悬液接种于BALB/c (nu/nu)小鼠右侧背部皮下,当肿瘤体积达100 mm 3时尾静脉注射siRNACY 5.5纳米复合物进行活体荧光成像。此外,于尾静脉注射负载siRNA Kras纳米复合物,蛋白印迹及免疫组织化学染色法观察肿瘤组织Kras蛋白表达水平。结果:1×107 cells/300μL 接种成瘤率达100%,成瘤时间<2周。荧光呈像及组织学检查显示载siRNA纳米微粒可靶向

  16. The Conspiracy Of Silence

    OpenAIRE

    Cohen, Saul

    1980-01-01

    The issue of the impaired physician is compounded by not only mass denial of the problem, but also a 'conspiracy of silence' among many groups associated with the physician. The conspirators—including the physician himself, his family, community, professional colleagues and nurses as well as hospital boards and administration—are unable to reconcile deteriorating performance due to alcohol or drugs with an otherwise gifted professional who should know the dangers of substance abuse. They may ...

  17. Silence in Intercultural communication

    Institute of Scientific and Technical Information of China (English)

    Guo Jun

    2012-01-01

    In communication, most of people's attention focuses on verbal communication, nonverbal language as a means of exchange is often ignored. However, nonverbal language continues sending signals, and most of these signals are sent to conversational partners unconsciously. So correct understanding of these signals will help people improve effectiveness of communication. This article will focus on silence, a major part of nonverbal communication, exploring its communicative functions and cultural differences.

  18. Silence in the Communication or Communicating through Silence: Silence in Psychoanalysis

    Directory of Open Access Journals (Sweden)

    Rita Marta

    2014-10-01

    Full Text Available This paper is a reflection upon the meaning and importance of silence in the psychoanalytical relationship. Beginning with the silence in the “normal” relationship between people, we show how silence can be experienced as confortable or unconfortable, and how it can be used to achieve a bigger proximity or distance in the relationship with others. We show these same aspects in the psychoanalytical relationship, and the evolution of the regard towards silence along the development of psychoanalysis. We end, presenting the Nacht’s thinking about silence, who emphasizes its integrative and fundamental role in the psychoanalytical relationship. Thus, only through silence certain affects can be born, and silence allows the patient to internalize the analyst.

  19. PERK silence inhibits glioma cell growth under low glucose stress by blockage of p-AKT and subsequent HK2's mitochondria translocation

    KAUST Repository

    Hou, Xu

    2015-03-12

    Glioma relies on glycolysis to obtain energy and sustain its survival under low glucose microenvironment in vivo. The mechanisms on glioma cell glycolysis regulation are still unclear. Signaling mediated by Double-stranded RNA-activated protein kinase (PKR) - like ER kinase (PERK) is one of the important pathways of unfolded protein response (UPR) which is comprehensively activated in cancer cells upon the hypoxic and low glucose stress. Here we show that PERK is significantly activated in human glioma tissues. PERK silencing results in decreased glioma cell viability and ATP/lactate production upon low glucose stress, which is mediated by partially blocked AKT activation and subsequent inhibition of Hexokinase II (HK2)\\'s mitochondria translocation. More importantly, PERK silenced glioma cells show decreased tumor formation capacity. Our results reveal that PERK activation is involved in glioma glycolysis regulation and may be a potential molecular target for glioma treatment.

  20. Breaking the silence

    DEFF Research Database (Denmark)

    Konradsen, Hanne; Kirkevold, Marit; McCallin, Antoinette;

    2012-01-01

    and individual interviews were analyzed using the grounded theory method. The findings revealed that the main concern of the patients was feeling isolated, which was resolved using a process of interactional integration. Interactional integration begins by breaking the silence to enable the progression from......Little is known about the psychosocial effects of facial disfigurement. We present the results of a qualitative study following 15 patients who had been surgically treated for head, neck, or eye cancer over the course of their first postoperative year. Taped nurse-patient conversations...

  1. An miRNA-mediated therapy for SCA6 blocks IRES-driven translation of the CACNA1A second cistron.

    Science.gov (United States)

    Miyazaki, Yu; Du, Xiaofei; Muramatsu, Shin-Ichi; Gomez, Christopher M

    2016-07-13

    Spinocerebellar ataxia type 6 (SCA6) is a dominantly inherited neurodegenerative disease characterized by slowly progressive ataxia and Purkinje cell degeneration. SCA6 is caused by a polyglutamine repeat expansion within a second CACNA1A gene product, α1ACT. α1ACT expression is under the control of an internal ribosomal entry site (IRES) present within the CACNA1A coding region. Whereas SCA6 allele knock-in mice show indistinguishable phenotypes from wild-type littermates, expression of SCA6-associated α1ACT (α1ACTSCA6) driven by a Purkinje cell-specific promoter in mice produces slowly progressive ataxia and cerebellar atrophy. We developed an early-onset SCA6 mouse model using an adeno-associated virus (AAV)-based gene delivery system to ectopically express CACNA1A IRES-driven α1ACTSCA6 to test the potential of CACNA1A IRES-targeting therapies. Mice expressing AAV9-mediated CACNA1A IRES-driven α1ACTSCA6 exhibited early-onset ataxia, motor deficits, and Purkinje cell degeneration. We identified miR-3191-5p as a microRNA (miRNA) that targeted CACNA1A IRES and preferentially inhibited the CACNA1A IRES-driven translation of α1ACT in an Argonaute 4 (Ago4)-dependent manner. We found that eukaryotic initiation factors (eIFs), eIF4AII and eIF4GII, interacted with the CACNA1A IRES to enhance α1ACT translation. Ago4-bound miR-3191-5p blocked the interaction of eIF4AII and eIF4GII with the CACNA1A IRES, attenuating IRES-driven α1ACT translation. Furthermore, AAV9-mediated delivery of miR-3191-5p protected mice from the ataxia, motor deficits, and Purkinje cell degeneration caused by CACNA1A IRES-driven α1ACTSCA6 We have established proof of principle that viral delivery of an miRNA can rescue a disease phenotype through modulation of cellular IRES activity in a mouse model. PMID:27412786

  2. Studies of the silencing of Baculovirus DNA binding protein

    NARCIS (Netherlands)

    Quadt, I.; Lent, van J.W.M.; Knebel-Morsdorf, D.

    2007-01-01

    Baculovirus DNA binding protein (DBP) binds preferentially single-stranded DNA in vitro and colocalizes with viral DNA replication sites. Here, its putative role as viral replication factor has been addressed by RNA interference. Silencing of DBP in Autographa californica multiple nucleopolyhedrovir

  3. Transcriptional silencing of multiple genes in trophozoites of Entamoeba histolytica.

    Directory of Open Access Journals (Sweden)

    Rivka Bracha

    2006-05-01

    Full Text Available In a previous work we described the transcriptional silencing of the amoebapore A (AP-A gene (Ehap-a of Entamoeba histolytica strain HM-1:IMSS. The silencing occurred following transfection with a plasmid containing a 5' upstream region (473 bp of Ehap-a that included a truncated segment (140 bp of a short interspersed nuclear element (SINE1. Silencing remained in effect even after removal of the plasmid (clone G3. Neither short interfering RNA nor methylated DNA were detected, but the chromatin domain of Ehap-a in the gene-silenced trophozoites was modified. Two other similar genes (Ehap-b and one encoding a Saposin-like protein, SAPLIP 1 also became silenced. In the present work we demonstrate the silencing of a second gene of choice, one that encodes the light subunit of the Gal/GalNAc inhibitable lectin (Ehlgl1 and the other, the cysteine proteinase 5 (EhCP-5. This silencing occurred in G3 trophozoites transfected with a plasmid in which the 473 bp 5' upstream Ehap-a fragment was directly ligated to the second gene. Transcriptional silencing occurred in both the transgene and the chromosomal gene. SINE1 sequences were essential, as was a direct connection between the Ehap-a upstream region and the beginning of the open reading frame of the second gene. Gene silencing did not occur in strain HM-1:IMSS with any of these plasmid constructs. The trophozoites with two silenced genes were virulence-attenuated as were those of clone G3. In addition, trophozoites not expressing Lgl1 and AP-A proteins had a significantly reduced ability to cap the Gal/GalNAc-lectin to the uroid region when incubated with antibodies against the heavy (170 kDa subunit of the lectin. Lysates of trophozoites lacking cysteine proteinase 5 and AP-A proteins had 30% less cysteine proteinase activity than those of HM-1:IMSS strain or the G3 clone. Silencing of other genes in G3 amoebae could provide a model to study their various functions. In addition, double gene-silenced

  4. Bodies, Spaces, Voices, Silences

    Directory of Open Access Journals (Sweden)

    Donatella Mazzoleni

    2013-07-01

    Full Text Available A good architecture should not only allow functional, formal and technical quality for urban spaces, but also let the voice of the city be perceived, listened, enjoyed. Every city has got its specific sound identity, or “ISO” (R. O. Benenzon, made up of a complex texture of background noises and fluctuation of sound figures emerging and disappearing in a game of continuous fadings. For instance, the ISO of Naples is characterized by a spread need of hearing the sound return of one’s/others voices, by a hate of silence. Cities may fall ill: illness from noise, within super-crowded neighbourhoods, or illness from silence, in the forced isolation of peripheries. The proposal of an urban music therapy denotes an unpublished and innovative enlarged interdisciplinary research path, where architecture, music, medicine, psychology, communication science may converge, in order to work for rebalancing spaces and relation life of the urban collectivity, through the care of body and sound dimensions.

  5. Edwin Hubble's Silence

    Science.gov (United States)

    Lago, D.

    2013-04-01

    In late 1928 Edwin Hubble was right in the middle of using V. M. Slipher's redshift data to prove that the universe is expanding, when Hubble's boss, George Hale, directed him to drop everything and rush to the Grand Canyon and test it as a possible site for Hale's planned 200-inch telescope. On his way, Hubble stopped at Lowell Observatory and met with V. M. Slipher. The letters both men wrote about this visit suggest that Hubble never said a word about his being in the middle of using Slipher's research to transform the universe. At the least, this silence is symbolic of the silence with which astronomical history has often treated Slipher's work. A survey of the historical literature suggests several reasons for this. Theorists and observers in astronomy (and other sciences) have long had different perspectives about how science works, and those who place more importance on theory have tended to credit the idea of the expanding universe to the theorists. Also, many sources indicate that Edwin Hubble was not a modest man or generous about sharing credit.

  6. siRNA沉默LKB1基因激活Hedgehog信号通路及对人乳腺癌裸鼠移植瘤模型生长的实验研究%Silencing LKB1 by siRNA activated Hedgehog signaling pathway and the growth of xenografted breast carcinoma in nude mice

    Institute of Scientific and Technical Information of China (English)

    庄志刚; 成小林; 蒋蓓琦; 傅韵; 李正东; 罗建民; 金伟

    2011-01-01

    目的 探讨应用小分子干扰RNA(small interfering RNA,siRNA)沉默抑癌基因LKB1对人乳腺癌细胞MDA-MB-231中Hedgehog信号通路相关因子的表达及人乳腺癌裸鼠移植瘤模型的肿瘤生长的影响.方法 构建LKB1基因siRNA质粒LKB1-siRNA;建立LKB1表达抑制的MDA-MB-435细胞模型;裸鼠乳晕皮下接种,建立人乳腺癌裸鼠移植瘤动物模型;成瘤后,观察肿瘤体积变化、裸鼠生存时间;并用Western印迹法检测瘤组织中LKB1和Hedgehog信号通路中信号肽Shh、Sufu、膜受体Ptch、Smo、转录因子Gli1、Hip 蛋白表达的变化.结果 LKB1-siRNA质粒组裸鼠的肿瘤体积明显增长(P<0.05);肿瘤内LKB1基因表达水平明显下降,而Hedgehog信号通路相关因子Shh、Gli1、Ptch、Smo的表达升高,Hedgehog信号通路抑制因子Sufu、Hip表达下降.结论 LKB1基因siRNA能够明显抑制人乳腺癌裸鼠移植模型的LKB1基因的表达,上调Hedgehog信号通路相关因子的表达,促进肿瘤生长.LKB1基因和Hedgehog信号通路在乳腺癌细胞中呈现负相关表达.%Objective To investigate the effect of silencing LKB1 by small interfering RNA(siR-NA) on the expression of the correlation factor of Hedgehog signaling pathways in human breast cancer MDA-MB-231 cells and the growth of xenografted breast carcinoma in nude mice. Methods Plasmids of siRNA for LKB1 gene were constructed. RNA interference technique was used to silence LKB1 gene in breast carcinoma cells,xenografted tumor model was established in nude mice by subcutaneous inoculation of MDA-MB-231 cells. The tumor volume and survival time of nude mice were recorded. The expression of LKB1, Shh, Sufu.Gli 1 ,Ptch,Smo and Hip was measured by Western blotting. Results The tumor size was significantly increased in LKBl-siRNA treated group(P <0. 01). Western blotting analysis showed that the expression of LKB1 in xenografted tumor was markedly decreased and the correlation factor of Hedgehog signaling pathways was

  7. Stability of Barley stripe mosaic virus-induced gene silencing in barley

    DEFF Research Database (Denmark)

    Bruun-Rasmussen, Marianne; Madsen, Christian Toft; Jessing, Stine;

    2007-01-01

    for barley and wheat; however, silencing using this vector is generally transient, with efficient silencing often being confined to the first two or three systemically infected leaves. To investigate this further, part of the barley Phytoene desaturase (PDS) gene was inserted into BSMV and the...... length influenced stability but not efficiency of VIGS. Silencing was transient in most cases; however, the decrease in PDS mRNA levels measured by qRT-PCR began earlier and lasted longer than the photobleaching. Occasionally, silencing persisted and could be transmitted through seed as well as via......Virus-induced gene silencing (VIGS) can be used as a powerful tool for functional genomics studies in plants. With this approach, it is possible to target most genes and downregulate the messenger (m)RNA in a sequence-specific manner. Barley stripe mosaic virus (BSMV) is an established VIGS vector...

  8. RNAi-induced silencing of embryonic tryptophan oxygenase in the Pyralid moth, Plodia interpunctella.

    Science.gov (United States)

    Fabrick, Jeffrey A; Kanost, Michael R; Baker, James E

    2004-01-01

    Gene silencing through the introduction of double-stranded RNA (RNA interference, RNAi) provides a powerful tool for the elucidation of gene function in many systems, including those where genomics and proteomics are incomplete. The use of RNAi technology for gene silencing in Lepidoptera has lacked significant attention compared to other systems. To demonstrate that RNAi can be utilized in the lepidopteran, Plodia interpunctella, we cloned a cDNA for tryptophan oxygenase, and showed that silencing of tryptophan oxygenase through RNAi during embryonic development resulted in loss of eye-color pigmentation. The complete amino acid sequence of Plodia tryptophan oxygenase can be accessed through NCBI Protein Database.

  9. RNA interference mediated inhibition of dengue virus multiplication and entry in HepG2 cells.

    Directory of Open Access Journals (Sweden)

    Mohammed Abdelfatah Alhoot

    Full Text Available BACKGROUND: Dengue virus-host cell interaction initiates when the virus binds to the attachment receptors followed by endocytic internalization of the virus particle. Successful entry into the cell is necessary for infection initiation. Currently, there is no protective vaccine or antiviral treatment for dengue infection. Targeting the viral entry pathway has become an attractive therapeutic strategy to block infection. This study aimed to investigate the effect of silencing the GRP78 and clathrin-mediated endocytosis on dengue virus entry and multiplication into HepG2 cells. METHODOLOGY/PRINCIPAL FINDINGS: HepG2 cells were transfected using specific siRNAs to silence the cellular surface receptor (GRP78 and clathrin-mediated endocytosis pathway. Gene expression analysis showed a marked down-regulation of the targeted genes (87.2%, 90.3%, and 87.8% for GRP78, CLTC, and DNM2 respectively in transfected HepG2 cells when measured by RT-qPCR. Intracellular and extracellular viral RNA loads were quantified by RT-qPCR to investigate the effect of silencing the attachment receptor and clathrin-mediated endocytosis on dengue virus entry. Silenced cells showed a significant reduction of intracellular (92.4% and extracellular viral RNA load (71.4% compared to non-silenced cells. Flow cytometry analysis showed a marked reduction of infected cells (89.7% in silenced HepG2 cells compared to non-silenced cells. Furthermore, the ability to generate infectious virions using the plaque assay was reduced 1.07 log in silenced HepG2 cells. CONCLUSIONS/SIGNIFICANCE: Silencing the attachment receptor and clathrin-mediated endocytosis using siRNA could inhibit dengue virus entry and multiplication into HepG2 cells. This leads to reduction of infected cells as well as the viral load, which might function as a unique and promising therapeutic agent for attenuating dengue infection and prevent the development of dengue fever to the severe life-threatening DHF or DSS

  10. SAT in silence.

    Science.gov (United States)

    Brockdorff, Neil

    2009-04-01

    X chromosome inactivation triggered by Xist RNA can only occur in specific developmental contexts. In this issue of Developmental Cell, Agrelo et al. show that the nuclear matrix protein SATB1 is a critical determinant of Xist responsiveness. PMID:19386254

  11. An image-based, dual fluorescence reporter assay to evaluate the efficacy of shRNA for gene silencing at the single-cell level [v1; ref status: indexed, http://f1000r.es/2tt

    Directory of Open Access Journals (Sweden)

    Shin-ichiro Kojima

    2014-02-01

    Full Text Available RNA interference (RNAi is widely used to suppress gene expression in a specific manner. The efficacy of RNAi is mainly dependent on the sequence of small interfering RNA (siRNA in relation to the target mRNA. Although several algorithms have been developed for the design of siRNA, it is still difficult to choose a really effective siRNA from among multiple candidates. In this article, we report the development of an image-based, quantitative, ratiometric fluorescence reporter assay to evaluate the efficacy of RNAi at the single-cell level. Two fluorescence reporter constructs are used. One expresses the candidate small hairpin RNA (shRNA together with an enhanced green fluorescent protein (EGFP; the other expresses a 19-nt target sequence inserted into a cassette expressing a red fluorescent protein (either DsRed or mCherry. Effectiveness of the candidate shRNA is evaluated as the extent to which it knocks down expression of the red fluorescent protein. Thus, the red-to-green fluorescence intensity ratio (appropriately normalized to controls is used as the read-out for quantifying the siRNA efficacy at the individual cell level. We tested this dual fluorescence assay and compared predictions to actual endogenous knockdown levels for three different genes (vimentin, lamin A/C and Arp3 and twenty different shRNAs. For each of the genes, our assay successfully predicted the target sequences for effective RNAi. To further facilitate testing of RNAi efficacy, we developed a negative selection marker (ccdB method for construction of shRNA and red fluorescent reporter plasmids that allowed us to purify these plasmids directly from transformed bacteria without the need for colony selection and DNA sequencing verification.

  12. An image-based, dual fluorescence reporter assay to evaluate the efficacy of shRNA for gene silencing at the single-cell level [v2; ref status: indexed, http://f1000r.es/39j

    Directory of Open Access Journals (Sweden)

    Shin-ichiro Kojima

    2014-05-01

    Full Text Available RNA interference (RNAi is widely used to suppress gene expression in a specific manner. The efficacy of RNAi is mainly dependent on the sequence of small interfering RNA (siRNA in relation to the target mRNA. Although several algorithms have been developed for the design of siRNA, it is still difficult to choose a really effective siRNA from among multiple candidates. In this article, we report the development of an image-based, quantitative, ratiometric fluorescence reporter assay to evaluate the efficacy of RNAi at the single-cell level. Two fluorescence reporter constructs are used. One expresses the candidate small hairpin RNA (shRNA together with an enhanced green fluorescent protein (EGFP; the other expresses a 19-nt target sequence inserted into a cassette expressing a red fluorescent protein (either DsRed or mCherry. Effectiveness of the candidate shRNA is evaluated as the extent to which it knocks down expression of the red fluorescent protein. Thus, the red-to-green fluorescence intensity ratio (appropriately normalized to controls is used as the read-out for quantifying the siRNA efficacy at the individual cell level. We tested this dual fluorescence assay and compared predictions to actual endogenous knockdown levels for three different genes (vimentin, lamin A/C and Arp3 and twenty different shRNAs. For each of the genes, our assay successfully predicted the target sequences for effective RNAi. To further facilitate testing of RNAi efficacy, we developed a negative selection marker (ccdB method for construction of shRNA and red fluorescent reporter plasmids that allowed us to purify these plasmids directly from transformed bacteria without the need for colony selection and DNA sequencing verification.

  13. Post-transcriptional gene silencing signal could move rapidly and bidirectionally in grafted Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    LI Ming; JIANG Shiling; WANG Youqun; LIU Guoqin

    2006-01-01

    RNA interference (RNAi), one of the newly found ways for post-transcriptional gene silencing, has been widely used to investigate gene functions through transgenic methods for introducing an RNA silencing signal into plants. In the present study, we constructed a dexamethazone (DEX)-inducible RNAi binary vector harboring a specific sequence fragment (168-bp) homologous to KatB and KatC, two kinesin isoform genes of Arabidopsis, which were proved to result in the post-transcriptional gene silencing of KatB and KatC in DEX-induced transgenic plants. RT-PCR and Northern blot analysis on transgenic homozygous Arabidopsis (termed as RNAi-type plants) showed that DEX inducement causes KatB and KatC mRNA degradation. With a simplified method, Arabidopsis grafting was effectively performed between RNAi-type and wild-type lines. The target gene mRNA levels were tested based on semi-quantitative RT-PCR. Our results demonstrateed that DEX-induced gene silencing signals could result in a reduction in KatB and KatC mRNA in the wild-type rootstocks or scions, indicating that silencing signals of RNAi could be transmitted bidirectionally across the graft junction whether RNAi-plants were scions or stocks. In contrast to the previously reported results on grafted tobacco, the transmission of post- transcriptional gene silencing signals caused by RNAi in grafted Arabidopsis is more effective than that in tobacco.

  14. Hepatitis C Virus (HCV) NS5B Nonnucleoside Inhibitors Specifically Block Single-Stranded Viral RNA Synthesis Catalyzed by HCV Replication Complexes In Vitro▿

    OpenAIRE

    Yang, Wengang; Sun, Yongnian; Phadke, Avinash; Deshpande, Milind; Huang, Mingjun

    2006-01-01

    Replication complexes of hepatitis C virus synthesized two major species of viral RNA in vitro, double stranded and single stranded. NS5B nonnucleoside inhibitors inhibited dose dependently the synthesis of single-stranded RNA but not double-stranded RNA. Moreover, replication complexes carrying a mutation resistant to a nonnucleoside inhibitor lost their susceptibilities to the inhibitor.

  15. RNA interference: an exciting new target validation tool of drug action and therapeutic approach on cardiovascular diseases

    Institute of Scientific and Technical Information of China (English)

    HeMing

    2005-01-01

    RNA interference (RNAi) is an evolutionarily conserved mechanism for silencing gene expression by targeted degradation of mRNA. Short double-stranded RNAs, known as small interfering RNAs (siRNA), are incorporated into an RNA-induced silencing complex that directs degradation of RNA containing a homologous sequence.

  16. A combinatorial code for splicing silencing: UAGG and GGGG motifs.

    Directory of Open Access Journals (Sweden)

    Kyoungha Han

    2005-05-01

    Full Text Available Alternative pre-mRNA splicing is widely used to regulate gene expression by tuning the levels of tissue-specific mRNA isoforms. Few regulatory mechanisms are understood at the level of combinatorial control despite numerous sequences, distinct from splice sites, that have been shown to play roles in splicing enhancement or silencing. Here we use molecular approaches to identify a ternary combination of exonic UAGG and 5'-splice-site-proximal GGGG motifs that functions cooperatively to silence the brain-region-specific CI cassette exon (exon 19 of the glutamate NMDA R1 receptor (GRIN1 transcript. Disruption of three components of the motif pattern converted the CI cassette into a constitutive exon, while predominant skipping was conferred when the same components were introduced, de novo, into a heterologous constitutive exon. Predominant exon silencing was directed by the motif pattern in the presence of six competing exonic splicing enhancers, and this effect was retained after systematically repositioning the two exonic UAGGs within the CI cassette. In this system, hnRNP A1 was shown to mediate silencing while hnRNP H antagonized silencing. Genome-wide computational analysis combined with RT-PCR testing showed that a class of skipped human and mouse exons can be identified by searches that preserve the sequence and spatial configuration of the UAGG and GGGG motifs. This analysis suggests that the multi-component silencing code may play an important role in the tissue-specific regulation of the CI cassette exon, and that it may serve more generally as a molecular language to allow for intricate adjustments and the coordination of splicing patterns from different genes.

  17. Apparent ploidy effects on silencing are post-transcriptional at HML and telomeres in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Jenny M McLaughlan

    Full Text Available The repression of genes in regions of heterochromatin is known as transcriptional silencing. It occurs in a wide range of organisms and can have importance in adaptation to the environment, developmental changes and disease. The model organism Saccharomyces cerevisiae has been used for many years to study transcriptional silencing, but until recently no study has been made in relation to ploidy. The aim of this work was to compare transcriptional silencing in haploids and diploids at both telomeres and the hidden mating-type (HM loci. Transcriptional silencing was assayed, by growth on 5-fluoroorotic acid (5-FOA media or by flow cytometry, on strains where a telomere or HM locus was marked. RNA levels were measured by quantitative RT-PCR to confirm that effects were transcriptional. 5-FOA assays and flow cytometry were consistent with transcriptional silencing at telomeres and at HML being reduced as ploidy increases which agreed with conclusions in previous publications. However, QRT-PCR revealed that transcriptional silencing was unaffected by ploidy and thus protein levels were increasing independently of RNA levels. At telomere XI left (XI-L, changes in protein level were strongly influenced by mating-type, whereas at HML mating-type had much less influence. The post-transcriptional effects seen in this study, illustrate the often ignored need to measure RNA levels when assaying transcriptional silencing in Saccharomyces cerevisiae.

  18. Offeree silence and contractual agreement.

    OpenAIRE

    Austen-Baker, Richard

    2006-01-01

    This article considers the mechanics of acceptance in the law of contract, focusing on the question of whether the silence of an offeree can effect acceptance. It finds the rule in Felthouse v Bindley (1862) alive and well, but creating problems for judges anxious to find agreements in place. The article considers whether there is, as sometimes suggested, a ‘duty to speak’ and dismisses this, along with any other supposed exceptions to the rule, concluding that silence cannot effect acceptanc...

  19. Silencing of MicroRNA-21 confers the sensitivity to tamoxifen and fulvestrant by enhancing autophagic cell death through inhibition of the PI3K-AKT-mTOR pathway in breast cancer cells.

    Science.gov (United States)

    Yu, Xinfeng; Li, Ruilian; Shi, Wenna; Jiang, Tao; Wang, Yufei; Li, Cong; Qu, Xianjun

    2016-02-01

    Tamoxifen (TAM) and fulvestrant (FUL) represent the major adjuvant therapy to estrogen receptor-alpha positive (ER(+)) breast cancer patients. However, endocrine resistance to TAM and FUL is a great impediment for successful treatment. We hypothesized that miR-21 might alter the sensitivity of breast cancer cells to TAM or FUL by regulating cell autophagy. Using the ER(+) breast cancer cells, we knockdown miR-21.by transfection with miR-21 inhibitor, then the cells were exposed to TAM or FUL and the percentages of apoptosis and autophagy were determined. Knockdown of miR-21 significantly increased the TAM or FUL-induced apoptosis in ER(+) breast cancer cells. Further, silencing of miR-21 in MCF-7 cells enhanced cell autophagy at both basal and TAM or FUL-induced level. The increase of autophagy in miR-21-knockdown MCF-7 cells was also indicated by increase of beclin-1, LC3-II and increased GFP-LC3 dots. Importantly, knockdown of miR-21 contributed to autophagic cell death, which is responsible for part of TAM induced cell death in miR-21 inhibitor-transfected cells. Further analysis suggested that miR-21 inhibitor enhance autophagic cell death through inhibition of PI3K-AKT-mTOR pathway. MiR-21 coordinated the function of autophagy and apoptosis by targeting Phosphatase and tensin homolog (PTEN) through inhibition of PI3K-AKT-mTOR pathway. In conclusion, silencing of miR-21 increased the sensitivity of ER(+) breast cancer cells to TAM or FUL by increasing autophagic cell death. Targeting autophagy-related miRNAs is a potential strategy for overcoming endocrine resistance to TAM and FUL.

  20. Post-transcriptional Gene Silencing Induced by Short Interfering RNAs in Cultured Transgenic Plant Cells

    Institute of Scientific and Technical Information of China (English)

    Wei Tang; Vanessa Samuels; Nicki Whitley; Nicole Bloom; Tinya DeLaGarza; Ronald J. Newton

    2004-01-01

    Short interfering RNA (siRNA) is widely used for studying post-transcriptional gene silencing and holds great promise as a tool for both identifying function of novel genes and validating drug targets. Two siRNA fragments (siRNA-a and -b),which were designed against different specific areas of coding region of the same target green fluorescent protein (GFP) gene, were used to silence GFP expression in cultured gfp transgenic cells of rice (Oryza sativa L.; OS), cotton (Gossypium hirsutum L.; GH), Fraser fir [Abies fraseri (Pursh) Poir; AF], and Virginia pine (Pinus virginiana Mill.; PV). Differential gene silencing was observed in the bombarded transgenic cells between two siRNAs, and these results were consistent with the inactivation of GFP confirmed by laser scanning microscopy, Northern blot,and siRNA analysis in tested transgenic cell cultures. These data suggest that siRNA-mediated gene inactivation can be the siRNA specific in different plant species. These results indicate that siRNA is a highly specific tool for targeted gene knockdown and for establishing siRNA-mediated gene silencing, which could be a reliable approach for large-scale screening of gene function and drug target validation.

  1. Gene transfer engineering for astrocyte-specific silencing in the CNS.

    Science.gov (United States)

    Merienne, N; Delzor, A; Viret, A; Dufour, N; Rey, M; Hantraye, P; Déglon, N

    2015-10-01

    Cell-type-specific gene silencing is critical to understand cell functions in normal and pathological conditions, in particular in the brain where strong cellular heterogeneity exists. Molecular engineering of lentiviral vectors has been widely used to express genes of interest specifically in neurons or astrocytes. However, we show that these strategies are not suitable for astrocyte-specific gene silencing due to the processing of small hairpin RNA (shRNA) in a cell. Here we develop an indirect method based on a tetracycline-regulated system to fully restrict shRNA expression to astrocytes. The combination of Mokola-G envelope pseudotyping, glutamine synthetase promoter and two distinct microRNA target sequences provides a powerful tool for efficient and cell-type-specific gene silencing in the central nervous system. We anticipate our vector will be a potent and versatile system to improve the targeting of cell populations for fundamental as well as therapeutic applications. PMID:26109254

  2. Targeting choline phospholipid metabolism: GDPD5 and GDPD6 silencing decrease breast cancer cell proliferation, migration, and invasion.

    Science.gov (United States)

    Cao, Maria Dung; Cheng, Menglin; Rizwan, Asif; Jiang, Lu; Krishnamachary, Balaji; Bhujwalla, Zaver M; Bathen, Tone F; Glunde, Kristine

    2016-08-01

    Abnormal choline phospholipid metabolism is associated with oncogenesis and tumor progression. We have investigated the effects of targeting choline phospholipid metabolism by silencing two glycerophosphodiesterase genes, GDPD5 and GDPD6, using small interfering RNA (siRNA) in two breast cancer cell lines, MCF-7 and MDA-MB-231. Treatment with GDPD5 and GDPD6 siRNA resulted in significant increases in glycerophosphocholine (GPC) levels, and no change in the levels of phosphocholine or free choline, which further supports their role as GPC-specific regulators in breast cancer. The GPC levels were increased more than twofold during GDPD6 silencing, and marginally increased during GDPD5 silencing. DNA laddering was negative in both cell lines treated with GDPD5 and GDPD6 siRNA, indicating absence of apoptosis. Treatment with GDPD5 siRNA caused a decrease in cell viability in MCF-7 cells, while GDPD6 siRNA treatment had no effect on cell viability in either cell line. Decreased cell migration and invasion were observed in MDA-MB-231 cells treated with GDPD5 or GDPD6 siRNA, where a more pronounced reduction in cell migration and invasion was observed under GDPD5 siRNA treatment as compared with GDPD6 siRNA treatment. In conclusion, GDPD6 silencing increased the GPC levels in breast cancer cells more profoundly than GDPD5 silencing, while the effects of GDPD5 silencing on cell viability/proliferation, migration, and invasion were more severe than those of GDPD6 silencing. Our results suggest that silencing GDPD5 and GDPD6 alone or in combination may have potential as a new molecular targeting strategy for breast cancer treatment. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27356959

  3. shRNA沉默芳香烃受体表达对心肌细胞分化过程中WNT信号通路的影响%Effect of shRNA silenced aromatic hydrocarbon receptor on WNT signaling pathway during the differentiation of cardiac myocytes

    Institute of Scientific and Technical Information of China (English)

    范志刚; 朱春; 余章斌; 胡晓山; 李萌萌; 朱金改; 朱莎莎; 韩树萍

    2013-01-01

      目的观察芳香烃受体(AHR)基因沉默后对P19细胞向心肌分化进程中WNT信号通路的影响。方法根据小鼠AHR基因序列设计并构建AHR基因的shRNA真核表达载体,将AHR干扰质粒转染至P19细胞,通过G418筛选出稳定的AHR基因沉默表达的阳性细胞株,应用实时荧光定量RT-PCR技术检测P19细胞分化过程中WNT信号通路中关键基因GSK3β、β-catenin mRNA的表达水平。结果构建的AHR-shRNA表达质粒能显著抑制AHR基因的表达;随着P19细胞向心肌细胞分化,实验组β-catenin基因的相对表达量低于对照组,而GSK3β基因的相对表达量则高于对照组,差异均有统计学意义(P均<0.01)。结论干扰AHR基因表达对心脏发育过程中WNT信号通路产生调节作用。%Objective To explore the effect of shRNA silenced aromatic hydrocarbon receptor (AHR) on WNT signaling path-way during the differentiation of P19 cells into cardiac myocytes. Methods The eukaryotic expression vector of mouse AHR gene was designed and constructed. The interference plasmid was transfected into P19 cell and the positive stains to AHR gene silencing were screened by G418. The mRNA expression of important genes GSK3βandβ-catenin were evaluated by real-time fluorescent quantita-tive PCR during the differentiation of P19 cells. Results The constructed AHR-shRNA plasmid significantly inhibited the expression of AHR gene. Along with the differentiation of P19 cell into cardiac myocytes, in the interference group the expression ofβ-catenin gene was lower whereas the expression of GSK3βgene was elevated than those of control group with significant differences (all P<0.01). Conclusions The interference of AHR gene expression can regulate WNT signaling pathway in the development of heart.

  4. The effects of age-in-block on RNA-seq analysis of archival formalin-fixed paraffin-embedded (FFPE) samples

    Science.gov (United States)

    Archival samples represent a vast resource for identification of chemical and pharmaceutical targets. Previous use of formalin-fixed paraffin-embedded (FFPE) samples has been limited due to changes in RNA introduced by fixation and embedding procedures. Recent advances in RNA-seq...

  5. Hydrolytic charge-reversal of PEGylated polyplexes enhances intracellular un-packaging and activity of siRNA.

    Science.gov (United States)

    Werfel, Thomas A; Swain, Corban; Nelson, Christopher E; Kilchrist, Kameron V; Evans, Brian C; Miteva, Martina; Duvall, Craig L

    2016-04-01

    Hydrolytically degrading nano-polyplexes (HDG-NPs) that reverse charge through conversion of tertiary amines to carboxylic acids were investigated to improve intracellular un-packaging of siRNA and target gene silencing compared to a non-degradable analog (non-HDG-NPs). Both NP types comprised reversible addition-fragmentation chain-transfer (RAFT) synthesized diblock copolymers of a poly(ethylene glycol) (PEG) corona-forming block and a cationic block for nucleic acid packaging that incorporated butyl methacrylate (BMA) and either dimethylaminoethyl methacrylate (DMAEMA, non-HDG-NPs) or dimethylaminoethyl acrylate (DMAEA, HDG-NPs). HDG-NPs decreased significantly in size and released significantly more siRNA (∼40%) than non-HDG-NPs after 24 h in aqueous solution. While both HDG-NPs and non-HDG-NPs had comparable uptake and cytotoxicity up to 150 nM siRNA doses, HDG-NPs achieved significantly higher target gene silencing of the model gene luciferase in vitro. High resolution FRET confocal microscopy was used to monitor the intracellular un-packaging of siRNA. Non-HDG-NPs had significantly higher FRET efficiency than HDG-NPs, indicating that siRNA delivered from HDG-NPs was more fully un-packaged and therefore had improved intracellular bioavailability.

  6. Antisense Gene Silencing: Therapy for Neurodegenerative Disorders?

    Directory of Open Access Journals (Sweden)

    Troels T. Nielsen

    2013-09-01

    Full Text Available Since the first reports that double-stranded RNAs can efficiently silence gene expression in C. elegans, the technology of RNA interference (RNAi has been intensively exploited as an experimental tool to study gene function. With the subsequent discovery that RNAi could also be applied to mammalian cells, the technology of RNAi expanded from being a valuable experimental tool to being an applicable method for gene-specific therapeutic regulation, and much effort has been put into further refinement of the technique. This review will focus on how RNAi has developed over the years and how the technique is exploited in a pre-clinical and clinical perspective in relation to neurodegenerative disorders.

  7. Silencing leptin with short interfering RNA reveals the role of leptin in rat firrosis%小干扰RNA沉默瘦素揭示了瘦素在大鼠肝纤维化中的作用

    Institute of Scientific and Technical Information of China (English)

    薛秀兰; 邢玉刚; 吴晓鹭; 刘家俊

    2014-01-01

    目的:观察靶向瘦素(leptin)小干扰 RNA(interference ,siRNA)真核细胞表达质粒对大鼠肝纤维化的影响。方法采用已构建的重组 Leptin-siRNA 真核细胞表达质粒,用脂质体包埋法通过腹腔注射将其导入大鼠肝纤维化模型体内。通过 HE染色观察肝脏病理形态学变化;Western Blot 检测 leptin、I 型胶原和 STAT3蛋白表达变化;半定量 RT-PCR 检测leptin mRNA表达。结果与正常对照组相比,肝纤维化组(CCl4)和质粒空载体组(CCl4(+)K)肝纤维化程度较重,纤维化评分多为Ⅳ级(P<0.01),同时肝脏 leptin、I型胶原和 STAT3含量明显上升(P<0.05);而与肝纤维化组(CCl4)和质粒空载体组(CCl4(+)K)比,leptin-siRNA质粒转染组(The CCl4(+)L)肝纤维化程度减轻,纤维化程度多为Ⅰ-Ⅱ级,肝脏 leptin、I型胶原和 STAT3表达均显著抑制(P<0.05)。正常对照组和 leptin-siRNA 质粒组(The CCl4(+)L)在组织学及 leptin、I 型胶原和 STAT3基因转录和表达水平无统计学差异(P>0.05)。结论 leptin-siRNA 表达质粒降低 I 型胶原和 STAT3含量、抑制 leptin表达,阻抑肝纤维化;Leptin有望成为肝纤维化基因治疗的新靶位点。%Obj ective To observe the effects of leptin-siRNA expression plasmids on rat liver fibro-sis.Methods The constructed leptin-siRNA expression plasmids were injected into the abdominal cavity of rat liver fibrosis models by encapsulation with Lipofectmine 2000.The pathological grade was observed by HE staining.Leptin,collagen I and STAT3 were detected by Western blot.The mRNA expression of lep-tin was assessed by semi-RT-PCR .Results The fibrosis model CCl4 and CCl4(+)K groups showed severe fibrosis,including septal fibrosis,extensive bridging,and fatty degeneration.The expressions of leptin, collagen type I and STAT3 in mRNA and protein were also elevated in the livers from these groups(P0.05).Conclusion The leptin-siRNA plasmid can decrease collagen I and STAT3,inhibit

  8. Specific Tandem Repeats Are Sufficient for Paramutation-Induced Trans-Generational Silencing

    OpenAIRE

    Belele, Christiane L.; Lyudmila Sidorenko; Maike Stam; Rechien Bader; Arteaga-Vazquez, Mario A.; Chandler, Vicki L.

    2013-01-01

    Paramutation is a well-studied epigenetic phenomenon in which trans communication between two different alleles leads to meiotically heritable transcriptional silencing of one of the alleles. Paramutation at the b1 locus involves RNA-mediated transcriptional silencing and requires specific tandem repeats that generate siRNAs. This study addressed three important questions: 1) are the tandem repeats sufficient for paramutation, 2) do they need to be in an allelic position to mediate paramutati...

  9. Trans-specific gene silencing between host and parasitic plants.

    Science.gov (United States)

    Tomilov, Alexey A; Tomilova, Natalia B; Wroblewski, Tadeusz; Michelmore, Richard; Yoder, John I

    2008-11-01

    Species of Orobanchaceae parasitize the roots of nearby host plants to rob them of water and other nutrients. Parasitism can be debilitating to the host plant, and some of the world's most pernicious agricultural pests are parasitic weeds. We demonstrate here that interfering hairpin constructs transformed into host plants can silence expression of the targeted genes in the parasite. Transgenic roots of the hemi-parasitic plant Triphysaria versicolor expressing the GUS reporter gene were allowed to parasitize transgenic lettuce roots expressing a hairpin RNA containing a fragment of the GUS gene (hpGUS). When stained for GUS activity, Triphysaria roots attached to non-transgenic lettuce showed full GUS activity, but those parasitizing transgenic hpGUS lettuce lacked activity in root tissues distal to the haustorium. Transcript quantification indicated a reduction in the steady-state level of GUS mRNA in Triphysaria when they were attached to hpGUS lettuce. These results demonstrate that the GUS silencing signal generated by the host roots was translocated across the haustorium interface and was functional in the parasite. Movement across the haustorium was bi-directional, as demonstrated in double-junction experiments in which non-transgenic Triphysaria concomitantly parasitized two hosts, one transgenic for hpGUS and the other transgenic for a functional GUS gene. Observation of GUS silencing in the second host demonstrated that the silencing trigger could be moved from one host to another using the parasite as a physiological bridge. Silencing of parasite genes by generating siRNAs in the host provides a novel strategy for controlling parasitic weeds. PMID:18643992

  10. The donor substrate site within the peptidyl transferase loop of 23 S rRNA and its putative interactions with the CCA-end of N-blocked aminoacyl-tRNA(Phe)

    DEFF Research Database (Denmark)

    Porse, B T; Thi-Ngoc, H P; Garrett, R A

    1996-01-01

    was employed to test for Watson-Crick base-pairing interactions between the -CCA end of the P-site bound tRNA(Phe) and this region of the peptidyl-transferase loop. Single nucleotide substitutions were introduced into the -CCA end of tRNA(Phe) and the ability of the 3'-terminal pentanucleotide fragments to act...

  11. Construction of lentiviral vectors carrying small interference RNA of alpha-fetoprotein gene and its efficiency of target gene silencing%携带甲胎蛋白基因小干扰RNA慢病毒载体的构建及其对靶基因的沉默效率

    Institute of Scientific and Technical Information of China (English)

    程木华; 李建芳; 张峰; 曾凤伟; 谢良骏

    2012-01-01

    Objective To construct lentiviral vectors carrying small interference RNA (siRNA) of alpha-fetoprotein(AFP) followed by transfection into hepatocellular carcinoma cells and to assess the efficiency of AFP gene silencing.Methods The AFP-specific positive siRNA and non-specific negative siRNA were designed and constructed prior to insertion to the recombinant lentiviral vectors carrying green fluorescent protein (GFP) gene.This was followed by transfection into human hepatocellular carcinoma cells HepG2 and subsequent screening of cell strains with GFP expression,and were assigned into lentivirustransfected positive siRNA group and lentivirus-transfected negative siRNA group respectively.Furthermore,liposome-transfected positive siRNA group,liposome-transfected negative siRNA group,siRNA group which treated with AFP siRNA but without transfection reagent,and blank control group were established simultaneously.The efficiency of transfection was assessed under fluorescent microscope,and the relative expression of AFP mRNA and protein in HepG2 cells was determined via fluorescent quantitative polymerase chain reaction and immunoblotting assay.In addition,the inhibition rate of AFP gene expression was compared among all groups.Results Lentiviral vectors were associated with effective transfection of siRNA into HepG2 cells.The siRNA transfected with lentivirus yielded considerably higher inhibition rate of AFP mRNA than that with liposomes,as evidenced by fluorescent quantitative polymerase chain reaction (92.1%vs 74.3%,P<0.05).Immunoblotting assay showed that higher inhibition rate of AFP protein was in favor of siRNA transfected with lentivirus,but not with liposomes (88.2% vs 63.7%,P<0.05).Conclusion AFP siRNA transfected with lentivirus is associated with more effective inhibition of AFP expression in HepG2 cells.%目的 构建携带甲胎蛋白(AFP)基因小干扰RNA(siRNA)的慢病毒载体并转染肝癌细胞,评价其

  12. Branched RNA: A New Architecture for RNA Interference

    Directory of Open Access Journals (Sweden)

    Anna Aviñó

    2011-01-01

    Full Text Available Branched RNAs with two and four strands were synthesized. These structures were used to obtain branched siRNA. The branched siRNA duplexes had similar inhibitory capacity as those of unmodified siRNA duplexes, as deduced from gene silencing experiments of the TNF-α protein. Branched RNAs are considered novel structures for siRNA technology, and they provide an innovative tool for specific gene inhibition. As the method described here is compatible with most RNA modifications described to date, these compounds may be further functionalized to obtain more potent siRNA derivatives and can be attached to suitable delivery systems.

  13. The doublesex splicing enhancer components Tra2 and Rbp1 also repress splicing through an intronic silencer.

    Science.gov (United States)

    Qi, Junlin; Su, Shihuang; Mattox, William

    2007-01-01

    The activation of sex-specific alternative splice sites in the Drosophila melanogaster doublesex and fruitless pre-mRNAs has been well studied and depends on the serine-arginine-rich (SR) splicing factors Tra, Tra2, and Rbp1. Little is known, however, about how SR factors negatively regulate splice sites in other RNAs. Here we examine how Tra2 blocks splicing of the M1 intron from its own transcript. We identify an intronic splicing silencer (ISS) adjacent to the M1 branch point that is sufficient to confer Tra2-dependent repression on another RNA. The ISS was found to function independently of its position within the intron, arguing against the idea that bound repressors function by simply interfering with branch point accessibility to general splicing factors. Conserved subelements of the silencer include five short repeated sequences that are required for Tra2 binding but differ from repeated binding sites found in Tra2-dependent splicing enhancers. The ISS also contains a consensus binding site for Rbp1, and this protein was found to facilitate repression of M1 splicing both in vitro and in Drosophila larvae. In contrast to the cooperative binding of SR proteins observed on the doublesex splicing enhancer, we found that Rbp1 and Tra2 bind to the ISS independently through distinct sequences. Our results suggest that functionally synergistic interactions of these SR factors can cause either splicing activation or repression.

  14. Molecular deregulation induced by silencing of the high mobility group protein A2 gene in retinoblastoma cells

    OpenAIRE

    Venkatesan, Nalini; Krishnakumar, Subramanian; Deepa, Perinkulam Ravi; Deepa, Murali; Khetan, Vikas; Reddy, M. Ashwin

    2012-01-01

    Aim To explore the molecular mechanisms deregulated by high mobility group protein A2 (HMGA2) gene silencing in retinoblastoma (RB) cells. Methods Synthetic anti-HMGA2 short interfering RNA (siRNA) was used to silence the HMGA2 gene in cultured Y79 RB cells that were subjected to whole genome microarray analysis. The expression of differentially regulated key genes was confirmed with quantitative reverse-transcriptase polymerase chain reaction (qRT–PCR) in post-silenced RB cell lines (Y79 and...

  15. Anticipating and blocking HIV-1 escape by second generation antiviral shRNAs

    Directory of Open Access Journals (Sweden)

    Berkhout Ben

    2010-06-01

    Full Text Available Abstract Background RNA interference (RNAi is an evolutionary conserved gene silencing mechanism that mediates the sequence-specific breakdown of target mRNAs. RNAi can be used to inhibit HIV-1 replication by targeting the viral RNA genome. However, the error-prone replication machinery of HIV-1 can generate RNAi-resistant variants with specific mutations in the target sequence. For durable inhibition of HIV-1 replication the emergence of such escape viruses must be controlled. Here we present a strategy that anticipates HIV-1 escape by designing 2nd generation short hairpin RNAs (shRNAs that form a complete match with the viral escape sequences. Results To block the two favorite viral escape routes observed when the HIV-1 integrase gene sequence is targeted, the original shRNA inhibitor was combined with two 2nd generation shRNAs in a single lentiviral expression vector. We demonstrate in long-term viral challenge experiments that the two dominant viral escape routes were effectively blocked. Eventually, virus breakthrough did however occur, but HIV-1 evolution was skewed and forced to use new escape routes. Conclusion These results demonstrate the power of the 2nd generation RNAi concept. Popular viral escape routes are blocked by the 2nd generation RNAi strategy. As a consequence viral evolution was skewed leading to new escape routes. These results are of importance for a deeper understanding of HIV-1 evolution under RNAi pressure.

  16. Switching off small RNA regulation with trap-mRNA

    DEFF Research Database (Denmark)

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

    2009-01-01

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

  17. Construction of lentiviral vector of shRNA specific for rat PAX2 and the silencing effect%大鼠PAX2基因RNA干扰慢病毒载体的构建及沉默效果

    Institute of Scientific and Technical Information of China (English)

    李里; 吴玉斌

    2014-01-01

    Objective To observe the influence of lentiviral vector-mediated RNA interference on expression of rat PAX 2 gene in rat normal renal tubular epithelial cells (NRK52E).Methods The effective RNA interference sequence targeting PAX 2 gene was screened and determined according to previous study.The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed,synthesized and cloned into pGCSIL-GFP vector to construct a lentiviral vector which expressed PAX 2 shRNA,subsequently confirmed by PCR and DNA sequencing analysis.293 T cells were co-transfected with the plasmids using liposome transfection methods ,and packaged to produce lentiviral particles.The recombinant lentiviruses were transfected into NRK52 E cells, and the PAX2 mRNA and protein expression were examined by RT-PCR and Western blot .The results were compared with those of the non-transfected and blank vector trans-fected NRK52E cells.Results PCR analysis and DNA sequencing confirmed that the PAX 2 shRNA sequences were successfully inserted in-to the lentiviral vectors.After transfection with PAX2 shRNA,the PAX2 expression in NRK52E cells was significantly inhibited at both mR-NA and protein levels compared with that in non-transfected and empty vector transfected NRK 52E cells.Conclusions The lentiviral RNAi vectors targeting PAX2 gene have been successfully constructed ,and can effectively inhibit the expression of PAX 2 gene in NRK52E cells.It paves a way for PAX2-targeted gene therapy of obstructive nephropathy.%目的:构建PAX2特异性shRNA 干扰慢病毒载体并观察其对大鼠肾小管上皮细胞( NRK52E)中PAX2基因的沉默效果。方法采用PAX2基因 RNAi靶点序列合成靶序列的 Oligo DNA,退火形成双链 DNA,与pGCSIL-GFP载体连接产生 shRNA慢病毒载体,经 PCR筛选阳性克隆进行 DNA 测序鉴定。用脂质体转染法将质粒共转染293T 细胞,将包装产生的慢病毒颗粒感染NRK52E

  18. Communicative Silences: Forms and Functions

    Science.gov (United States)

    Bruneau, Thomas J.

    1973-01-01

    The nature of silence is discussed as an imposition of mind, as an interdependent signification ground for speech signs, as a relationship to mental time (as opposed to artificial time), and as it relates to sensation, perception and metaphorical movement. (Author)

  19. Silence in the creative process

    Directory of Open Access Journals (Sweden)

    Francesco Wilhelm

    2015-01-01

    Full Text Available A research about silence as the moment where creative thinking takes place, as an art object and as a way of interpreting artworks, is the main issue of the graduation thesis named Il silenzio nel processo creativo (2014. Here is proposed an offprint, which summarizes its fundamental steps.

  20. Heart Block

    Science.gov (United States)

    ... the signal causes the heart to contract and pump blood. Heart block occurs if the electrical signal is ... degree heart block limits the heart's ability to pump blood to the rest of the body. This type ...

  1. Population Blocks.

    Science.gov (United States)

    Smith, Martin H.

    1992-01-01

    Describes an educational game called "Population Blocks" that is designed to illustrate the concept of exponential growth of the human population and some potential effects of overpopulation. The game material consists of wooden blocks; 18 blocks are painted green (representing land), 7 are painted blue (representing water); and the remaining…

  2. siRNA, miRNA and HIV: promises and challenges

    Institute of Scientific and Technical Information of China (English)

    Man Lung YEUNG; Yamina BENNASSER; Shu Yun LE; Kuan Teh JEANG

    2005-01-01

    Small interfering RNA (siRNA) and microRNA (miRNA) are small RNAs of 18-25 nucleotides (nt) in length that play important roles in regulating gene expression. They are incorporated into an RNA-induced silencing complex (RISC) and serve as guides for silencing their corresponding target mRNAs based on complementary base-pairing.The promise of gene silencing has led many researchers to consider siRNA as an anti-viral tool. However, in long-term settings, many viruses appear to escape from this therapeutical strategy. An example of this may be seen in the case of human immunodeficiency virus type-1 (HIV-1) which is able to evade RNA silencing by either mutating the siRNAtargeted sequence or by encoding for a partial suppressor of RNAi (RNA interference). On the other hand, because miRNA targeting does not require absolute complementarity of base-pairing, mutational escape by viruses from miRNAspecified silencing may be more difficult to achieve. In this review, we discuss stratagems used by various viruses to avoid the cells' antiviral si/mi-RNA defenses and notions of how viruses might control and regulate host cell genes by encoding viral miRNAs (vmiRNAs).

  3. Double-mismatched siRNAs enhance selective gene silencing of a mutant ALS-causing allele

    Institute of Scientific and Technical Information of China (English)

    Chang-ming GENG; Hong-liu DING

    2008-01-01

    Aim: Our previous study demonstrated an siRNA-mediated, allele-specific silenc-ing of mutant genes that cause amyotrophic lateral sclerosis. To improve siRNA design for better therapeutic use of RNA interference, we systematically tested the base-pairing mismatch strategy in the design of asymmetric siRNA. Methods: A naturally symmetric siRNA that targets the human Cu Zn superoxide dismutase G85R mutant allele was modified by placing either 1 or 2 mismatches at the end of the siRNA from position 1 to 4 at each time. The target preference and silencing efficacy of modified siRNA were measured using a modified dual luciferase system. Results: The modification of single base-pairing mismatch successfully achieved the conversion of the siRNA that was originally favored to the antisense of the mutant allele to the one that was favored to the sense strand of the gene. Com-pared to the single-mismatched siRNA, those with double-mismatch at one end demonstrated an increased asymmetry, and thus, an enhanced specificity and efficacy of gene silencing. In addition, the siRNA with double-mismatch at both ends remained in symmetry. Conclusion: Our results suggest the effectiveness of converting a symmetric siRNA to an asymmetric one by introducing mismatches into its structure, and the superiority of double-mismatched siRNA to single-mismatched siRNA in producing selective gene silencing resulting from the dis-ruption of siRNA symmetry. The double-mismatch strategy is an improvement of the single-mismatch method and could be useful in the design of effective siRNAs for the treatment of diseases caused by dominant, gain-of-function gene mutations, such as ALS.

  4. Induction of Silencing in Plants by High-Pressure Spraying of In vitro-Synthesized Small RNAs.

    Science.gov (United States)

    Dalakouras, Athanasios; Wassenegger, Michèle; McMillan, John N; Cardoza, Vinitha; Maegele, Ira; Dadami, Elena; Runne, Miriam; Krczal, Gabi; Wassenegger, Michael

    2016-01-01

    In this report, we describe a method for the delivery of small interfering RNAs (siRNAs) into plant cells. In vitro synthesized siRNAs that were designed to target the coding region of a GREEN FLUORESCENT PROTEIN (GFP) transgene were applied by various methods onto GFP-expressing transgenic Nicotiana benthamiana plants to trigger RNA silencing. In contrast to mere siRNA applications, including spraying, syringe injection, and infiltration of siRNAs that all failed to induce RNA silencing, high pressure spraying of siRNAs resulted in efficient local and systemic silencing of the GFP transgene, with comparable efficiency as was achieved with biolistic siRNA introduction. High-pressure spraying of siRNAs with sizes of 21, 22, and 24 nucleotides (nt) led to local GFP silencing. Small RNA deep sequencing revealed that no shearing of siRNAs was detectable by high-pressure spraying. Systemic silencing was basically detected upon spraying of 22 nt siRNAs. Local and systemic silencing developed faster and more extensively upon targeting the apical meristem than spraying of mature leaves. PMID:27625678

  5. Application of RNA interference in treating human diseases

    Indian Academy of Sciences (India)

    S. Abdolhamid Angaji; Sara Sadate Hedayati; Reihane Hosein Poor; Safoura Madani; Sanaz Samad Poor; Samin Panahi

    2010-12-01

    Gene silencing can occur either through repression of transcription, termed transcriptional gene silencing (TGS), or through translation repression and mRNA degradation, termed posttranscriptional gene silencing (PTGS). PTGS results from sequence-specific mRNA degradation in the cytoplasm without dramatic changes in transcription of corresponding gene in nucleus. Both TGS and PTGS are used to regulate endogenous genes. Interestingly, mechanisms for gene silencing also protect the genome from transposons and viruses. In this paper, we first review RNAi mechanism and then focus on some of its applications in biomedical research such as treatment for HIV, viral hepatitis, cardiovascular and cerebrovascular diseases, metabolic disease, neurodegenerative disorders and cancer.

  6. The effects of silencing of ski gene expression by small interfering RNA on proliferation and cell cycle of human retinal pigment epithelial cells%干扰ski基因对视网膜色素上皮细胞增生和周期的影响

    Institute of Scientific and Technical Information of China (English)

    郭斌; 王莉; 刘晓娟; 范钦华

    2015-01-01

    目的 观察siRNA干扰c-ski对视网膜色素上皮(RPE)细胞增生和周期的影响.方法 RPE细胞分为3组:空白对照组(转染时加脂质体)、阴性对照组(无关序列非特异性siRNA转染)和ski-siRNA转染组,另设阳性对照转染组(采用β-actin siRNA转染).采用蛋白电泳检测转染24h和48 h时的c-ski和Cyclin D1蛋白表达变化;比色法检测转染1、2、3、4d时细胞的增生速率;流式细胞术检测转染24 h和48 h细胞周期的变化.结果 与空白对照组和阴性对照组相比,siRNA转染hRPE细胞增生在各个时间点均受到明显抑制(P<0.05);3组间RPE细胞c-ski和Cyclin D1蛋白表达比较,差异有统计学意义(P<0.05),与空白对照组和阴性对照组相比,c-ski siRNA转染组c-ski、Cyclin D1蛋白表达下降(P<0.05).各组G0/G1、S和G2/M期RPE细胞比例比较,差异有统计学意义(P<0.01),c-ski siRNA组G0/G1期细胞比例高于空白对照组和阴性对照组siRNA组(P<0.05),S期细胞比例低于空白对照组和阴性对照组siRNA组(P<0.05).结论 c-ski基因干扰可以有效地抑制RPE细胞增生,该分子机制可能是抑制RPE细胞G1期的调节子cyclin D,造成G0/G1期阻滞,不能进入S期.%Objective To investigate the effect of silencing c-ski gene with small interfering RNA (siRNA) on cell proliferation and cell cycle of human retinal pigment epithelial (RPE) cells.Methods The RPE cells were divided into 3 groups : blank control group, negative control group and ski siRNA group.Lipofectamine 2000 was utilized to transfect RPE cells in Ski siRNA and control siRNA group.Western blotting was used to detect the protein expressions of ski and Cyclin D1 on 24 h and 48 h after transfected.MTT colorimetric assay and flow cytometry were used to examine cell proliferation on 1 d, 2 d, 3 d and 4 d after transfected and cell cycle on 24 h and 48 h after transfected, respectively.Results The proliferation of RPE cells transfected with ski-siRNA was

  7. A vector library for silencing central carbon metabolism genes with antisense RNAs in Escherichia coli.

    Science.gov (United States)

    Nakashima, Nobutaka; Ohno, Satoshi; Yoshikawa, Katsunori; Shimizu, Hiroshi; Tamura, Tomohiro

    2014-01-01

    We describe here the construction of a series of 71 vectors to silence central carbon metabolism genes in Escherichia coli. The vectors inducibly express antisense RNAs called paired-terminus antisense RNAs, which have a higher silencing efficacy than ordinary antisense RNAs. By measuring mRNA amounts, measuring activities of target proteins, or observing specific phenotypes, it was confirmed that all the vectors were able to silence the expression of target genes efficiently. Using this vector set, each of the central carbon metabolism genes was silenced individually, and the accumulation of metabolites was investigated. We were able to obtain accurate information on ways to increase the production of pyruvate, an industrially valuable compound, from the silencing results. Furthermore, the experimental results of pyruvate accumulation were compared to in silico predictions, and both sets of results were consistent. Compared to the gene disruption approach, the silencing approach has an advantage in that any E. coli strain can be used and multiple gene silencing is easily possible in any combination.

  8. Plasma hydrogenated cationic detonation nanodiamonds efficiently deliver to human cells in culture functional siRNA targeting the Ewing sarcoma junction oncogene.

    Science.gov (United States)

    Bertrand, Jean-Rémi; Pioche-Durieu, Catherine; Ayala, Juan; Petit, Tristan; Girard, Hugues A; Malvy, Claude P; Le Cam, Eric; Treussart, François; Arnault, Jean-Charles

    2015-03-01

    The expression of a defective gene can lead to major cell dysfunctions among which cell proliferation and tumor formation. One promising therapeutic strategy consists in silencing the defective gene using small interfering RNA (siRNA). In previous publications we showed that diamond nanocrystals (ND) of primary size 35 nm, rendered cationic by polyethyleneimine-coating, can efficiently deliver siRNA into cell, which further block the expression of EWS/FLI-1 oncogene in a Ewing sarcoma disease model. However, a therapeutic application of such nanodiamonds requires their elimination by the organism, particularly in urine, which is impossible for 35 nm particles. Here, we report that hydrogenated cationic nanodiamonds of primary size 7 nm (ND-H) have also a high affinity for siRNA and are capable of delivering them in cells. With siRNA/ND-H complexes, we measured a high inhibition efficacy of EWS/FLI-1 gene expression in Ewing sarcoma cell line. Electron microscopy investigations showed ND-H in endocytosis compartments, and especially in macropinosomes from which they can escape before siRNA degradation occurred. In addition, the association of EWS/FLI-1 silencing by the siRNA/ND-H complex with a vincristine treatment yielded a potentiation of the toxic effect of this chemotherapeutic drug. Therefore ND-H appears as a promising delivery agent in anti-tumoral gene therapy. PMID:25662499

  9. ADME studies of [5‐3H]‐2′‐O‐methyluridine nucleoside in mice: a building block in siRNA therapeutics

    OpenAIRE

    Lozac'h, Frederic; Christensen, Jesper; Faller, Thomas; van de Kerkhof, Esther; Krauser, Joel; Garnier, Maxime; Litherland, Karine; Catoire, Alexandre; Natt, Francois; Hunziker, Jurg; Swart, Piet

    2016-01-01

    Abstract The chemical modification 2′‐O‐methyl of nucleosides is often used to increase siRNA stability towards nuclease activities. However, the metabolic fate of modified nucleosides remains unclear. Therefore, the aim of this study was to determine the mass balance, pharmacokinetic, and absorption, distribution, metabolism, and excretion (ADME)‐properties of tritium‐labeled 2′‐O‐methyluridine, following a single intravenous dose to male CD‐1 mice. The single intravenous administration of [...

  10. Long noncoding RNA ANRIL indicates a poor prognosis of gastric cancer and promotes tumor growth by epigenetically silencing of miR-99a/miR-449a.

    Science.gov (United States)

    Zhang, Er-bao; Kong, Rong; Yin, Dan-dan; You, Liang-hui; Sun, Ming; Han, Liang; Xu, Tong-peng; Xia, Rui; Yang, Jin-song; De, Wei; Chen, Jin fei

    2014-04-30

    Long noncoding RNAs are involved in diseases including cancer. Here, we reported that ANRIL (CDKN2B-AS1), a 3.8-kb long noncoding RNA, recruiting and binding to PRC2, was generally upregulated in human gastric cancer (GC) tissues. In a cohort of 120 GC patients, the higher expression of ANRIL was significantly correlated with a higher TNM stage (P=0.041) and tumor size (P=0.001). Multivariate analyses revealed that ANRIL expression served as an independent predictor for overall survival (P=0.036). Further experiments revealed that ANRIL knockdown significantly repressed the proliferation both in vitro and in vivo. We also showed that E2F1 could induce ANRIL and ANRIL-mediated growth promotion is in part due to epigenetic repression of miR-99a/miR-449a in Trans (controlling the targets--mTOR and CDK6/E2F1 pathway) by binding to PRC2, thus forming a positive feedback loop, continuing to promote GC cell proliferation. To our knowledge, this is the first report showed that the role of ANRIL in the progression of GC and ANRIL could crosstalk with microRNAs in epigenetic level. Our results suggest that ANRIL, as a growth regulator, may serve as a candidate prognostic biomarker and target for new therapies in human gastric cancer. PMID:24810364

  11. 利用RNAi技术沉默东方粘虫V-ATP酶H亚基研究%Silencing of V-ATPase Subunit H of Mythimna separata through RNA Interference

    Institute of Scientific and Technical Information of China (English)

    张淑静; 王高振; 刘爽; 吴文君; 祁志军

    2015-01-01

    昆虫V ATP酶(vacular-type ATPase)是以ATP为能量跨膜转运H+的质子泵,对其亚基进行干扰都会影响ATPase的活性,进而影响昆虫的生理生化指标.本研究利用特异性引物通过RT-PCR法扩增东方粘虫(Mythimna sepa rata)V-ATPase H亚基的2个片段,合成相应dsRNA,通过显微注射导入3龄粘虫体内,观察试虫表型及存活情况,RT PCR检测试虫体内H亚基的表达.结果表明,注射粘虫V-ATPase H亚基的dsRNA能够显著降低粘虫中肠V-ATPase H亚基的表达水平,直至导致目的基因沉默和粘虫死亡.本项研究成功实现东方粘虫体内V-ATPase H亚基基因沉默.

  12. Blocking the Wnt/β-Catenin Pathway by Lentivirus-Mediated Short Hairpin RNA Targeting β-Catenin Gene Suppresses Silica-Induced Lung Fibrosis in Mice

    OpenAIRE

    Xin Wang; Wujing Dai; Yanrang Wang; Qing Gu; Deyi Yang; Ming Zhang

    2015-01-01

    Silicosis is a form of occupational lung disease caused by inhalation of crystalline silica dust. While the pathogenesis of silicosis is not clearly understood, the Wnt/β-catenin signaling pathway is thought to play a major role in lung fibrosis. To explore the role of Wnt/β-catenin pathway in silicosis, we blocked Wnt/β-catenin pathway both in silica-treated MLE-12 cells (a mouse pulmonary epithelial cell line) and in a mouse silicosis model by using a lentiviral vector expressing a short ha...

  13. Silencing of the rotavirus NSP4 protein decreases the incidence of biliary atresia in murine model.

    Directory of Open Access Journals (Sweden)

    Jiexiong Feng

    Full Text Available Biliary atresia is a common disease in neonates which causes obstructive jaundice and progressive hepatic fibrosis. Our previous studies indicate that rotavirus infection is an initiator in the pathogenesis of experimental biliary atresia (BA through the induction of increased nuclear factor-kappaB and abnormal activation of the osteopontin inflammation pathway. In the setting of rotavirus infection, rotavirus nonstructural protein 4 (NSP4 serves as an important immunogen, viral protein 7 (VP7 is necessary in rotavirus maturity and viral protein 4 (VP4 is a virulence determiner. The purpose of the current study is to clarify the roles of NSP4, VP7 and VP4 in the pathogenesis of experimental BA. Primary cultured extrahepatic biliary epithelia were infected with Rotavirus (mmu18006. Small interfering RNA targeting NSP4, VP7 or VP4 was transfected before rotavirus infection both in vitro and in vivo. We analyzed the incidence of BA, morphological change, morphogenesis of viral particles and viral mRNA and protein expression. The in vitro experiments showed NSP4 silencing decreased the levels of VP7 and VP4, reduced viral particles and decreased cytopathic effect. NSP4-positive cells had strongly positive expression of integrin subunit α2. Silencing of VP7 or VP4 partially decreased epithelial injury. Animal experiments indicated after NSP4 silencing, mouse pups had lower incidence of BA than after VP7 or VP4 silencing. However, 33.3% of VP4-silenced pups (N = 6 suffered BA and 50% of pups (N = 6 suffered biliary injury after VP7 silencing. Hepatic injury was decreased after NSP4 or VP4 silencing. Neither VP4 nor VP7 were detected in the biliary ducts after NSP4. All together, NSP4 silencing down-regulates VP7 and VP4, resulting in decreased incidence of BA.

  14. Reversal effect of short hairpin RNA targeting forkhead box protein Q1 gene silencing on epithelial-mesenchymal transition in bladder cancer%短发夹RNA靶向沉默叉头框蛋白Q1基因对膀胱癌上皮间质转化的逆转作用

    Institute of Scientific and Technical Information of China (English)

    朱智能; 朱朝辉; 庞自力; 兰东阳; 王海鹏

    2013-01-01

    Objective To investigate the reversal effect of epithelial-mesenchymal transition (EMT) by silencing transcription factor forkhead box protein Q1 (FOXQ1) in human bladder cancer cell line T24,and study its role in mvasion and metastasis of T24 cells.Methods Short hairpin RNA (shRNA) eukaryotic expression vector (FOXQ1-shRNA) targeting human FOXQ1 gene was transfected into T24 cells with high metastatic potential by lipofectamine 2000.The expression of FOXQ1 and epithelial mesenchymal markers (E-Cadherin,N-Cadherin,vimentin) was detected by using reverse transcriptionpolymerase chain reaction (RT-PCR) and Westem blotting after transfection for 48 h.The migration and metastatic potentials of T24 cells were examined by cell wound model and Transwell chamber assay in vitro respectively,and the proliferation of T24 cells was determined by using MTT assay.Acridine orange/ethidium bromide (AO/EB) fluorescent staining was performed to detect apoptosis.Results In the FOXQ1shRNA transfection group,the expression of FOXQ1,N-Cadherin and Vimentin was decreased in the T24 cells remarkably,and the expression of E-Cadherin was significantly up-regulated (P < 0.05).The morphology of T24 cells was transformed into a normal epithelial phenotype,and the motility and invasion of T24 cells [(15.4± 1.4)% vs (76.5 ± 1.1)%,P < 0.05] were inhibited,the abilities of proliferation were inhibited significantly (57.8% vs 82.7%,P< 0.05) in vitro,and apoptosis rate (37.6% vs 14.2%,P < 0.05) was increased notablely as compared with the negative control group (NC-shRNA).Conclusion The silencing of FOXQ1 may reverse mesenchymal morphology into a normal epithelial phenotype,inhibit cancer cell migration and invasion,and suppress tumor metastatic potential.%目的 观察转录因子叉头框蛋白Q1(FOXQl)基因沉默对膀胱癌T24细胞上皮间质转化(EMT)的逆转,探讨其促进肿瘤侵袭转移的作用机制.方法 构建针对FOXQ1基因的短发卡RNA(shRNA)

  15. Endocytic pathway mediates refractoriness of insect Bactrocera dorsalis to RNA interference.

    Science.gov (United States)

    Li, Xiaoxue; Dong, Xiaolong; Zou, Cong; Zhang, Hongyu

    2015-01-01

    RNA interference (RNAi) is a powerful and convenient tool for sequence-specific gene silencing, and it is triggered by double-stranded RNA (dsRNA). RNAi can be easily achieved in many eukaryotes by either injecting or feeding dsRNAs. This mechanism has demonstrated its potential in fundamental research on genetics, medicine and agriculture. However, the possibility that insects might develop refractoriness to RNAi remains unexplored. In this study, we report that the oriental fruit fly, Bactrocera dorsalis, became refractory to RNAi using orally administered dsRNA targeting endogenous genes. Furthermore, refractoriness to RNAi is not gene-specific, and its duration depends on the dsRNA concentration. RNAi blockage requires the endocytic pathway. Fluorescence microscopy indicated that in RNAi refractory flies, dsRNA uptake is blocked. Genes involved in the entry of dsRNAs into cells, including chc, cog3, light and others, are down-regulated in RNAi refractory flies. Increasing the endocytic capacity by improving F-actin polymerization disrupts RNAi refractoriness after both primary and secondary dsRNA exposures. Our results demonstrate that an insect can become refractory to RNAi by preventing the entry of dsRNA into its cells. PMID:25731667

  16. Silencing of CXCR2 and CXCR7 protects against esophageal cancer.

    Science.gov (United States)

    Wu, Kai; Cui, Lingling; Yang, Yang; Zhao, Jia; Zhu, Dengyan; Liu, Donglei; Zhang, Chunyang; Qi, Yu; Li, Xiangnan; Li, Weihao; Zhao, Song

    2016-01-01

    This study was aimed to investigate the functional roles of cytokine receptor (CXCR) CXCR2 and CXCR7 in esophageal cancer (EC). Specific small interfering RNAs (siRNA) against CXCR2 and CXCR7 were transfected into EC cell lines TE-1, EC9706, and EC109 cells. Expression of CXCR2 and CXCR7 was validated, along with cell viability, chemotaxis, apoptosis rate, and ERK1/2 pathways associated protein after transfection. Moreover, EC9706 cells treated with or without CXCR2/7 siRNA were injected into athymic nude mice. Tumor volumes were measured. Besides, immunohistochemical (IHC) staining was performed to investigate the expression of CXCR2/7 in adjacent normal tissues and tumor tissues from esophageal squamous cell carcinoma (ESCC) patients. Also, the associations between CXCR2/7 expression and clinicopathological features and progression were explored. The mRNA levels of CXCR2 and CXCR7 were significantly reduced after transfection. Silencing of CXCR2 and CXCR7 statistically decreased cell viability and chemotaxis, and increased apoptotic rate. Cells invasion was significantly reduced by silencing of CXCR2, however, no significance was found in silencing of CXCR7. The protein levels of pERK1/2 were significantly decreased by silencing of CXCR2 and CXCR7. Besides, silencing of CXCR2 and CXCR7 significantly reduced tumor growth in vivo, and associated with clinicopathological features and progression. Silencing of CXCR2 and CXCR7 protects against EC by inhibiting cell growth and chemotaxis, and inducing apoptosis though ERK1/2 pathways. Silencing of CXCR2 and CXCR7 has potentially therapeutic target for EC. PMID:27648130

  17. Specific tandem repeats are sufficient for paramutation-induced trans-generational silencing.

    Directory of Open Access Journals (Sweden)

    Christiane L Belele

    Full Text Available Paramutation is a well-studied epigenetic phenomenon in which trans communication between two different alleles leads to meiotically heritable transcriptional silencing of one of the alleles. Paramutation at the b1 locus involves RNA-mediated transcriptional silencing and requires specific tandem repeats that generate siRNAs. This study addressed three important questions: 1 are the tandem repeats sufficient for paramutation, 2 do they need to be in an allelic position to mediate paramutation, and 3 is there an association between the ability to mediate paramutation and repeat DNA methylation levels? Paramutation was achieved using multiple transgenes containing the b1 tandem repeats, including events with tandem repeats of only one half of the repeat unit (413 bp, demonstrating that these sequences are sufficient for paramutation and an allelic position is not required for the repeats to communicate. Furthermore, the transgenic tandem repeats increased the expression of a reporter gene in maize, demonstrating the repeats contain transcriptional regulatory sequences. Transgene-mediated paramutation required the mediator of paramutation1 gene, which is necessary for endogenous paramutation, suggesting endogenous and transgene-mediated paramutation both require an RNA-mediated transcriptional silencing pathway. While all tested repeat transgenes produced small interfering RNAs (siRNAs, not all transgenes induced paramutation suggesting that, as with endogenous alleles, siRNA production is not sufficient for paramutation. The repeat transgene-induced silencing was less efficiently transmitted than silencing induced by the repeats of endogenous b1 alleles, which is always 100% efficient. The variability in the strength of the repeat transgene-induced silencing enabled testing whether the extent of DNA methylation within the repeats correlated with differences in efficiency of paramutation. Transgene-induced paramutation does not require extensive

  18. Silencing of CXCR2 and CXCR7 protects against esophageal cancer

    Science.gov (United States)

    Wu, Kai; Cui, Lingling; Yang, Yang; Zhao, Jia; Zhu, Dengyan; Liu, Donglei; Zhang, Chunyang; Qi, Yu; Li, Xiangnan; Li, Weihao; Zhao, Song

    2016-01-01

    This study was aimed to investigate the functional roles of cytokine receptor (CXCR) CXCR2 and CXCR7 in esophageal cancer (EC). Specific small interfering RNAs (siRNA) against CXCR2 and CXCR7 were transfected into EC cell lines TE-1, EC9706, and EC109 cells. Expression of CXCR2 and CXCR7 was validated, along with cell viability, chemotaxis, apoptosis rate, and ERK1/2 pathways associated protein after transfection. Moreover, EC9706 cells treated with or without CXCR2/7 siRNA were injected into athymic nude mice. Tumor volumes were measured. Besides, immunohistochemical (IHC) staining was performed to investigate the expression of CXCR2/7 in adjacent normal tissues and tumor tissues from esophageal squamous cell carcinoma (ESCC) patients. Also, the associations between CXCR2/7 expression and clinicopathological features and progression were explored. The mRNA levels of CXCR2 and CXCR7 were significantly reduced after transfection. Silencing of CXCR2 and CXCR7 statistically decreased cell viability and chemotaxis, and increased apoptotic rate. Cells invasion was significantly reduced by silencing of CXCR2, however, no significance was found in silencing of CXCR7. The protein levels of pERK1/2 were significantly decreased by silencing of CXCR2 and CXCR7. Besides, silencing of CXCR2 and CXCR7 significantly reduced tumor growth in vivo, and associated with clinicopathological features and progression. Silencing of CXCR2 and CXCR7 protects against EC by inhibiting cell growth and chemotaxis, and inducing apoptosis though ERK1/2 pathways. Silencing of CXCR2 and CXCR7 has potentially therapeutic target for EC. PMID:27648130

  19. Effects ShRNA-mediated Gene Silencing Smad4 on the Myofibrogenesis of C2C12 Myoblasts%ShRNA介导的Smad4基因沉默对C2C12成肌细胞纤维化的影响

    Institute of Scientific and Technical Information of China (English)

    陈始秋; 陈疾忤; 陈世益; 李宏云; 尚西亮; 蒋佳

    2011-01-01

    Objective To study the effect of Smad4-shRNA on the myofibrosis process. Methods (1) The most efficient silencing Smad4-shRNA was designed and used to produce lentivirus-Smad4 shRNA to infect C2C12 myoblasts. The Smad4 expression was examined. (2) According to the model that TGF-β1 induced the myofibrosis process of the C2C12 myoblasts, the lentivirus mediated cells were divided into four groups: group A, B, C, and D, respectively presenting the normal C2C12 cells group, the TGF-β1 induced C2C12 cells group, the lentivirus-mediated cells group and the TGF-β1 induced lentivirus-mediated cells group. Results (1) The mRNA and protein expression of Smad4 showed a lower level in Smad4-shRNAl Lentivirus-mediated C2C12 cells (P 0.05 ) . Conclusion Smad4 down-regulation could effectively suppress myofibrosis induced by TGF-β1, and Smad4 inhibition was a potential method for anti-fibrosis of the injured skeletal muscle.%目的:研究慢病毒介导的Smad4-shRNA对TGF-β1诱导的纤维化进程的影响.方法:(1)设计并选择抑制效能最高的Smad4-shRNA,包装生产慢病毒,转染C2C12成肌细胞,并检测转染后细胞的Smad4表达;(2)根据TGF-β1诱导C2C12成肌细胞向成肌纤维细胞分化的模型,以慢病毒介导的Smad4-shRNA转染细胞,将不同处理的细胞分为A、B、C、D四组,分别为C2C12细胞组、TGF-β1诱导组、C2C12细胞转染组和TGF-β1诱导后转染组.通过荧光Realtime-PCR和Westerblot检测各组collagen Ⅰ和α-SMA表达水平.结果:(1)慢病毒介导Smad4-shRNA1转染C2C12细胞后,其Smad4 mRNA和蛋白表达显著低于未转染组(P<0.05);(2) TGF-β1诱导组α-SMA和Collagen Ⅰ mRNA及蛋白表达均显著高于C2C12细胞组(P<0.05);(3) TGF-β1诱导后转染组α-SMA与collagen Ⅰ mRNA及蛋白表达显著低于TGF-β1诱导组(P<0.05),与C2C12细胞组和C2C12细胞转染组相比则无明显差异(P>0.05).结论:降低Smad4表达能有效抑制成肌细胞及受TGF-β1诱导成肌纤维

  20. Hepatocyte-specific delivery of siRNAs conjugated to novel non-nucleosidic trivalent N-acetylgalactosamine elicits robust gene silencing in vivo.

    Science.gov (United States)

    Rajeev, Kallanthottathil G; Nair, Jayaprakash K; Jayaraman, Muthusamy; Charisse, Klaus; Taneja, Nate; O'Shea, Jonathan; Willoughby, Jennifer L S; Yucius, Kristina; Nguyen, Tuyen; Shulga-Morskaya, Svetlana; Milstein, Stuart; Liebow, Abigail; Querbes, William; Borodovsky, Anna; Fitzgerald, Kevin; Maier, Martin A; Manoharan, Muthiah

    2015-04-13

    We recently demonstrated that siRNAs conjugated to triantennary N-acetylgalactosamine (GalNAc) induce robust RNAi-mediated gene silencing in the liver, owing to uptake mediated by the asialoglycoprotein receptor (ASGPR). Novel monovalent GalNAc units, based on a non-nucleosidic linker, were developed to yield simplified trivalent GalNAc-conjugated oligonucleotides under solid-phase synthesis conditions. Synthesis of oligonucleotide conjugates using monovalent GalNAc building blocks required fewer synthetic steps compared to the previously optimized triantennary GalNAc construct. The redesigned trivalent GalNAc ligand maintained optimal valency, spatial orientation, and distance between the sugar moieties for proper recognition by ASGPR. siRNA conjugates were synthesized by sequential covalent attachment of the trivalent GalNAc to the 3'-end of the sense strand and resulted in a conjugate with in vitro and in vivo potency similar to that of the parent trivalent GalNAc conjugate design.

  1. Construction of TLR4 gene-specific RNA interference recombinant vectors and comparison on their silence effects%TLR4基因RNA干扰载体的构建及其抑制效率比较

    Institute of Scientific and Technical Information of China (English)

    姜泓; 王平忠; 张野; 徐哲; 王九平; 彭梅娟; 黄长形; 白雪帆

    2009-01-01

    目的 构建含有Toll样受体4(TLR4)基因特异性RNA干扰序列的pSliencer4.1-CMV neo载体,比较其对TLR4的抑制效率.方法 通过Ambion公司的在线软件设计TLR4基因(GenBank:NM138554)的RNA干扰(RNAi)序列,按siRNA设计的优化原则筛选靶序列,再选取其中部分序列进行合成,经退火、连接,构建含有TLR4基因特异性RNA干扰序列的pSliencer4.1-CMVneo载体.用脂质体SPort XP-1分别将载体及阳性和阴性对照质粒转染血管内皮细胞系EVC-304细胞,提取细胞总RNA,进行RT-PCR反应,筛选抑制效率较高的载体.结果 共设计出可供选择的序列228条,经BLAST比对后,有30条序列有较好的序列特异性,并根据其在基因中的相对位置选择9条作为siRNA靶序列.RT-PCR结果表明其中有5个载体具有较高的抑制效率,以TS4,TS6,TS8抑制效率最高,分别为89%,90%,88%,其余siRNA的抑制效率均未超过50%.抑制效果较好的5条序列在TLR4基因中的位置分别为444、1 203、1 389、2 774、3 409nt处.结论 成功构建了抑制效率较高的TLR4基因特异性RNA干扰序列的pSliencer4.1-CMV neo载体,筛选出特异性抑制TLR4表达的序列,为TLR4信号转导的研究奠定了基础.

  2. SHARP-2 gene silence by lentiviral-based short hairpin RNA interference prolongs survival time of rat kidney transplant recipients%基于慢病毒的SHARP-2特异的RNA干扰延长肾移植受者大鼠存活时间

    Institute of Scientific and Technical Information of China (English)

    寿张飞; 陈江华; 肖华婴; 徐义芳; 王远志; 杨毅; 姜虹; 王慧萍; 陈莹; 李一文

    2009-01-01

    Objective To investigate if rat enhancer of split- and hairy-related protein-2 (SHARP-2) short hairpin RNA interference (shRNAi) prolongs the survival time of rat kidney transplant recipients. Methods Gene recombinant procedures, transfection and co-transfection were carried out to introduce short hairpin RNA interference sequences target for SHARP-2 into 3rd generation self-inactivated lentiviral-ViraPower packaging mix. Limiting dilution method was used for viral titration. Real-time PCR was employed for quantification of gene expression. Rat kidney transplantation was utilized to investigate the effect of SHARP-2 gene silence on the recipient survival. Results A lentiviral-based shRNAi construct LV-SHARP-2iC showed 84% SHARP-2 gene silence efficiency in normal rat kidney cells. At multiplicity of infection 20, 57% T cells could be transfected by lentivirus with spinoculation method. In activated T cells, SHARP-2 g ene silence resulted in 61.3% and 68.7% reduction of intedeukin 2 (IL-2) and interferon γ (IFN-γ) gene expression. When donor kidney was perfused with 5×107 TU LV-SHARP-2iC, the median survival time prolonged for 4-5 days as compared to blank and scramble control groups. Conclusions A recombinant lentivirus LV-SHARP-2iC that effectively silence SHARP-2 gene expression is constructed successfully, leading to the inhibition of IL-2 and IFN-γ. LV-SHARP-2iC treatment can prolong the survival time of rat kidney transplant recipients.%目的 研究SHARP-2(rat enhancer of split-and hairy-related protein-2)特异性基因沉默后对白细胞介素2(IL-2)和γ干扰素(IFN-γ)基因表达及肾移植受者大鼠存活时间的影响.方法 采用基因重组、转染、共转染等分子生物学方法将针对SHARP-2的短发夹式RNA干扰序列导入到第3代自失活慢病毒载体ViraPower packaging mix,以构建重组慢病毒;有限稀释法确定病毒滴度;实时定量PCR法研究基因表达变化;原位大鼠肾移植模型研究SHARP-2基因

  3. RNAi-induced silencing of embryonic tryptophan oxygenase in the Pyralid moth, Plodia interpunctella

    Directory of Open Access Journals (Sweden)

    Jeffrey A. Fabrick

    2004-05-01

    Full Text Available Gene silencing through the introduction of double-stranded RNA (RNA interference, RNAi provides a powerful tool for the elucidation of gene function in many systems, including those where genomics and proteomics are incomplete. The use of RNAi technology for gene silencing in Lepidoptera has lacked significant attention compared to other systems. To demonstrate that RNAi can be utilized in the lepidopteran, Plodia interpunctella, we cloned a cDNA for tryptophan oxygenase, and showed that silencing of tryptophan oxygenase through RNAi during embryonic development resulted in loss of eye-color pigmentation. The complete amino acid sequence of Plodia tryptophan oxygenase can be accessed through NCBI Protein Database under NCBI Accession # AY427951.

  4. Evidence for large complex networks of plant short silencing RNAs.

    Directory of Open Access Journals (Sweden)

    Daniel MacLean

    Full Text Available BACKGROUND: In plants and animals there are many classes of short RNAs that carry out a wide range of functions within the cell; short silencing RNAs (ssRNAs of 21-25 nucleotides in length are produced from double-stranded RNA precursors by the protein Dicer and guide nucleases and other proteins to their RNA targets through base pairing interactions. The consequence of this process is degradation of the targeted RNA, suppression of its translation or initiation of secondary ssRNA production. The secondary ssRNAs in turn could then initiate further layers of ssRNA production to form extensive cascades and networks of interacting RNA [1]. Previous empirical analysis in plants established the existence of small secondary ssRNA cascade [2], in which a single instance of this event occurred but it was not known whether there are other more extensive networks of secondary sRNA production. METHODOLOGY/PRINCIPAL FINDINGS: We generated a network by predicting targets of ssRNA populations obtained from high-throughput sequencing experiments. The topology of the network shows it to have power law connectivity distribution, to be dissortative, highly clustered and composed of multiple components. We also identify protein families, PPR and ULP1, that act as hubs within the network. Comparison of the repetition of genomic sub-sequences of ssRNA length between Arabidopsis and E.coli suggest that the network structure is made possible by the underlying repetitiveness in the genome sequence. CONCLUSIONS/SIGNIFICANCE: Together our results provide good evidence for the existence of a large, robust ssRNA interaction network with distinct regulatory function. Such a network could have a massive effect on the regulation of gene expression via mediation of transcript levels.

  5. [A gun silencer of a special kind].

    Science.gov (United States)

    Schyma, C; Schyma, P; Milbradt, H

    2000-01-01

    The authors report about a small bore pistol with silencer. In addition, the silencer can be sealed up by a rubber plate. This leads to a false estimation of the shooting distance. Also at close range shots the shot with the silencer and the rubber seal leaves hardly gunshot residues. The bullet wipe persists but microradiography shows his changed morphological composition. The use of the rubber seal leads predominantly to atypical bullet holes. PMID:10829239

  6. The Caenorhabditis elegans RDE-10/RDE-11 complex regulates RNAi by promoting secondary siRNA amplification

    NARCIS (Netherlands)

    Zhang, Chi; Montgomery, Taiowa A; Fischer, Sylvia E J; Garcia, Susana M D A; Riedel, Christian G; Fahlgren, Noah; Sullivan, Christopher M; Carrington, James C; Ruvkun, Gary

    2012-01-01

    BACKGROUND: In nematodes, plants, and fungi, RNAi is remarkably potent and persistent due to the amplification of initial silencing signals by RNA-dependent RNA polymerases (RdRPs). In Caenorhabditis elegans (C. elegans), the interaction between the RNA-induced silencing complex (RISC) loaded with p

  7. Effects of seven RNA silencing suppressors on heterologous expression of green fluorescence protein expression mediated by a plant virus-based system in Nicotiana benthamiana%7种RNA沉默抑制子对植物病毒载体表达系统表达水平的影响

    Institute of Scientific and Technical Information of China (English)

    王盛; 董洁; 曹慜; 穆红珍; 丁国平; 张虹

    2012-01-01

    Objective To test the effects of 7 virus-encoded RNA silencing suppressors (RSSs) for enhancement of a plant virus-based vector system-mediated heterologous expression of green fluorescence protein (GFP) in Nicotiana benthamiana. Methods Seven transient expression vectors for the 7 RSSs were constructed and co-inoculated on the leaves of Nicotiana benthamiana with PVXdt-GFP vector, a novel Potato virus X-based plant expression vector, through agroinfiltration. The protein and mRNA expression levels of the reporter gene GFP in the co-inoculated Nicotiana leaves were examined by Western blotting, ELISA and RT-qPCR to assess the effect of the RSSs for GFP expression enhancement. Results The 7 RSSs differed in the degree and duration of enhancement of heterologous GFP expression, and the pl9 protein of Tomato bushy stunt virus (TBSV) induced the highest expression of GFP. African cassava mosaic virus AC2 protein and Rice yellow mettle virus PI protein produced no obvious enhancement GFP expression. Conclusion Transient co-expression of RSSs suppresses host silencing response to allow high-level and long-term expression of heterologous genes in plant, but the optimal RSS has to be identified for each plant virus-based expression vector system.%目的 探索不同植物病毒RNA沉默抑制子对植物病毒载体表达系统重组蛋白表达水平的作用,为合理高效地利用这一新型的外源基因表达平台奠定基础.方法 构建了7种不同的RNA沉默抑制子瞬时表达载体,以农杆菌渗滤法,与马铃薯X病毒表达载体PVXdt-GFP共侵染寄主植物本明烟,通过对报告基因绿色荧光蛋白(GFP)的荧光观察,并以Western blotting、ELISA 和RT-qPCR等测定GFP在烟草中的表达情况,分析不同RNA沉默抑制子对植物中外源基因表达水平的作用和特点.结果 7种病毒RNA沉默抑制子对外源基因GFP在烟草中表达水平的作用效果和持续时间存在差异,其中,番茄丛矮病毒的P19蛋白的

  8. [E. M. Jellinek's silenced and silencing transgenerational story].

    Science.gov (United States)

    Kelemen, Gábor; Márk, Mónika

    2013-01-01

    Jellinek is a kind of archetypal character for future generations in the field of addiction studies. His implosion in the arena of alcoholism around the age of 50 was an unexpected challenge to medical science. We know very little about his own role models giving an intellectual and moral compass to his pragmatic creativity. More than 30 years has passed since Jellinek's death when an American sociologist Ron Roizen started unearthing his silent story. Roizen discerned that there are a lot of unsaid and muted issues in his personal Hungarian past. Our paper, based on the authors' research in Hungarian archives and other sources reveals that not just Jellinek's personal but his transgenerational narrative has been not-yet-said. This silenced and silencing history appears an unfinished business of acculturation of the family, which started prior to four generations. Authors have been concluding that the issue of religious conversion is a critical point in the process of acculturation. They examine the counter move of loyalty to family values and driving force of assimilation making their story unspeakable.

  9. [E. M. Jellinek's silenced and silencing transgenerational story].

    Science.gov (United States)

    Kelemen, Gábor; Márk, Mónika

    2013-01-01

    Jellinek is a kind of archetypal character for future generations in the field of addiction studies. His implosion in the arena of alcoholism around the age of 50 was an unexpected challenge to medical science. We know very little about his own role models giving an intellectual and moral compass to his pragmatic creativity. More than 30 years has passed since Jellinek's death when an American sociologist Ron Roizen started unearthing his silent story. Roizen discerned that there are a lot of unsaid and muted issues in his personal Hungarian past. Our paper, based on the authors' research in Hungarian archives and other sources reveals that not just Jellinek's personal but his transgenerational narrative has been not-yet-said. This silenced and silencing history appears an unfinished business of acculturation of the family, which started prior to four generations. Authors have been concluding that the issue of religious conversion is a critical point in the process of acculturation. They examine the counter move of loyalty to family values and driving force of assimilation making their story unspeakable. PMID:24443572

  10. RNA interference in Lepidoptera

    DEFF Research Database (Denmark)

    Terenius, Ole; Papanicolaou, Alexie; Garbutt, Jennie S.;

    2011-01-01

    Gene silencing through RNA interference (RNAi) has revolutionized the study of gene function, particularly in non-model insects. However, in Lepidoptera (moths and butterflies) RNAi has many times proven to be difficult to achieve. Most of the negative results have been anecdotal and the positive...... public database at http://insectacentral.org/RNAi will continue to gather information on RNAi experiments. 2010 Elsevier Ltd. All rights reserved....

  11. Delayed translational silencing of ceruloplasmin transcript in gamma interferon-activated U937 monocytic cells: role of the 3' untranslated region

    Science.gov (United States)

    Mazumder, B.; Fox, P. L.

    1999-01-01

    Ceruloplasmin (Cp) is an acute-phase protein with ferroxidase, amine oxidase, and pro- and antioxidant activities. The primary site of Cp synthesis in human adults is the liver, but it is also synthesized by cells of monocytic origin. We have shown that gamma interferon (IFN-gamma) induces the synthesis of Cp mRNA and protein in monocytic cells. We now report that the induced synthesis of Cp is terminated by a mechanism involving transcript-specific translational repression. Cp protein synthesis in U937 cells ceased after 16 h even in the presence of abundant Cp mRNA. RNA isolated from cells treated with IFN-gamma for 24 h exhibited a high in vitro translation rate, suggesting that the transcript was not defective. Ribosomal association of Cp mRNA was examined by sucrose centrifugation. When Cp synthesis was high, i.e., after 8 h of IFN-gamma treatment, Cp mRNA was primarily associated with polyribosomes. However, after 24 h, when Cp synthesis was low, Cp mRNA was primarily in the nonpolyribosomal fraction. Cytosolic extracts from cells treated with IFN-gamma for 24 h, but not for 8 h, contained a factor which blocked in vitro Cp translation. Inhibitor expression was cell type specific and present in extracts of human cells of myeloid origin, but not in several nonmyeloid cells. The inhibitory factor bound to the 3' untranslated region (3'-UTR) of Cp mRNA, as shown by restoration of in vitro translation by synthetic 3'-UTR added as a "decoy" and detection of a binding complex by RNA gel shift analysis. Deletion mapping of the Cp 3'-UTR indicated an internal 100-nucleotide region of the Cp 3'-UTR that was required for complex formation as well as for silencing of translation. Although transcript-specific translational control is common during development and differentiation and global translational control occurs during responses to cytokines and stress, to our knowledge, this is the first report of translational silencing of a specific transcript following cytokine

  12. Bidirectional transfer of RNAi between honey bee and Varroa destructor: Varroa gene silencing reduces Varroa population.

    Science.gov (United States)

    Garbian, Yael; Maori, Eyal; Kalev, Haim; Shafir, Sharoni; Sela, Ilan

    2012-12-01

    The mite Varroa destructor is an obligatory ectoparasite of the honey bee (Apis mellifera) and is one of the major threats to apiculture worldwide. We previously reported that honey bees fed on double-stranded RNA (dsRNA) with a sequence homologous to that of the Israeli acute paralysis virus are protected from the viral disease. Here we show that dsRNA ingested by bees is transferred to the Varroa mite and from mite on to a parasitized bee. This cross-species, reciprocal exchange of dsRNA between bee and Varroa engendered targeted gene silencing in the latter, and resulted in an over 60% decrease in the mite population. Thus, transfer of gene-silencing-triggering molecules between this invertebrate host and its ectoparasite could lead to a conceptually novel approach to Varroa control.

  13. Bidirectional transfer of RNAi between honey bee and Varroa destructor: Varroa gene silencing reduces Varroa population.

    Directory of Open Access Journals (Sweden)

    Yael Garbian

    2012-12-01

    Full Text Available The mite Varroa destructor is an obligatory ectoparasite of the honey bee (Apis mellifera and is one of the major threats to apiculture worldwide. We previously reported that honey bees fed on double-stranded RNA (dsRNA with a sequence homologous to that of the Israeli acute paralysis virus are protected from the viral disease. Here we show that dsRNA ingested by bees is transferred to the Varroa mite and from mite on to a parasitized bee. This cross-species, reciprocal exchange of dsRNA between bee and Varroa engendered targeted gene silencing in the latter, and resulted in an over 60% decrease in the mite population. Thus, transfer of gene-silencing-triggering molecules between this invertebrate host and its ectoparasite could lead to a conceptually novel approach to Varroa control.

  14. Transgene-based anthocyanin hyper-pigmentation as a visual reporter of gene silencing in plants

    Science.gov (United States)

    “Co-suppression” associated loss of flower pigmentation in transgenic petunia plants was one of the first clear indicators of the natural process of RNA-associated gene silencing in plants. We have been exploring the use of genetically engineered anthocyanin over-production in vegetative tissues as...

  15. Effects of siRNA CD31-mediated PECAM-1 gene silencing on proliferation and VEGF expression in vascular endothelial cells%siRNA CD31沉默PECAM-1对鼠源血管内皮瘤细胞VEGF表达的影响

    Institute of Scientific and Technical Information of China (English)

    欧阳金生; 陈成水; 李玉苹; 蔡畅; 叶民; 林全; 陈俊杰

    2014-01-01

    目的:研究siRNA CD31靶向沉默血管内皮细胞中血小板内皮细胞黏附分子1(platelet endothelialcell adhesion molecule 1,PECAM-1)基因对鼠源性血管内皮瘤(murine hemangioendothelioma,EOMA)细胞增殖及其VEGF表达的影响.方法:实验分为裸siRNA CD31组、siRNA CD31-FAM组、稳定阴性对照(SNC)组、空白对照(Opti-Med)组,以阳离子脂质体(RNAi-mate)为载体将化学合成的2'-O-甲基修饰的siRNA CD31转染体外培养的EOMA细胞,以激光共聚焦显微镜观察siRNA CD31的转染效果,以四甲基偶氮唑蓝(MTT)法检测siRNA CD31对EOMA细胞增殖的影响,以RT-PCR、Western blotting分别检测EOMA细胞中PECAM-1、VEGF的表达水平.结果:与SNC组和空白对照组比较,裸siRNA CD31和siRNACD31-FAM转染的EOMA细胞中的PECAM-1 mRNA和蛋白、VEGF mRNA和蛋白的表达量均显著降低(均P<0.01).与SNC组比较,裸siRNA CD31组、siRNA CD31-FAM组的EOMA细胞增殖抑制率明显上升[(18.82±1.46)%、(18.91±2.21)%vs(0.61±1.06)%,均P<0.01].结论:采用siRNA CD31-脂质体复合物沉默EOMA细胞中的PECAM-1基因可抑制VEGF mRNA和蛋白的表达,从而抑制EOMA细胞的增殖.

  16. Bone marrow mesenchymal stem cells with Nogo-66 receptor gene silencing for repair of spinal cord injury

    OpenAIRE

    Li, Zhiyuan; Zhang, Zhanxiu; Zhao, Lili; LI Hui; Wang, Suxia; Shen, Yong

    2014-01-01

    We hypothesized that RNA interference to silence Nogo-66 receptor gene expression in bone marrow mesenchymal stem cells before transplantation might further improve neurological function in rats with spinal cord transection injury. After 2 weeks, the number of neurons and BrdU-positive cells in the Nogo-66 receptor gene silencing group was higher than in the bone marrow mesenchymal stem cell group, and significantly greater compared with the model group. After 4 weeks, behavioral performance ...

  17. TTP and BRF proteins nucleate processing body formation to silence mRNAs with AU-rich elements

    OpenAIRE

    Franks, Tobias M.; Lykke-Andersen, Jens

    2007-01-01

    In mammalian cells, mRNAs with AU-rich elements (AREs) are targeted for translational silencing and rapid degradation. Here we present evidence that in human cells the proteins Tristetraprolin (TTP) and BRF-1 deliver ARE-mRNAs to processing bodies (PBs), cytoplasmic assemblies of mRNAs, and associated factors that promote translational silencing and mRNA decay. First, depletion of endogenous TTP and BRF proteins, or overexpression of dominant-negative mutant TTP proteins, impairs the localiza...

  18. Structure And Gene Silencing Activities of Monovalent And Pentavalent Cationic Lipid Vectors Complexed With Sirna

    Energy Technology Data Exchange (ETDEWEB)

    Bouxsein, N.F.; McAllister, C.S.; Ewert, K.K.; Samuel, C.E.; Safinya, C.R.; /UC, Santa Barbara

    2007-07-03

    Small interfering RNAs (siRNAs) of 19-25 bp mediate the cleavage of complementary mRNA, leading to post-transcriptional gene silencing. We examined cationic lipid (CL)-mediated delivery of siRNA into mammalian cells and made comparisons to CL-based DNA delivery. The effect of lipid composition and headgroup charge on the biophysical and biological properties of CL-siRNA vectors was determined. X-ray diffraction revealed that CL-siRNA complexes exhibited lamellar and inverted hexagonal phases, qualitatively similar to CL-DNA complexes, but also formed other nonlamellar structures. Surprisingly, optimally formulated inverted hexagonal 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) CL-siRNA complexes exhibited high toxicity and much lower target-specific gene silencing than lamellar CL-siRNA complexes even though optimally formulated, inverted hexagonal CL-DNA complexes show high transfection efficiency in cell culture. We further found that efficient silencing required cationic lipid/nucleic acid molar charge ratios (chg) nearly an order of magnitude larger than those yielding efficiently transfecting CL-DNA complexes. This second unexpected finding has implications for cell toxicity. Multivalent lipids (MVLs) require a smaller number of cationic lipids at a given chg of the complex. Consistent with this observation, the pentavalent lipid MVL5 exhibited lower toxicity and superior silencing efficiency over a large range in both the lipid composition and chg when compared to monovalent DOTAP. Most importantly, MVL5 achieved much higher total knockdown of the target gene in CL-siRNA complex regimes where toxicity was low. This property of CL-siRNA complexes contrasts to CL-DNA complexes, where the optimized transfection efficiencies of multivalent and monovalent lipids are comparable.

  19. Effect of siRNA-mediated silencing Bmi-1 gene expression on the proliferation of lung cancer cell line A549 in vitro and in vivo%Bmi-1-siRNA对肺腺癌A549细胞体内外增殖能力的影响

    Institute of Scientific and Technical Information of China (English)

    郑翔宇; 朱杰; 王艺芳; 刘纯青; 刘奔; 杨春辉; 刘丹丹; 孟秀香

    2013-01-01

    sequence was chosen from four Bmi-1 siRNA sequences which were designed by our lab, and one random sequence was chosen as a negative control. In short, the chemically synthesized siRNA and control sequences were connected to a retrovirus expressing vector, pSUPERretro-Neo plasmid, and then transfected into A549 cells. The stably transfected cells were cultured and passed. The level of mRNA and protein of Bmi-1 in A549 cells were assessed by RT-PCR and Western blot respectively. The proliferations of A549 cells in vivo was analyzed with MTT, trypan blue exclusion and plate colony forming methods. Flow cytometry was used for cell cycle analysis. The potency of tumorigenesis was observed in nude mouse through hypodermic inoculation of A549 cells. The expressions of cyclin D, p21/27, p-AKT and PTEN were analyzed by Western blot. Results:Compared to A549-ctr and A549-wt cells, Bmi-1 mRNA and protein levels all signiifcantly reduced in A549-Bmi-1-siRNA cells. Bmi-1-siRNA inhibited the growth, colony formation in vitro and tumorigenesis in vitro of A549 cells, and the interference cells cell cycle arrested in G1 phase. In A549-Bmi-1-siRNA cells, p-AKT and cyclinD1 expression were down-regulated while p21/p27 and PTEN were up-regulated. Conclusion:Silencing Bmi-1 gene inhibits the proliferation of A549 cells through G1 phase arrest, which involves the downregulation of cyclin D/p-AKT and upregulation of p21/p27/PTEN.

  20. Zitongxi Block

    Institute of Scientific and Technical Information of China (English)

    1996-01-01

    @@ Zitongxi Block (Western Zitong Block), is located in Zitong County, northwest of Sichuan Province (as shown on Fig. 8 ). Geologically. it is situated in the Zitong Depression, southwest of the middle Longmenshan faulted and folded belt, covering an area of 1 830 km2. Transportation is very convenient. A crisscross network of highways run through the block and the Baocheng railway is nearby. The climate is moderate. Most area belongs to hilly land with the elevation of 500-600 m.The Tongjiang River runs across the area.

  1. Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents.

    Science.gov (United States)

    Stein, C A; Hansen, J Bo; Lai, Johnathan; Wu, SiJian; Voskresenskiy, Anatoliy; Høg, Anja; Worm, Jesper; Hedtjärn, Maj; Souleimanian, Naira; Miller, Paul; Soifer, Harris S; Castanotto, Daniella; Benimetskaya, Luba; Ørum, Henrik; Koch, Troels

    2010-01-01

    For the past 15-20 years, the intracellular delivery and silencing activity of oligodeoxynucleotides have been essentially completely dependent on the use of a delivery technology (e.g. lipofection). We have developed a method (called 'gymnosis') that does not require the use of any transfection reagent or any additives to serum whatsoever, but rather takes advantage of the normal growth properties of cells in tissue culture in order to promote productive oligonucleotide uptake. This robust method permits the sequence-specific silencing of multiple targets in a large number of cell types in tissue culture, both at the protein and mRNA level, at concentrations in the low micromolar range. Optimum results were obtained with locked nucleic acid (LNA) phosphorothioate gap-mers. By appropriate manipulation of oligonucleotide dosing, this silencing can be continuously maintained with little or no toxicity for >240 days. High levels of oligonucleotide in the cell nucleus are not a requirement for gene silencing, contrary to long accepted dogma. In addition, gymnotic delivery can efficiently deliver oligonucleotides to suspension cells that are known to be very difficult to transfect. Finally, the pattern of gene silencing of in vitro gymnotically delivered oligonucleotides correlates particularly well with in vivo silencing. The establishment of this link is of particular significance to those in the academic research and drug discovery and development communities.

  2. siRNA silencing the expression of HLA-A2 gene in hMSCs decreases the secretion of IFN- γ and IL-2 in cocultured allogeneic human T lymphocytes%siRNA沉默HLA-A2表达的hMSCs对人异体T淋巴细胞分泌IFN-γ和IL-2功能的影响

    Institute of Scientific and Technical Information of China (English)

    郑仕杰; 于洋; 单伟; 郑德宇; 秦书俭; 包翠芬

    2011-01-01

    目的 探讨应用siRNA技术下调HLA-A2表达的人骨髓间充质干细胞(human mesenchymal stem ceIts,hMSCs)对人异体T淋巴细胞分泌功能的调节作用.方法 从成人骨髓中分离和培养hMSCs,并且通过免疫细胞化学检测其表面标志;利用人工合成HLA-A2靶向小分子干扰RNA转染至第3代hMSCs,然后将转染前后的hMSCs,与经植物血凝素(PHA)刺激的人异体T淋巴细胞共同培养.用ELISA分别检测各培养上清液中T淋巴细胞分泌IFN-γ和IL-2的水平.结果 原代培养3d后,约70%hMSCs贴壁生长,7d后细胞迅速增殖,进入对数生长期,14d左右进入平台期,第3代细胞多呈梭形生长,形态比较均一,hMSCs表面抗原CD44、CD166阳性.HLA-A2靶向小分子干扰RNA转染3天后,可见少量细胞死亡,免疫荧光染色可见细胞核和部分胞浆呈阳性表达.转染HLA-A2靶向小分子干扰RNA的和未转染的hMSCs均能抑制人T淋巴细胞分泌IFN-γ和IL-2,转染后的hMSCs抑制作用强于未转染的hMSCs(P<0.05).结论 体外沉默HLA-A2基因表达可增强hMSCs抑制PHA刺激人T淋巴细胞分泌因子.%Objective To investigate the effect of small interfering RNA (siRNA) silencing the expression of HLA-A2 gene in human mesenchymal stem cells(hMSCs) on the secretion of IFN- γ and IL-2 in allogeneic human T lymphocytes cocultured with the hMSCs in vitro. Methods hMSCs from human marrow cells were isolated and expanded,then cultured and purified by sticking plastic bottle. Using synthetic HLA-A2 targeting siRNA transfected into the 3rd generation hMSCs, the levels of INF- γand IL-2 secretion in T lymphocytes in different culture conditions were determined by ELISA after human allogeneic T lymphocyte proliferation stimulated by phytohemagglutinin(PHA). Results 70% hMSCs were adhered to wall following 3 days primary culture, hMSCs were amplified at day 7 and entered into logarithmic phase and into platform at day 14. hMSCs at passage 3 grew fast with spindle

  3. Cytokine-dependent and–independent gene expression changes and cell cycle block revealed in Trypanosoma cruzi-infected host cells by comparative mRNA profiling

    Directory of Open Access Journals (Sweden)

    Burleigh Barbara A

    2009-05-01

    Full Text Available Abstract Background The requirements for growth and survival of the intracellular pathogen Trypanosoma cruzi within mammalian host cells are poorly understood. Transcriptional profiling of the host cell response to infection serves as a rapid read-out for perturbation of host physiology that, in part, reflects adaptation to the infective process. Using Affymetrix oligonucleotide array analysis we identified common and disparate host cell responses triggered by T. cruzi infection of phenotypically diverse human cell types. Results We report significant changes in transcript abundance in T. cruzi-infected fibroblasts, endothelial cells and smooth muscle cells (2852, 2155 and 531 genes respectively; fold-change ≥ 2, p-value T. cruzi-infected fibroblasts and endothelial cells transwell plates were used to distinguish cytokine-dependent and -independent gene expression profiles. This approach revealed the induction of metabolic and signaling pathways involved in cell proliferation, amino acid catabolism and response to wounding as common themes in T. cruzi-infected cells. In addition, the downregulation of genes involved in mitotic cell cycle and cell division predicted that T. cruzi infection may impede host cell cycle progression. The observation of impaired cytokinesis in T. cruzi-infected cells, following nuclear replication, confirmed this prediction. Conclusion Metabolic pathways and cellular processes were identified as significantly altered at the transcriptional level in response to T. cruzi infection in a cytokine-independent manner. Several of these alterations are supported by previous studies of T. cruzi metabolic requirements or effects on the host. However, our methods also revealed a T. cruzi-dependent block in the host cell cycle, at the level of cytokinesis, previously unrecognized for this pathogen-host cell interaction.

  4. Mild and severe cereal yellow dwarf viruses differ in silencing suppressor efficiency of the P0 protein.

    Science.gov (United States)

    Almasi, Reza; Miller, W Allen; Ziegler-Graff, Véronique

    2015-10-01

    Viral pathogenicity has often been correlated to the expression of the viral encoded-RNA silencing suppressor protein (SSP). The silencing suppressor activity of the P0 protein encoded by cereal yellow dwarf virus-RPV (CYDV-RPV) and -RPS (CYDV-RPS), two poleroviruses differing in their symptomatology was investigated. CYDV-RPV displays milder symptoms in oat and wheat whereas CYDV-RPS is responsible for more severe disease. We showed that both P0 proteins (P0(CY-RPV) and P0(CY-RPS)) were able to suppress local RNA silencing induced by either sense or inverted repeat transgenes in an Agrobacterium tumefaciens-mediated expression assay in Nicotiana benthamiana. P0(CY-RPS) displayed slightly higher activity. Systemic spread of the silencing signal was not impaired. Analysis of short-interfering RNA (siRNA) abundance revealed that accumulation of primary siRNA was not affected, but secondary siRNA levels were reduced by both CYDV P0 proteins, suggesting that they act downstream of siRNA production. Correlated with this finding we showed that both P0 proteins partially destabilized ARGONAUTE1. Finally both P0(CY-RPV) and P0(CY-RPS) interacted in yeast cells with ASK2, a component of an E3-ubiquitin ligase, with distinct affinities.

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

    Science.gov (United States)

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

  6. ADME studies of [5-(3)H]-2'-O-methyluridine nucleoside in mice: a building block in siRNA therapeutics.

    Science.gov (United States)

    Lozac'h, Frederic; Christensen, Jesper; Faller, Thomas; van de Kerkhof, Esther; Krauser, Joel; Garnier, Maxime; Litherland, Karine; Catoire, Alexandre; Natt, Francois; Hunziker, Jurg; Swart, Piet

    2016-02-01

    The chemical modification 2'-O-methyl of nucleosides is often used to increase siRNA stability towards nuclease activities. However, the metabolic fate of modified nucleosides remains unclear. Therefore, the aim of this study was to determine the mass balance, pharmacokinetic, and absorption, distribution, metabolism, and excretion (ADME)-properties of tritium-labeled 2'-O-methyluridine, following a single intravenous dose to male CD-1 mice. The single intravenous administration of [5-(3)H]-2'-O-methyluridine was well tolerated in mice. Radioactivity was rapidly and widely distributed throughout the body and remained detectable in all tissues investigated throughout the observation period of 48 h. After an initial rapid decline, blood concentrations of total radiolabeled components declined at a much slower rate. [(3)H]-2'-O-Methyluridine represented a minor component of the radioactivity in plasma (5.89% of [(3)H]-AUC 0-48 h). Three [(3)H]-2'-O-methyluridine metabolites namely uridine (M1), cytidine (M2), and uracil (M3) were the major circulating components representing 32.8%, 8.11%, and 23.6% of radioactivity area under the curve, respectively. The highest concentrations of total radiolabeled components and exposures were observed in kidney, spleen, pineal body, and lymph nodes. The mass balance, which is the sum of external recovery of radioactivity in excreta and remaining radioactivity in carcass and cage wash, was complete. Renal excretion accounted for about 52.7% of the dose with direct renal excretion of the parent in combination with metabolism to the endogenous compounds cytidine, uracil, cytosine, and cytidine. PMID:26977299

  7. EGFR antisense RNA blocks expression of the epidermal growth factor receptor and partially reverse the malignant phenotype of human breast cancer MDA—MB—231 cells

    Institute of Scientific and Technical Information of China (English)

    FANWENHONG; YINGLINLU; 等

    1998-01-01

    The effects of human EGFR to the malignant phenotype of human breast cancer cell line MDA-MB-231 were investigated experimentally.A retroviral vector containing a 5'1350bp fragment of the human EGFR cDNA in the antisense orientation was transfected into targeted cells by lipofectamine.The effects on cell proliferation,cell cycle and adherent ability to extracellular matrix(ECM) components were studied after the expression of antisense transcripts to EGFR5'1350bp fragment in target cells,In vitro studies showed that the growth ability of the transfected cells was partialy inhibited in comparison to parental cells and to cells transfected with the plasmid containing the neomycin resistance gene only.It was found that EGF(10ng/ml) had an augmenation effect on the growth of transfected MDA-AS10 cells but not MDA-MB-231 cells.Flow cytometric analysis showed that the cell cycle of the transfected cells was abnormal with a decrease of cells in G2/M and S phases and an increase of cells in G1 phase,indicating a blockage in phase G1.Immunofluorescence of EGFR expression in transfectants stained with an antiEGFR antibody was decreased and their growth in soft agarose was also severely imparired.The transfected cells showed less adherence to laminin(LN) and fibronectin (FN).In short,EGFR antisense RNA decreases the expression of EGFR on MDA-MB-231 cells and partially reverses their malignant phenotype as well.

  8. Silence in Cross-cultural Communication

    Institute of Scientific and Technical Information of China (English)

    常利娜; 郭芳芳; 郝继亭; 顾庆媛; 孙胜强

    2008-01-01

    In academic field, silence is classified as part of the nonverbal communication. Edward T. Hall regarded silence as a key standard of dividing high-context cultures and low-context cultures (毕 46). In high-context cultures, information is provided through gestures, the use of space, and even silence. Little information is explicitly explained by words. Chinese, Japanese, Native American cultures are high-context ones. However, "in low-context cultures, the verbal message contains most of the information and very little is embedded in the context." (Samovar, A. Larry, Richard E. Porter and Lisa A. Stefani 79). How do people from different cultures respond to silence? How their values affect their attitudes? What causes silence?

  9. In Vivo Imaging of RNA Interference

    OpenAIRE

    Hong, Hao; Zhang, Yin; Cai, Weibo

    2010-01-01

    RNA interference (RNAi), an effective technique for regulating/silencing specific genes, can be applied to treat various diseases. Multiple clinical trials using RNAi are ongoing and molecular imaging can serve as a powerful tool in RNAi-based therapies. This brief review will highlight the current progress on in vivo imaging of RNAi delivery and silencing effects. Incorporation of suitable molecular imaging techniques into future RNAi-based clinical trials will provide more pieces of the puz...

  10. Silencing of Hsp27 and Hsp72 in glioma cells as a tool for programmed cell death induction upon temozolomide and quercetin treatment

    Energy Technology Data Exchange (ETDEWEB)

    Jakubowicz-Gil, Joanna, E-mail: jjgil@poczta.umcs.lublin.pl [Department of Comparative Anatomy and Anthropology, Maria Curie-Sklodowska University, Akademicka 19, 20-033 Lublin (Poland); Langner, Ewa [Department of Medical Biology, Institute of Agricultural Medicine, Jaczewskiego 2, 20-950 Lublin (Poland); Bądziul, Dorota [Department of Comparative Anatomy and Anthropology, Maria Curie-Sklodowska University, Akademicka 19, 20-033 Lublin (Poland); Wertel, Iwona [1st Department of Gynaecology, University School of Medicine, Staszica 16, 20-081 Lublin (Poland); Rzeski, Wojciech [Department of Medical Biology, Institute of Agricultural Medicine, Jaczewskiego 2, 20-950 Lublin (Poland); Department of Immunology and Virology, Maria Curie-Sklodowska University, Akademicka 19, 20-033 Lublin (Poland)

    2013-12-15

    The aim of the present study was to investigate whether silencing of Hsp27 or Hsp72 expression in glioblastoma multiforme T98G and anaplastic astrocytoma MOGGCCM cells increases their sensitivity to programmed cell death induction upon temozolomide and/or quercetin treatment. Transfection with specific siRNA was performed for the Hsp gene silencing. As revealed by microscopic observation and flow cytometry, the inhibition of Hsp expression was correlated with severe apoptosis induction upon the drug treatment studied. No signs of autophagy were detected. This was correlated with a decreased mitochondrial membrane potential, increased level of cytochrome c in the cytoplasm, and activation of caspase 3 and caspase 9. All these results suggest that the apoptotic signal was mediated by an internal pathway. Additionally, in a large percentage of cells treated with temozolomide, with or without quercetin, granules within the ER system were found, which was accompanied by an increased level of caspase 12 expression. This might be correlated with ER stress. Quercetin and temozolomide also changed the shape of nuclei from circular to “croissant like” in both transfected cell lines. Our results indicate that blocking of Hsp27 and Hsp72 expression makes T98G cells and MOGGCCM cells extremely vulnerable to apoptosis induction upon temozolomide and quercetin treatment and that programmed cell death is initiated by an internal signal. - Highlights: • Hsps gene silencing induced severe apoptosis upon temozolomide–quercetin treatment • Apoptosis in transfected glioma cells was initiated by internal signal • Programmed cell death was preceded by ER stress • Temozolomide–quercetin treatment changed nuclei shape in transfected glioma cells.

  11. Silencing BMI1 eliminates tumor formation of pediatric glioma CD133+ cells not by affecting known targets but by down-regulating a novel set of core genes.

    Science.gov (United States)

    Baxter, Patricia A; Lin, Qi; Mao, Hua; Kogiso, Mari; Zhao, Xiumei; Liu, Zhigang; Huang, Yulun; Voicu, Horatiu; Gurusiddappa, Sivashankarappa; Su, Jack M; Adesina, Adekunle M; Perlaky, Laszlo; Dauser, Robert C; Leung, Hon-chiu Eastwood; Muraszko, Karin M; Heth, Jason A; Fan, Xing; Lau, Ching C; Man, Tsz-Kwong; Chintagumpala, Murali; Li, Xiao-Nan

    2014-01-01

    Clinical outcome of children with malignant glioma remains dismal. Here, we examined the role of over-expressed BMI1, a regulator of stem cell self-renewal, in sustaining tumor formation in pediatric glioma stem cells. Our investigation revealed BMI1 over-expression in 29 of 54 (53.7%) pediatric gliomas, 8 of 8 (100%) patient derived orthotopic xenograft (PDOX) mouse models, and in both CD133+ and CD133- glioma cells. We demonstrated that lentiviral-shRNA mediated silencing of suppressed cell proliferation in vitro in cells derived from 3 independent PDOX models and eliminated tumor-forming capacity of CD133+ and CD133- cells derived from 2 PDOX models in mouse brains. Gene expression profiling showed that most of the molecular targets of BMI1 ablation in CD133+ cells were different from that in CD133- cells. Importantly, we found that silencing BMI1 in CD133+ cells derived from 3 PDOX models did not affect most of the known genes previously associated with the activated BMI1, but modulated a novel set of core genes, including RPS6KA2, ALDH3A2, FMFB, DTL, API5, EIF4G2, KIF5c, LOC650152, C20ORF121, LOC203547, LOC653308, and LOC642489, to mediate the elimination of tumor formation. In summary, we identified the over-expressed BMI1 as a promising therapeutic target for glioma stem cells, and suggest that the signaling pathways associated with activated BMI1 in promoting tumor growth may be different from those induced by silencing BMI1 in blocking tumor formation. These findings highlighted the importance of careful re-analysis of the affected genes following the inhibition of abnormally activated oncogenic pathways to identify determinants that can potentially predict therapeutic efficacy.

  12. Exon silencing by UAGG motifs in response to neuronal excitation.

    Directory of Open Access Journals (Sweden)

    Ping An

    2007-02-01

    Full Text Available Alternative pre-mRNA splicing plays fundamental roles in neurons by generating functional diversity in proteins associated with the communication and connectivity of the synapse. The CI cassette of the NMDA R1 receptor is one of a variety of exons that show an increase in exon skipping in response to cell excitation, but the molecular nature of this splicing responsiveness is not yet understood. Here we investigate the molecular basis for the induced changes in splicing of the CI cassette exon in primary rat cortical cultures in response to KCl-induced depolarization using an expression assay with a tight neuron-specific readout. In this system, exon silencing in response to neuronal excitation was mediated by multiple UAGG-type silencing motifs, and transfer of the motifs to a constitutive exon conferred a similar responsiveness by gain of function. Biochemical analysis of protein binding to UAGG motifs in extracts prepared from treated and mock-treated cortical cultures showed an increase in nuclear hnRNP A1-RNA binding activity in parallel with excitation. Evidence for the role of the NMDA receptor and calcium signaling in the induced splicing response was shown by the use of specific antagonists, as well as cell-permeable inhibitors of signaling pathways. Finally, a wider role for exon-skipping responsiveness is shown to involve additional exons with UAGG-related silencing motifs, and transcripts involved in synaptic functions. These results suggest that, at the post-transcriptional level, excitable exons such as the CI cassette may be involved in strategies by which neurons mount adaptive responses to hyperstimulation.

  13. Silencing MicroRNA-155 Ameliorates Experimental Autoimmune Encephalomyelitis

    OpenAIRE

    Murugaiyan, Gopal; Beynon, Vanessa; Mittal, Akanksha; Joller, Nicole; Howard L Weiner

    2011-01-01

    IFN-γ–producing Th1 and IL-17–producing Th17 cells are the key participants in various autoimmune diseases, including multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). Although both of these T cell subsets are known to be regulated by specific transcription factors and cytokines, the role of microRNAs that control these two inflammatory T cell subsets and whether targeting microRNAs can have therapeutic effects are not known. In this study, we show that...

  14. Silencing Huntington's chorea: Is RNA Interference a Potential Cure?

    OpenAIRE

    Metz, Gerlinde A.; Ian Q Whishaw; Afra Foroud; Jadavji, Nafisa M.

    2006-01-01

    In 1872, George Huntington described Huntington's disease as characterized by motor, cognitive and psychiatric impairments. Huntington's disease is a dominant and autosomal mutation on chromosome 4 featuring the insertion of numerous CAG repeats. CAG codes for the amino acid, glutmanine that forms part of the Huntingtin protein (htt). Excess glutamine attachments make htt prone to accumulate in neurons. Three genes can be considered when developing therapies for Huntington's disease. They inc...

  15. Gene silencing by RNAi in mouse Sertoli cells

    Directory of Open Access Journals (Sweden)

    del Mazo Jesús

    2008-07-01

    Full Text Available Abstract Background RNA interference (RNAi is a valuable tool in the investigation of gene function. The purpose of this study was to examine the availability, target cell types and efficiency of RNAi in the mouse seminiferous epithelium. Methods The experimental model was based on transgenic mice expressing EGFP (enhanced green fluorescent protein. RNAi was induced by in vivo transfection of plasmid vectors encoding for short hairpin RNAs (shRNAs targeting EGFP. shRNAs were transfected in vivo by microinjection into the seminiferous tubules via the rete testis followed by square wave electroporation. As a transfection reporter, expression of red fluorescent protein (HcRed 1 was used. Cell types, the efficiency of both transfections and RNAi were all evaluated. Results Sertoli cells were the main transfected cells. A reduction of about 40% in the level of EGFP protein was detected in cells successfully transfected both in vivo and in vitro. However, the efficiency of in vivo transfection was low. Conclusion In adult seminiferous epithelial cells, in vivo post-transcriptional gene silencing mediated by RNAi via shRNA is efficient in Sertoli cells. Similar levels of RNAi were detected both in vivo and in vitro. This also indicates that Sertoli cells have the necessary silencing machinery to repress the expression of endogenous genes via RNAi.

  16. Investigation of transcriptional gene silencing and mechanism induced by shRNAs targeted to RUNX3 in vitro

    Institute of Scientific and Technical Information of China (English)

    Xue-Zhi Feng; Xiu-Sheng He; Ying-Zhi Zhuang; Qiao Luo; Jun-Hao Jiang; Shuai Yang; Xue-Fang Tang; Ju-Lin Liu; Tao Chen

    2008-01-01

    AIM: To investigate transcriptional gene silencing induced by short hairpin RNAs (shRNAs) that target gene prompter regions of RUNX3 gene, and whether shRNAs homologous to DNA sequences may serve as initiators for methylation.METHODS: According to the principle of RNAi design,pSilencer3.1-H1-shRNA/RUNX3 expression vector was constructed, The recombinant plasmid shRNA was transfected into human stomach carcinoma cell line SGC7901 with Lipofectamine 2000. Then, the positive cell clones were screened by G418. The mRNA and protein expression level of RUNX3 in the stable transfected cell line SGC7901 were determined by RT-PCR, Western blotting and immunocytochemistry. Characteristics of the cell lines including SGC7901, pSilencer3.1-H1/SGC7901 and pSilencer3.1-H1-shRNA/RUNX3/SGC7901 were analyzed with growth curves, clone formation rate and cell-cycle distribution. The activated level of RUNX3 was examined after treatment with the different density of 5'-aza-2'-deoxycytidine (5-Aza-CdR) by using semiquantitative RT-PCR and Western blotting.RESULTS: In the cell line SGC7901 transfected with pSilencer3.1-Hl-shRNA/RUNX3, mRNA and protein expression of the RUNX3 gene was lost identified by RTPCR, Western blotting and immunocytochemistry assay.The growth of pSilencer3.1-H1-shRNA/RUNX3/SGC7901cells without expression of RUNX3 was the fastest (P < 0.05), its rate of clone formation was the highest (P <0.01), and the cell distribution in G0/G1 and S/M phases was lowest and highest, respectively (P < 0.05),compared with that of the transfected pSilencer3.1-H1 and non-transfected cells. Through RT-PCR and Western blot assay, inactivated RUNX3 could not be reactivated by 5-Aza-CdR.CONCLUSION: We found that, although shRNAs targeted to gene prompter regions of RUNX3 could effectively induce transcriptional repression with chromatic changes characteristic of inaction promoters, this was independent of DNA methylation, and the presence of RNA-dependent transcriptional silencing

  17. Ghost Block

    OpenAIRE

    Webb, Neil

    2011-01-01

    Filmed on the English south coast 'Ghost Block' depicts the uncanny and eerie atmosphere at the site of a WW2 coastal defence line. The concrete cubes were used as an anti-invasion blockade against potential landing forces. This protection line now slowly decaying and becoming enmeshed into the environment still acts as a defence to repel unwanted visitors. The area is a natural reserve to nesting birds that often lay eggs directly onto the beach surface. The blocks act as a final barrier ...

  18. A cytoplasmic pathway for gapmer antisense oligonucleotide-mediated gene silencing in mammalian cells

    Science.gov (United States)

    Castanotto, Daniela; Lin, Min; Kowolik, Claudia; Wang, LiAnn; Ren, Xiao-Qin; Soifer, Harris S.; Koch, Troels; Hansen, Bo Rode; Oerum, Henrik; Armstrong, Brian; Wang, Zhigang; Bauer, Paul; Rossi, John; Stein, C.A.

    2015-01-01

    Antisense oligonucleotides (ASOs) are known to trigger mRNA degradation in the nucleus via an RNase H-dependent mechanism. We have now identified a putative cytoplasmic mechanism through which ASO gapmers silence their targets when transfected or delivered gymnotically (i.e. in the absence of any transfection reagent). We have shown that the ASO gapmers can interact with the Ago-2 PAZ domain and can localize into GW-182 mRNA-degradation bodies (GW-bodies). The degradation products of the targeted mRNA, however, are not generated by Ago-2-directed cleavage. The apparent identification of a cytoplasmic pathway complements the previously known nuclear activity of ASOs and concurrently suggests that nuclear localization is not an absolute requirement for gene silencing. PMID:26433227

  19. Tumor Selective Silencing Using an RNAi-Conjugated Polymeric Nanopharmaceutical.

    Science.gov (United States)

    Svenson, Sonke; Case, Roy I; Cole, Roderick O; Hwang, Jungyeon; Kabir, Sujan R; Lazarus, Douglas; Lim Soo, Patrick; Ng, Pei-Sze; Peters, Christian; Shum, Pochi; Sweryda-Krawiec, Beata; Tripathi, Snehlata; van der Poll, Derek; Eliasof, Scott

    2016-03-01

    Small interfering RNA (siRNA) therapeutics have potential advantages over traditional small molecule drugs such as high specificity and the ability to inhibit otherwise "undruggable" targets. However, siRNAs have short plasma half-lives in vivo, can induce a cytokine response, and show poor cellular uptake. Formulating siRNA into nanoparticles offers two advantages: enhanced siRNA stability against nuclease degradation beyond what chemical modification alone can provide; and improved site-specific delivery that takes advantage of the enhanced permeability and retention (EPR) effect. Existing delivery systems generally suffer from poor delivery to tumors. Here we describe the formation and biological activity of polymeric nanopharmaceuticals (PNPs) based on biocompatible and biodegradable poly(lactic-co-glycolic acid) (PLGA) conjugated to siRNA via an intracellular cleavable disulfide linker (PLGA-siRNA). Additionally, these PNPs contain (1) PLGA conjugated to polyethylene glycol (PEG) for enhanced pharmacokinetics of the nanocarrier; (2) a cation for complexation of siRNA and charge compensation to avoid high negative zeta potential; and (3) neutral poly(vinyl alcohol) (PVA) to stabilize the PNPs and support the PEG shell to prevent particle aggregation and protein adsorption. The biological data demonstrate that these PNPs achieve prolonged circulation, tumor accumulation that is uniform throughout the tumor, and prolonged tumor-specific knockdown. PNPs employed in this study had no effect on body weight, blood cell count, serum chemistry, or cytokine response at doses >10 times the effective dose. PNPs, therefore, constitute a promising solution for achieving durable siRNA delivery and gene silencing in tumors. PMID:26835715

  20. The gifts of silence and solitude.

    Science.gov (United States)

    Schmidt Bunkers, Sandra

    2008-01-01

    In this column the author describes the importance of finding silence and solitude amid the noise and technology present today in the teaching-learning academy. Three gifts of silence and solitude are identified: the gift of comforting aloneness, the gift of vision for new horizons, and the gift of a sense of freedom. A humanbecoming perspective is used to explore the implications of these gifts. This column introduces a column by Diana Vander Woude describing her teaching-learning experience in leadership focusing on silence and solitude. PMID:18096981

  1. Epidural block

    Science.gov (United States)

    ... Drugs & Supplements Videos & Tools Español You Are Here: Home ... It numbs or causes a loss of feeling in the lower half your body. This lessens the pain of contractions during childbirth. An epidural block may also be used to ...

  2. Induction and maintenance of DNA methylation in plant promoter sequences by apple latent spherical virus-induced transcriptional gene silencing

    OpenAIRE

    Tatsuya eKon; Nobuyuki eYoshikawa

    2014-01-01

    Apple latent spherical virus (ALSV) is an efficient virus-induced gene silencing vector in functional genomics analyses of a broad range of plant species. Here, an Agrobacterium-mediated inoculation (agroinoculation) system was developed for the ALSV vector, and virus-induced transcriptional gene silencing (VITGS) is described in plants infected with the ALSV vector. The cDNAs of ALSV RNA1 and RNA2 were inserted between the CaMV 35S promoter and the NOS-T sequences in a binary vector pCAMBIA1...

  3. MAGNUM OPUS: CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS BIOLOGY AND PROKARYOTIC GENE SILENCING

    Directory of Open Access Journals (Sweden)

    Prem Saran Tirumalai

    2013-01-01

    Full Text Available Gene Silencing was a technology that was established in eukaryotic system a decade ago and is being used as a research tool widely. However, prokaryotic gene silencing was not workable, till recently a team of researchers from the University of Georgia have proved it possible. Where they have shown that short motif sequences determines the targets of the prokaryotic Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR defence system is regulated by RNA guided Cas protein complex. Thus role of CRISPR system in microbial defense against foreign genetic material (Plasmid or Phages is an important milestone in the field of microbial molecular biology/biotechnology. These findings will make it easier to understand the significance of a gene, metabolically or physiologically. The revelation by this novel finding by core group of researcher is indeed, Mangum opus. This article is a commentary, to bring to light, prokaryotic gene silencing as one of the latest advances in prokaryotic science.

  4. Development of Studies on RNA Interference

    Institute of Scientific and Technical Information of China (English)

    Li ZHANG; Yaqiong ZHANG; Lina SHE; Wenting XU; Yangying JIA; Shiqing XIE; WenliSUN; Quan LIANG

    2012-01-01

    RNA interference (RNAi), caused by endogenous or exogenous double- stranded RNA (dsRNA) homologous with target genes, refers to gene silencing widely existing in animals and plants. It was first found in plants, and now it has developed into a kind of biotechnology as well as an important approach in post- genome era. This paper is to summarize the achievements of studies on RNAi tech- nology in basic biology, medicine, pharmacy, botany and other fields.

  5. Initiation, establishment, and maintenance of heritable MuDR transposon silencing in maize are mediated by distinct factors.

    Directory of Open Access Journals (Sweden)

    Margaret Roth Woodhouse

    2006-10-01

    Full Text Available Paramutation and transposon silencing are two epigenetic phenomena that have intrigued and puzzled geneticists for decades. Each involves heritable changes in gene activity without changes in DNA sequence. Here we report the cloning of a gene whose activity is required for the maintenance of both silenced transposons and paramutated color genes in maize. We show that this gene, Mop1 (Mediator of paramutation1 codes for a putative RNA-dependent RNA polymerase, whose activity is required for the production of small RNAs that correspond to the MuDR transposon sequence. We also demonstrate that although Mop1 is required to maintain MuDR methylation and silencing, it is not required for the initiation of heritable silencing. In contrast, we present evidence that a reduction in the transcript level of a maize homolog of the nucleosome assembly protein 1 histone chaperone can reduce the heritability of MuDR silencing. Together, these data suggest that the establishment and maintenance of MuDR silencing have distinct requirements.

  6. IARS2 silencing induces non-small cell lung cancer cells proliferation inhibition, cell cycle arrest and promotes cell apoptosis.

    Science.gov (United States)

    Yin, J; Liu, W; Li, R; Liu, J; Zhang, Y; Tang, W; Wang, K

    2016-01-01

    The purpose of this study was to investigate the potential role of Ileucyl-tRNA synthetase (IARS2) silencing in non-small cell lung cancer (NSCLC). The silencing of IARS2 in H1299 cells and A549 cells were performed by lentivirus encoding shRNAs. The efficiency of IARS2 silencing was detected by quantitative real time PCR and western blot. The effects of IARS2 silencing on cell growth, cell apoptosis, cell cycle and cell colony formation ability were assessed by cells counting, MTT assay, flow cytometer analysis and soft agar colony formation assay, respectively. Compared with negative control group, IARS2 was significantly knockdown by transfection with lentivirus encoding shRNA of IARS2. The IARS2 silencing significantly inhibited the cells proliferation and cells colony formation ability, induced cell cycle arrest at G1/S phase and promoted cell apoptosis. IARS2 silencing induced NSCLC cells growth inhibition, cell cycle arrest and promoted cell apoptosis. These results suggest that IARS2 may be a novel target for the treatment of NSCLC. PMID:26639235

  7. Effect of RNAi-mediated silencing of Livin gene on biological properties of colon cancer cell line LoVo.

    Science.gov (United States)

    Zou, A M; Wang, H F; Zhu, W F; Wang, F X; Shen, J J

    2014-05-16

    This study aimed to investigate the effect of RNAi-mediated silencing of the Livin gene on biological properties of the colon cancer cell line LoVo. Interference vectors pSilencer4.1-Ll and pSilencer4.1-L2 targeting the Livin gene were constructed and transfected into LoVo cells. The expression of the Livin gene was determined by RT-PCR and Western blotting. The apoptosis, cell cycle, colony formation, proliferation of LoVo cells, as well as their sensitivity to cisplatin, were detected by flow cytometry, colony formation assay and MTT. Livin mRNA and protein expression in LoVo cells could be effectively silenced by pSilencer4.1-Ll but not pSilencer4.1-L2. In the pSilencer4.1-Ll transfection group, the apoptosis rate of LoVo cells was significantly higher than in the control group (24.2 ± 3.2 vs 8.1 ± 1.4%, P LoVo colon cancer cells, inhibit cell proliferation and colony formation, induce apoptosis, and enhance sensitivity to cisplatin.

  8. Simultaneous gene silencing of Bcl-2, XIAP and Survivin re-sensitizes pancreatic cancer cells towards apoptosis

    International Nuclear Information System (INIS)

    Pancreatic ductal adenocarcinoma shows a distinct apoptosis resistance, which contributes significantly to the aggressive nature of this tumor and constrains the effectiveness of new therapeutic strategies. Apoptosis resistance is determined by the net balance of the cells pro-and anti-apoptotic 'control mechanisms'. Numerous dysregulated anti-apoptotic genes have been identified in pancreatic cancer and seem to contribute to the high anti-apoptotic buffering capacity. We aimed to compare the benefit of simultaneous gene silencing (SGS) of several candidate genes with conventional gene silencing of single genes. From literature search we identified the anti-apoptotic genes XIAP, Survivin and Bcl-2 as commonly upregulated in pancreatic cancer. We performed SGS and silencing of single candidate genes using siRNA molecules in two pancreatic cancer cell lines. Effectiveness of SGS was assessed by qRT-PCR and western blotting. Apoptosis induction was measured by flow cytometry and caspase activation. Simultaneous gene silencing reduced expression of the three target genes effectively. Compared to silencing of a single target or control, SGS of these genes resulted in a significant higher induction of apoptosis in pancreatic cancer cells. In the present study we performed a subliminal silencing of different anti-apoptotic target genes simultaneously. Compared to silencing of single target genes, SGS had a significant higher impact on apoptosis induction in pancreatic cancer cells. Thereby, we give further evidence for the concept of an anti-apoptotic buffering capacity of pancreatic cancer cells

  9. The proliferative effect on silencing of endogenetic mdr1 gene expression by RNA interference in ovarian cancer resistant strain SKOV3/TAXOL%RNAi沉默卵巢癌耐药细胞株SKOV3/TAXOL内源mdr1基因对细胞增生的影响

    Institute of Scientific and Technical Information of China (English)

    冯继良; 王红梅; 姚焕玲; 马传侠; 杜秀平

    2009-01-01

    Objective To construct the short hairpin RNA recombinant plasmids targeting mdr1 gene which expresses highly in ovarian cancer resistance strain SKOV3/TAXOL to silence endogenefic mdr1 gene expression and investigate the role of mdr1 gene in the development of resistant ovarian cancer. Methods The pGPU6/GFP/Neo-mdr1 were constructed by gene clone technology. The influence on proliferation and apoptosis were investigated by CCK-8 in SKOV3/TAXOL after transfected pGPU6/GFP/Neo-mdr1. Results The expression against mdr1 proteins were inhibited by pGPU6/GFP/Neo-mdr1. The cell proliferation were inhibited after transfected pGPU6/GFP/Neo-mdr1 by CCK-8. The apoptosis were observed in DAB experiments and the apoptosis rate increased. Conclusion mdr1 plays an important role in proliferation of resistant ovarian cancer and the short hairpin RNA of mdr1 can efficiently suppress mdr1 expression and enhance the apoptosis in SKOV3/ATAXOL.%目的 构建靶向于卵巢癌耐药细胞株中高表达的mdr1基因的短发夹状RNA重组质粒,研究其沉默卵巢癌耐药细胞株SKOV3/TAXOL内源mdr1基因对细胞增生的抑制作用.方法 运用基因克隆技术构建靶向于mdr1基因的重组质粒pGPU6/GFP/Neo-mdr1并转染至卵巢癌耐药细胞株SKOV3/TAXOL,CCK-8检测对SKOV3/TAXOL细胞增生的抑制作用.结果 构建的重组质粒pGPU6/GFP/Neo-mdr1能明显抑制mdr1基因的表达.CCK-8检测结果表明,转染重组质粒pGPU6/GFP/Neo-mdr1的SKOV3/TAXOL靶细胞的抑制率比对照组显著提高(P<0.05).pGPU6/GFP/Neo-mdr1明显抑制SKOV3/TAXOL细胞的增生.细胞凋亡检测显示,pGPU6/GFP/Neo-mdr1转染SKOV3/T后细胞凋亡增加,通过pGPU6/GFP/Neo-mdr1转染SKOV3/T促进凋亡的发生.结论 mdr1基因在耐药的卵巢癌细胞增生与凋亡过程中发挥作用,重组质粒pGPU6/GFP/Neo-mdr1可有效地抑制SKOV3/TAXOL细胞内源mdr1基因的表达和mRNA的转录,并抑制细胞的增生和促进凋亡的发生.

  10. Bone marrow mesenchymal stem cells with Nogo-66 receptor gene silencing for repair of spinal cord injur y

    Institute of Scientific and Technical Information of China (English)

    Zhiyuan Li; Zhanxiu Zhang; Lili Zhao; Hui Li; Suxia Wang; Yong Shen

    2014-01-01

    We hypothesized that RNA interference to silence Nogo-66 receptor gene expression in bone marrow mesenchymal stem cells before transplantation might further improve neurological function in rats with spinal cord transection injury. After 2 weeks, the number of neurons and BrdU-positive cells in the Nogo-66 receptor gene silencing group was higher than in the bone marrow mesenchymal stem cell group, and significantly greater compared with the model group. After 4 weeks, behavioral performance was signiifcantly enhanced in the model group. Af-ter 8 weeks, the number of horseradish peroxidase-labeled nerve ifbers was higher in the Nogo-66 receptor gene silencing group than in the bone marrow mesenchymal stem cell group, and signiifcantly higher than in the model group. The newly formed nerve ifbers and myelinated ner ve ifbers were detectable in the central transverse plane section in the bone marrow mesenchymal stem cell group and in the Nogo-66 receptor gene silencing group.

  11. Huhe Block

    Institute of Scientific and Technical Information of China (English)

    1996-01-01

    @@ Huhe Block is located in the mid-west part of Inner Mogolia Autonomous Region, covering an area of 15 079km2, in the range of 109°40'-112°00'E and 39°23()-40°40'N. Topographically. the Fengzhen hill is to the east, the Yinshan Mounts is to the north, the Hetao Plain and Ordos Plateau are respectively in its west and south.The Yellow River flows across this block. The elevation is 1 000 m in the flat area and in the range of 1 000-1 300m. in the plateau area, good for the development of agriculture and industry as well as husbandry. It belongs to inland plateau climate with annually averaged temperature of 8℃, the minimum being -12℃ in winter and the maximum 22℃ in summer.

  12. Effects of silencing H-ras gene by RNA interference on cetuximab-sensitivity of cetuximab- resistant human nasopharyngeal carcinoma cells%RNA干扰H-ras基因对鼻咽癌细胞西妥昔单抗敏感性的影响

    Institute of Scientific and Technical Information of China (English)

    左强; 罗荣城

    2011-01-01

    Objective To explore the changes of the sensitivity of cetuximab-resistant human nasopharyngeal carcinoma ( hNPC ) cells 5-8F/Erbitux to cetuximab by silencing H-ras gene with RNA interference (RNAi). Methods The 5-8F/Erbitux cells were induced by stepwise exposure to increasing doses of cetuximab. Western blot was conducted to detect the protein levels of H-ras and K-ras. Real-time PCR was employed to detect the expression of H-ras and K-ras. H-ras-shRNA plasmids ( shRNA vector carrying the H-ras gene) were constructed and transferred into 5-8F/Erbitux cells. The gene and protein expression levels of H-ras and the changes of the sensitivity of 5-8F/Erbitux cells to cetuximab after transfection were measured, respectively. Results After treatment with cetuximab for 3 and 5 days, the resistance index (RI) of the 5-8F/Erbitux cells was 1.2 and 1.1, and the protein levels of H-ras and K-ras in 5-8F/Erbitux cells were 0. 798 ± 0. 019 and 0. 190 ± 0.011, respectively, significantly higher than that in the 5-8F cells (P <0.001 ). The gene expressions of H-ras and K-ras in 5-8F/Erbitux cells were 1. 260 ±0. 114 and 0. 850 ±0. 006, respectively. Compared with 5-8F cells, the former was higher ( P =0. 016) and the latter was lower (P =0. 000). After transfection with H-ras-shRNA plasmid, the 5-8F/Erbitux cells showed reduced levels of H-ras gene and protein, and the cell apoptosis and inhibition rates increased significantly (P < 0.001 ). Conclusions H-raa siRNA can reverse cetuximab-resistance of 5-8F/Erbitux cells through down-regulation of H-ras gene expression, indicating that the generation of cetuximab-resistance in 5-8F/Erbitux cells is associated with amplification and overexpression of the H-ras gene.%目的 探讨RNA干扰H-ras基因对鼻咽癌细胞西妥昔单抗敏感性的影响.方法 采用逐步增加剂量法诱导建立西妥昔单抗耐药人鼻咽癌细胞系5-8F/Erbitux.Western blot法检测H-ras和K-ras蛋白的表达,实时荧光定量PCR

  13. The Malignant Phenotype of Breast Cancer Cells Is Reduced by COX-2 Silencing

    Directory of Open Access Journals (Sweden)

    Ioannis Stasinopoulos

    2008-11-01

    Full Text Available The cyclooxygenase (COX pathway is currently targeted for therapeutic intervention in different cancers. We have previously shown that silencing of COX-2 in the poorly differentiated metastatic breast cell line MDA-MB-231 by RNA interference markedly delayed tumor onset and inhibited metastasis. To understand the functional effects of COX-2 silencing underlying the inhibition of tumor growth and metastasis previously reported, we investigated changes in these cells for a number of cancer-associated phenotypes. Cyclooxygenase-2-silenced cells were less able to acidify tissue culture medium, a response that could partly be attributed to decreased lactate production or export detected by reduced lactate in the medium. Consistent with the significantly reduced transcript levels of hyaluronan synthase 2, an enzyme responsible for the total level of hyaluronan secreted by these cells, COX-2 silencing resulted in lower hyaluronan levels secreted in culture medium. Inhibition of human umbilical vein endothelial cell network association in a coculture assay was also observed in COX-2-silenced cells. These data highlight the functional role of COX-2 in pathways that mediate increased malignancy.

  14. Titration and hysteresis in epigenetic chromatin silencing

    International Nuclear Information System (INIS)

    Epigenetic mechanisms of silencing via heritable chromatin modifications play a major role in gene regulation and cell fate specification. We consider a model of epigenetic chromatin silencing in budding yeast and study the bifurcation diagram and characterize the bistable and the monostable regimes. The main focus of this paper is to examine how the perturbations altering the activity of histone modifying enzymes affect the epigenetic states. We analyze the implications of having the total number of silencing proteins, given by the sum of proteins bound to the nucleosomes and the ones available in the ambient, to be constant. This constraint couples different regions of chromatin through the shared reservoir of ambient silencing proteins. We show that the response of the system to perturbations depends dramatically on the titration effect caused by the above constraint. In particular, for a certain range of overall abundance of silencing proteins, the hysteresis loop changes qualitatively with certain jump replaced by continuous merger of different states. In addition, we find a nonmonotonic dependence of gene expression on the rate of histone deacetylation activity of Sir2. We discuss how these qualitative predictions of our model could be compared with experimental studies of the yeast system under anti-silencing drugs. (paper)

  15. Effect of silencing Fas expression by RNA interference on mice brain microvascular endothelial cell line bEnd.3%RNA干扰抑制Fas基因的表达对小鼠脑微血管内皮细胞bEnd.3的影响

    Institute of Scientific and Technical Information of China (English)

    张春兵; 高峰; 张明顺; 滕凤猛

    2013-01-01

    Objective To investigate the impact of reduced Fas expression by RNA interference on the proliferation of mice brain microvascular endothelial cell line bend.3 and the signal transduction pathway of Fas-associated death domain-containing protein (FADD),FADD-like IL-1β-converting enzyme (FLIP),tumor necrosis factor receptor-associated factor (TRAF) and nuclear factor κB (NF-κB).Methods The siRNA fragment targeted to mice Fas gene was designed and synthesized,and transfected into bEnd.3 cells by Lipofection 2000.The cell proliferation was measured using cell counting kit-8 (CCK-8).The expression levels of Fas mRNA and Fas protein were measured by real-time quantitative PCR and western blot respectively.The levels of FADD,FLIP and TRAF protein were measured by western blot simultaneously.Results CCK-8 assay demonstrated that no significantly difference of cell proliferation was found between Fas-siRNA group and blank control group (t =1.805,P > 0.05).Quantification analysis showed that Fas mRNA expression levels were markedly decreased in Fas-228-,Fas-310-,Fas-427-and Fas-891-siRNA group compared with the blank control group (F =123.127,P < 0.05).Western blot indicated that Fas-siRNA significantly reduced the expression of Fas protein (t =12.101,P < 0.01).The relative expression levels of FADD,FLIP and TRAF were significantly decreased as compared with blank group(t =28.315,17.563 and 8.903,P < 0.05).Conclusion Fas-siRNA can effectively block the expression of Fas gene in bEnd.3 cell and decrease the protein levels of Fas downstream signal molecules FADD,FLIP and TRAF.%目的 用RNA干扰(RNAi)技术抑制小鼠Fas基因表达,观察其对脑微血管内皮细胞bEnd.3增殖及FADD-FLIP-TRAFNF-κB信号传导途径的影响,为后续FasL低剂量兴奋效应研究提供实验基础.方法 设计合成靶向小鼠Fas基因的小干扰RNA (siRNA)片段,用脂质体包埋转染bEnd.3细胞;CCK-8法检测细胞增殖;RT-PCR检测bEnd.3细胞Fas m

  16. Magnetic Resonance Spectroscopy of siRNA-Based Cancer Therapy

    Science.gov (United States)

    Penet, Marie-France; Chen, Zhihang; Mori, Noriko; Krishnamachary, Balaji; Bhujwalla, Zaver M.

    2016-01-01

    Small interfering RNA (siRNA) is routinely used as a biological tool to silence specific genes, and is under active investigation in cancer treatment strategies. Noninvasive magnetic resonance spectroscopy (MRS) provides the ability to assess the functional effects of siRNA-mediated gene silencing in cultured cancer cells, and following nanoparticle-based delivery in tumors in vivo. Here we describe the use of siRNA to downregulate choline kinase, a critical enzyme in choline phospholipid metabolism of cancer cells and tumors, and the use of 1H MRS of cells and 1H magnetic resonance spectroscopic imaging (MRSI) of tumors to assess the efficacy of the downregulation. PMID:26530913

  17. RNA interference and antiviral therapy

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    RNA interference (RNAi) is an evolutionally conserved gene silencing mechanism present in a variety of eukaryotic species. RNAi uses short double-stranded RNA (dsRNA) to trigger degradation or translation repression of homologous RNA targets in a sequence-specific manner. This system can be induced effectively in vitro and in vivo by direct application of small interfering RNAs (siRNAs), or by expression of short hairpin RNA (shRNA) with non-viral and viral vectors. To date, RNAi has been extensively used as a novel and effective tool for functional genomic studies, and has displayed great potential in treating human diseases, including human genetic and acquired disorders such as cancer and viral infections. In the present review, we focus on the recent development in the use of RNAi in the prevention and treatment of viral infections. The mechanisms,strategies, hurdles and prospects of employing RNAi in the pharmaceutical industry are also discussed.

  18. MicroRNA-16 Modulates HuR Regulation of Cyclin E1 in Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Xun Guo

    2015-03-01

    Full Text Available RNA binding protein (RBPs and microRNAs (miRNAs or miRs are post-transcriptional regulators of gene expression that are implicated in development of cancers. Although their individual roles have been studied, the crosstalk between RBPs and miRNAs is under intense investigation. Here, we show that in breast cancer cells, cyclin E1 upregulation by the RBP HuR is through specific binding to regions in the cyclin E1 mRNA 3' untranslated region (3'UTR containing U-rich elements. Similarly, miR-16 represses cyclin E1, dependent on its cognate binding sites in the cyclin E1 3'UTR. Evidence in the literature indicates that HuR can regulate miRNA expression and recruit or dissociate RNA-induced silencing complexes (RISC. Despite this, miR-16 and HuR do not affect the other’s expression level or binding to the cyclin E1 3'UTR. While HuR overexpression partially blocks miR-16 repression of a reporter mRNA containing the cyclin E1 3'UTR, it does not block miR-16 repression of endogenous cyclin E1 mRNA. In contrast, miR-16 blocks HuR-mediated upregulation of cyclin E1. Overall our results suggest that miR-16 can override HuR upregulation of cyclin E1 without affecting HuR expression or association with the cyclin E1 mRNA.

  19. HBXIP and LSD1 Scaffolded by lncRNA Hotair Mediate Transcriptional Activation by c-Myc.

    Science.gov (United States)

    Li, Yinghui; Wang, Zhen; Shi, Hui; Li, Hang; Li, Leilei; Fang, Runping; Cai, Xiaoli; Liu, Bowen; Zhang, Xiaodong; Ye, Lihong

    2016-01-15

    c-Myc is regarded as a transcription factor, but the basis for its function remains unclear. Here, we define a long noncoding RNA (lncRNA)/protein complex that mediates the transcriptional activation by c-Myc in breast cancer cells. Among 388 c-Myc target genes in human MCF-7 breast cancer cells, we found that their promoters could be occupied by the oncoprotein HBXIP. We confirmed that the HBXIP expression correlated with expression of the c-Myc target genes cyclin A, eIF4E, and LDHA. RNAi-mediated silencing of HBXIP abolished c-Myc-mediated upregulation of these target genes. Mechanistically, HBXIP interacted directly with c-Myc through the leucine zippers and recruited the lncRNA Hotair along with the histone demethylase LSD1, for which Hotair serves as a scaffold. Silencing of HBXIP, Hotair, or LSD1 was sufficient to block c-Myc-enhanced cancer cell growth in vitro and in vivo. Taken together, our results support a model in which the HBXIP/Hotair/LSD1 complex serves as a critical effector of c-Myc in activating transcription of its target genes, illuminating long-standing questions on how c-Myc drives carcinogenesis.

  20. MicroRNA-Mediated Downregulation of the Potassium Channel Kv4.2 Contributes to Seizure Onset

    Directory of Open Access Journals (Sweden)

    Christina Gross

    2016-09-01

    Full Text Available Seizures are bursts of excessive synchronized neuronal activity, suggesting that mechanisms controlling brain excitability are compromised. The voltage-gated potassium channel Kv4.2, a major mediator of hyperpolarizing A-type currents in the brain, is a crucial regulator of neuronal excitability. Kv4.2 expression levels are reduced following seizures and in epilepsy, but the underlying mechanisms remain unclear. Here, we report that Kv4.2 mRNA is recruited to the RNA-induced silencing complex shortly after status epilepticus in mice and after kainic acid treatment of hippocampal neurons, coincident with reduction of Kv4.2 protein. We show that the microRNA miR-324-5p inhibits Kv4.2 protein expression and that antagonizing miR-324-5p is neuroprotective and seizure suppressive. MiR-324-5p inhibition also blocks kainic-acid-induced reduction of Kv4.2 protein in vitro and in vivo and delays kainic-acid-induced seizure onset in wild-type but not in Kcnd2 knockout mice. These results reveal an important role for miR-324-5p-mediated silencing of Kv4.2 in seizure onset.

  1. HBXIP and LSD1 Scaffolded by lncRNA Hotair Mediate Transcriptional Activation by c-Myc.

    Science.gov (United States)

    Li, Yinghui; Wang, Zhen; Shi, Hui; Li, Hang; Li, Leilei; Fang, Runping; Cai, Xiaoli; Liu, Bowen; Zhang, Xiaodong; Ye, Lihong

    2016-01-15

    c-Myc is regarded as a transcription factor, but the basis for its function remains unclear. Here, we define a long noncoding RNA (lncRNA)/protein complex that mediates the transcriptional activation by c-Myc in breast cancer cells. Among 388 c-Myc target genes in human MCF-7 breast cancer cells, we found that their promoters could be occupied by the oncoprotein HBXIP. We confirmed that the HBXIP expression correlated with expression of the c-Myc target genes cyclin A, eIF4E, and LDHA. RNAi-mediated silencing of HBXIP abolished c-Myc-mediated upregulation of these target genes. Mechanistically, HBXIP interacted directly with c-Myc through the leucine zippers and recruited the lncRNA Hotair along with the histone demethylase LSD1, for which Hotair serves as a scaffold. Silencing of HBXIP, Hotair, or LSD1 was sufficient to block c-Myc-enhanced cancer cell growth in vitro and in vivo. Taken together, our results support a model in which the HBXIP/Hotair/LSD1 complex serves as a critical effector of c-Myc in activating transcription of its target genes, illuminating long-standing questions on how c-Myc drives carcinogenesis. PMID:26719542

  2. RNA干扰沉默骨桥蛋白对大鼠移植肾小管上皮细胞向间充质转分化的作用%Effect of silencing osteopontin by RNA interference upon EMT of tubular epithelial cells in rats with chronic allograft nephropathy

    Institute of Scientific and Technical Information of China (English)

    张挺; 徐东亮; 周强平; 鲁佩; 殷长军; 张炜; 徐正铨; 顾民

    2011-01-01

    Objective:To investigate the effect of in vivo shRNA plasmid vector gene transfer to silence OPN expression upon epithelial to mesenchymal transition of the tubular epithelial cells (TECs) in rat chronic allograft nephropathy (CAN) models, and to explore the possible role of OPN in CAN. Methods: Orthotopic renal-transplantation using F344 rats as donors and Lewis rats as recipients was operated to establish Allo group, while transplantation from Lewis to Lewis rats as Syn group. Hydromechanics based gene transfer method was used to establish RNAi and EV groups, for which OPN-shRNA or empty vector was injected into the renal vein. Rats in each group were sacrificed 12 weeks after the surgery. Allograft samples were collected and sectioned for HE staining, masson's trichrome staining, immunohislochemical study and Western blot assay. Results: The morphological changes of allograft in Allo/EV group consist with CAN. Compared with the Syn group,Western blot showed a significant rise of OPN expression. In the RNAi group, the OPN expression was significantly reduced(P<0.05) compared with Allo/EV group. The RNA interference inhibited EMT of TECs and the pathogenesis of CAN, which were confirmed by immunohistochemistry and HE/Masson's trichrome lightscopy. Conclusion:The OPN expression in rat CAN model was significantly up-regulated. The in vivo RNA interference inhibited the OPN expression. Inhibition of OPN can protect the allograft from pathogenesis of EMT and IF/TA in CAN.%目的:探讨骨桥蛋白(osteopontin,OPN)的shRNA干扰质粒在大鼠慢性移植肾肾病(chronic allograft nephropathy,CAN)模型中对大鼠移植肾上皮细胞向间充质转分化(epithelial to mesenchymal transition,EMT)的作用,初步探讨OPN在慢性移植物肾病中的作用及机制.方法:以F344大鼠为供体,Lewis大鼠为受体,行原位异体肾移植术,建立大鼠CAN模型(Allo组),以Lewis大鼠做供、受体作为同系移植对照(Syn组),以基于流体力学为基础

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

    Science.gov (United States)

    Fletcher, Bradley S

    2010-11-01

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

  4. Evaluation of BACE1 Silencing in Cellular Models

    Directory of Open Access Journals (Sweden)

    Malgorzata Sierant

    2009-01-01

    Full Text Available Beta-secretase (BACE1 is the major enzyme participating in generation of toxic amyloid-beta (Aβ peptides, identified in amyloid plaques of Alzheimer's disease (AD brains. Its downregulation results in decreasing secretion of Aβ. Thus, BACE1 silencing by RNAi represents possible strategy for antiamyloid therapy in the treatment of AD. In this study, a series of newly designed sequences of synthetic and vector-encoded siRNAs (pSilencer, pcPURhU6, and lentivirus were tested against overexpressed and endogenous BACE1 in several cell lines and in adult neural progenitor cells, derived from rat hippocampus. SiRNAs active in human, mouse, and rat cell models were shown to diminish the level of BACE1. In HCN A94 cells, two BACE1-specific siRNAs did not alter the expression of genes of BACE2 and several selected genes involved in neurogenesis (Synapsin I, βIII-Tubulin, Calbidin, NeuroD1, GluR2, CREB, MeCP2, PKR, however, remarkable lowering of SCG10 mRNA, coding protein of stathmin family, important in the development of nervous system, was observed.