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Results for “RNA interference”

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At least 667 records · Page 37Linked to original sources

PubMed2006

Inhibition of measles virus and subacute sclerosing panencephalitis virus by RNA interference.

Subacute sclerosing panencephalitis (SSPE) is a rare, but fatal outcome of measles virus (MeV) infection. SSPE develops after prolonged persistence of mutated MeV called SSPE virus. Although a combination therapy using interferon and inosiplex or ribavirin appears to prolong survival time to some extent, there is currently no effective treatment to completely cure SSPE and a new treatment strategy is greatly needed. In this study, we adopted RNA interference (RNAi) strategy and examined whether small interfering RNAs (siRNAs) can be used to inhibit replication of MeV and SSPE virus. We report here that siRNAs targeted against L mRNA of MeV, either synthetic siRNAs or those generated by pcPUR+U6i-based expression plasmids, effectively and specifically inhibited replication of both MeV and SSPE virus without exhibiting any cytotoxic effect. The L protein of MeV is a major component of RNA-dependent RNA polymerase that is essential for viral RNA replication, and yet it is least abundant among all the MeV proteins expressed. Therefore, mRNA encoding the L protein would be a good target for RNAi strategy. The present results imply the possibility that our siRNAs against MeV L mRNA are among the potential candidates to be used to treat patients with SSPE.

Animals↗

PubMedDate not supplied

The RNA interference of amino acid transporter LAT1 inhibits the growth of KB human oral cancer cells.

BACKGROUND: Amino acid transporters are essential for growth and proliferation in all living cells. Among the amino acid transporters, the system L amino acid transporters are the major nutrient transport system responsible for the Na+-independent transport of neutral amino acids, including several essential amino acids. The L-type amino acid transporter 1 (LAT1) is overexpressed to support cell growth in malignant tumors. Double-stranded RNA-mediated RNA interference (RNAi) analysis can be used in a wide variety of eukaryotes to induce the sequence-specific inhibition of gene expression. The current study attempted to investigate the effects of silencing LAT1 expression with small interfering RNA (siRNA) on cell growth in the KB human oral squamous cell carcinoma. MATERIALS AND METHODS: The effects of silencing LAT1 expression with siRNA KB on cell growth were examined using RT-PCR, Western blot analysis, amino acid transport measurement and the MTT assay. RESULTS: In the RT-PCR and Western blot analyses, the siRNA of LAT1 inhibited the expressions of LAT1 mRNA and protein. The uptake of [14C]L-leucine was also inhibited by the siRNA of LAT1. In the MTT assay, the siRNA of LAT1 inhibited the growth of the KB cells in a time-dependent manner, indicating that this growth inhibition was induced by the LAT1-mediated blocking of neutral amino acid transport. CONCLUSION: The transport of neutral amino acids, including several essential amino acids, into the KB human oral squamous cell carcinoma is mainly mediated by LAT1. Furthermore, LAT1 could be a new target for the inhibition of cancer cell growth.

Carbon Radioisotopes↗

PubMed2004

Inhibition of human immunodeficiency virus type 1 replication by RNA interference directed against human transcription elongation factor P-TEFb (CDK9/CyclinT1).

The human positive transcription elongation factor P-TEFb is composed of two subunits, cyclin T1 (hCycT1) and CDK9, and is involved in transcriptional regulation of cellular genes as well as human immunodeficiency virus type 1 (HIV-1) mRNA. Replication of HIV-1 requires the Tat protein, which activates elongation of RNA polymerase II at the HIV-1 promoter by interacting with hCycT1. To understand the cellular functions of P-TEFb and to test whether suppression of host proteins such as P-TEFb can modulate HIV infectivity without causing cellular toxicity or lethality, we used RNA interference (RNAi) to specifically knock down P-TEFb expression by degrading hCycT1 or CDK9 mRNA. RNAi-mediated gene silencing of P-TEFb in HeLa cells was not lethal and inhibited Tat transactivation and HIV-1 replication in host cells. We also found that CDK9 protein stability depended on hCycT1 protein levels, suggesting that the formation of P-TEFb CDK-cyclin complexes is required for CDK9 stability. Strikingly, P-TEFb knockdown cells showed normal P-TEFb kinase activity. Our studies suggest the existence of a dynamic equilibrium between active and inactive pools of P-TEFb in the cell and indicate that this equilibrium shifts towards the active kinase form to sustain cell viability when P-TEFb protein levels are reduced. The finding that a P-TEFb knockdown was not lethal and still showed normal P-TEFb kinase activity suggested that there is a critical threshold concentration of activated P-TEFb required for cell viability and HIV replication. These results provide new insights into the regulation of P-TEFb function and suggest the possibility that similar mechanisms for monitoring protein levels to modulate the activity of proteins may exist for the regulation of a variety of other enzymatic pathways.

Cell Line↗

PubMed2004

Inhibition of HLA-G production in JEG-3 choriocarcinoma cells by RNA interference.

Human leukocyte antigen-G (HLA-G) plays a major role in escape of trophoblast cells from maternal cytotoxicity. Unless its malignant transformation, Jeg-3 choriocarcinoma cell line maintained the capacity of HLA-G production. For the analysis of function and mechanisms of HLA-G-induced immune regulation, a human cellular model with suppressed HLA-G would be very helpful. RNA interference (RNAi) is a very elegant method for this approach, but the design of appropriate oligonucleotide sequences may provide difficulties. We designed oligonucleotides to interfere exclusively with HLA-G mRNA, which were applied at different concentrations to 50% confluent Jeg-3 cells. After 36 hr, the HLA-G content in Jeg-3 cells was analyzed by Western blots. Applying the described RNAi method and oligonucleotides the cellular content of HLA-G was dose-dependently reduced as assessed in several independent Western blots. This method provides a tool for extensive investigation of HLA-G functions in vitro and if vivo.

Cell Line, Tumor↗

PubMed2004

The long processes of short interfering RNAs--RNA interference and its implications in neuronal cells.

Reverse genetics has been greatly advanced by the discovery of RNA interference (RNAi). This intracellular RNA-mediated gene silencing pathway is partially conserved from plants to mammals and offers a new powerful tool for the analysis of gene function. We give a brief overview of the discovery of RNAi, the underlying mechanisms and probable intrinsic roles of the pathway. Recent reports utilizing RNAi for gene silencing approaches in neuronal cells are reviewed and possible delivery techniques for small interfering RNA/double-stranded RNA are discussed.

Animals↗

PubMed2004

Inhibition of porcine endogenous retroviruses by RNA interference: increasing the safety of xenotransplantation.

Transplantation of porcine xenografts into human recipients is a realistic option to overcome the growing worldwide shortage of suitable allogeneic organs. However, there remains the risk of infection by porcine endogenous retroviruses (PERVs) that cannot be eliminated like that by other microorganisms by breeding pigs under specified pathogen-free conditions. To reduce the release of PERVs by porcine transplants, a new approach, RNA interference (RNAi), was applied. Here, we show significant reduction of PERV expression by synthetic short interfering RNAs (siRNAs) corresponding to different parts of the viral genes gag, pol, and env. The most inhibitory sequences were selected and expressed as short hairpin RNAs (shRNAs) by a polymerase III vector system leading to persistent suppression of PERV replication. Cells or organs from transgenic pigs producing such shRNAs should increase the safety of xenotransplantation.

Animals↗

PubMed2006

RNA interference: a potential therapeutic tool for silencing splice isoforms linked to human diseases.

Alternative splicing of precursor messenger RNAs (pre-mRNAs) is one of the most important sources of protein diversity in vertebrates. An estimated 35%-70% of human genes generate transcripts that are alternatively spliced, and defects in this process are linked to numerous human genetic diseases and various forms of cancer. The discovery that 21-23 nucleotide RNA duplexes, known as small interfering RNAs (siRNAs), can knockdown the homologous mRNAs in mammalian cells has revolutionized many aspects of drug discovery including down-regulation of disease-associated splicing isoforms. In addition, RNA interference (RNAi)-mediated silencing of splicing regulators has the potential to define the complex network of alternative splicing regulation and to analyze gene function. In this review, I first provide a brief introduction to mRNA splicing and its relationship to human diseases. This is followed by a brief overview of RNAi. Finally I discuss the therapeutic potential of RNAi in targeting disease-linked splicing isoforms.

Animals↗

PubMed2005

Smad4 silencing in pancreatic cancer cell lines using stable RNA interference and gene expression profiles induced by transforming growth factor-beta.

The transforming growth factor-beta (TGF-beta)-Smad signaling pathway inhibits the growth of human epithelial cells and plays a role in tumor suppression. The Smad4 gene is mutated or deleted in 50% of pancreatic cancers. In this study, we succeeded in establishing Smad4 knockdown (S4KD) pancreatic cancer cell lines using the stable RNA interference (RNAi) method. Smad4 protein expression was reduced dramatically and TGF-beta-Smad signaling was markedly inhibited in the S4KD cell lines. The S4KD and control cells were stimulated with TGF-beta and analysed using a cDNA microarray that contained 3756 genes, in order to screen for target molecules downstream of TGF-beta. The microarray analysis revealed that 187 S4KD genes and 155 genes in the control cells were regulated immediately upon TGF-beta stimulation. Quantitative RT-PCR analysis on several of these genes produced results that corroborated the outcome of the microarray analysis. Most of the genes in the S4KD and control cells identified by the array differed, which suggests signaling pathways that differ according to Smad4 status. Of the identified genes, 246 have not been reported previously as genes that lie downstream of TGF-beta. Genes that are involved in cell proliferation, adhesion, and motility were found to be regulated differentially with respect to S4KD and control cells. Cell migration induced by TGF-beta was inhibited in the S4KD cells, which might be associated with a different regulation of integrin beta7. The knock down of a specific gene using stable RNAi appears to be a promising tool for analysing endogenous gene function.

Cell Line, Tumor↗

PubMed2005

Somatic misexpression of germline P granules and enhanced RNA interference in retinoblastoma pathway mutants.

Caenorhabditis elegans homologues of the retinoblastoma (Rb) tumour suppressor complex specify cell lineage during development. Here we show that mutations in Rb pathway components enhance RNA interference (RNAi) and cause somatic cells to express genes and elaborate perinuclear structures normally limited to germline-specific P granules. Furthermore, particular gene inactivations that disrupt RNAi reverse the cell lineage transformations of Rb pathway mutants. These findings suggest that mutations in Rb pathway components cause cells to revert to patterns of gene expression normally restricted to germ cells. Rb may act by a similar mechanism to transform mammalian cells.

Alleles↗

PubMed2004

Antisense and RNA interference approaches to target validation in pain research.

In recent years numerous potential new targets for the development of novel drugs to treat chronic pain have been identified. Antisense strategies provide a straightforward approach to validate and further analyze their function. Furthermore, they offer the possibility of investigating a single member of a closely related family of proteins that cannot be easily targeted using small-molecule pharmacological tools. The recent discovery that short double-stranded RNA molecules can be employed to induce RNA interference in mammalian systems has opened exciting new roads for functional genomics. Gene silencing by small interfering RNAs has been demonstrated in neurons, and several targets involved in pain perception have been addressed. Furthermore, promising in vivo results have recently been obtained with small interfering RNAs.

Animals↗

PubMed2005

Suppression of Epstein-Barr nuclear antigen 1 (EBNA1) by RNA interference inhibits proliferation of EBV-positive Burkitt's lymphoma cells.

PURPOSE: Epstein-Barr virus (EBV) is associated with the development of several lymphoid and epithelial malignancies, including Burkitt's lymphoma. The EBV latent protein, EBV Nuclear Antigen 1 (EBNA1), is detectable in almost all types of EBV-associated tumors and is essential for replication and maintenance of the latent episome of EBV. We here examined whether the RNA interference (RNAi) technique could be employed to suppress expression of EBNA1 in EBV-positive Burkitt's lymphoma cells. METHODS: A Raji cell line expressing small hairpin RNAs (shRNAs) against EBNA1 was established and EBNA1 mRNA level was determined by real-time RT-PCR analysis. We investigated the effects of EBNA1 silence on lymphoma cell growth and cell cycle progression. RESULTS: Transfection of an EBNA1 RNAi plasmid resulted in substantial loss of EBNA1 mRNA and significantly inhibited proliferation of Raji cells relative to the control plasmid case. Suppression of EBNA1 was also associated with downregulation of EBV oncogene EBNA2, a decreased PCNA labeling index and increased G0/G1 fraction in cell cycle analysis. CONCLUSIONS: These findings point to potential therapeutic applications for vector-mediated siRNA delivery to control EBV-associated malignant disorders.

Apoptosis↗

PubMed2000

EGO-1 is related to RNA-directed RNA polymerase and functions in germ-line development and RNA interference in C. elegans.

BACKGROUND: Cell-fate determination requires that cells choose between alternative developmental pathways. For example, germ cells in the nematode worm Caenorhabditis elegans choose between mitotic and meiotic division, and between oogenesis and spermatogenesis. Germ-line mitosis depends on a somatic signal that is mediated by a Notch-type signaling pathway. The ego-1 gene was originally identified on the basis of genetic interactions with the receptor in this pathway and was also shown to be required for oogenesis. Here, we provide more insight into the role of ego-1 in germ-line development. RESULTS: We have determined the ego-1 gene structure and the molecular basis of ego-1 alleles. Putative ego-1 null mutants had multiple, previously unreported defects in germ-line development. The ego-1 transcript was found predominantly in the germ line. The predicted EGO-1 protein was found to be related to the tomato RNA-directed RNA polymerase (RdRP) and to Neurospora crassa QDE-1, two proteins implicated in post-transcriptional gene silencing (PTGS). For a number of germ-line-expressed genes, ego-1 mutants were resistant to a form of PTGS called RNA interference. CONCLUSIONS: The ego-1 gene is the first example of a gene encoding an RdRP-related protein with an essential developmental function. The ego-1 gene is also required for a robust response to RNA interference by certain genes. Hence, a protein required for germ-line development in C. elegans may be a component of the RNA interference/PTGS machinery.

Amino Acid Sequence↗

PubMed2006

Inhibition of CD147 gene expression via RNA interference reduces tumor cell invasion, tumorigenicity and increases chemosensitivity to paclitaxel in HO-8910pm cells.

Overexpression of extracellular matrix metalloproteinase inducer (EMMPRIN or CD147), a member of the immunoglobulin family and a glycoprotein enriched on the surface of tumor cells, promotes invasion, metastasis, growth and survival of malignant cells, and confers resistance to some chemotherapeutic drugs. Here, we used a human U6 promoter-driven DNA template approach to induce short hairpin RNA (shRNA)-triggered RNA interference (RNAi) to block CD147 gene expression in the human ovarian cancer cell line HO-8910pm. Knockdown of CD147 by shRNA resulted in decrease of the HO-8910pm invasion activity in vitro and tumorigenicity in nude mice. The suppression of CD147 expression also sensitized cells to be more sensitive to paclitaxel. These results suggested that CD147 was an ovarian cancer-related gene and CD147 might be a potential target for therapeutic anti-cancer drugs.

Animals↗

PubMed2004

Silencing structural and nonstructural genes in baculovirus by RNA interference.

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.

Animals↗

PubMed2006

Prior infection of Manduca sexta with non-pathogenic Escherichia coli elicits immunity to pathogenic Photorhabdus luminescens: roles of immune-related proteins shown by RNA interference.

Prior infection of Manduca sexta caterpillars with the non-pathogenic bacterium Escherichia coli elicits effective immunity against subsequent infection by the usually lethal and highly virulent insect pathogen Photorhabdus luminescens TT01. Induction of this protective effect is associated with up-regulation of both microbial pattern recognition protein genes (hemolin, immulectin-2 and peptidoglycan recognition protein) and anti-bacterial effector genes (attacin, cecropin, lebocin, lysozyme and moricin). We used RNA interference to knock down over-transcription of members of both these sets of genes one at a time. Interfering with expression of individual recognition proteins had a drastic adverse effect on the E. coli elicited immunity. RNAi knock-down of immulectin-2 caused the greatest reduction in immunity, followed by hemolin and peptidoglycan recognition protein (PGRP) in that order, to the extent that knock-down of any one of these three proteins left the insects more susceptible to P. luminescens infection than insects that had not experienced prior infection with E. coli. Interfering with the expression of individual antibacterial effector proteins and peptides had a much less marked effect on immunity. Knock-down of attacin, cecropin or moricin caused treated insects to be more susceptible to P. luminescens infection than controls that had been pre-infected with E. coli but which had not received the specific RNAi reagents, but they were still less susceptible than insects that had not been pre-infected with E. coli. RNAi knock-down with expression of lebocin or lysozyme had no effect on E. coli-induced immunity to P. luminescens, indicating that these effectors are not involved in the response. By bleeding pre-infected caterpillars and growing the pathogen directly within cell-free insect haemolymph, we showed that at least part of the protection elicited by previous exposure to E. coli is due to the presence of factors within the blood plasma that inhibit the growth of P. luminescens. The production of these factors is inhibited by RNAi treatment with ds-RNA reagents that knock down hemolin, immulectin-2, and PGRP. These results demonstrate that the insect immune system can be effectively primed by prior infection with non-pathogenic bacteria against subsequent infection by a highly virulent pathogen. Given the continuous normal exposure of insects to environmental and symbiotic bacteria, we suggest that prior infection is likely to play a significant and underestimated role in determining the level of insect immunity found in nature.

Animals↗

PubMed2005

Identification of potential genes regulated by DNA methyltransferase 3B in a hepatocellular carcinoma cell line by RNA interference and microarray analysis.

Whether DNA methyltransferase 3B (DNMT3B) is deregulated in hepatocellular carcinoma cell lines is still unclear. The expression levels of DNMT3B protein in normal liver cell line, pericacinoma cell line and hepatocellular carcinoma cell lines were compared by both Western blotting and immunocytochemistry. Long-term downregulated DNMT3B in a hepatocellular carcinoma cell line SMMC-7721 was achieved using a RNAi recombinant plasmid. The suppression of DNMT3B induced by RNA interference was confirmed using semi-quantitative RT-PCR and Western blotting. High throughput cDNA microarray was used to analyze the expression profiling of downstream genes of DNMT3B displayed in the treated cell lines and control. In the result,DNMT3B in hepatocellular carcinoma cell lines was expressed at a significantly higher level compared to those in pericacinoma cell line and normal liver cell line. A specific DNMT3B siRNA stably expressed from a plasmid vector effectively suppressed the expression of DNMT3B in SMMC-7721 cell line. By microarray analysis,26 downregulated genes and 115 upregulated genes have been identified in the DNMT3B knockdown cell line,including some important developmental genes and tumor-related genes such as SNCG, NOTCH1, MBD3, WNT11, MAOA and FACL4. The discovery showed DNMT3B was over-expressed in most hepatocellular carcinoma cell lines examined and may be linked to the carcinogenesis of hepatocytes. An array of candidate genes that are involved in the action of DNMT3B have been identified,including those related to development.

Carcinoma, Hepatocellular↗

PubMed2005

RNA interference (RNAi)-dependent and RNAi-independent association of the Chp1 chromodomain protein with distinct heterochromatic loci in fission yeast.

The establishment of centromeric heterochromatin in the fission yeast Schizosaccharomyces pombe is dependent on the RNA interference (RNAi) pathway. Dicer cleaves centromeric transcripts to produce short interfering RNAs (siRNAs) that actively recruit components of heterochromatin to centromeres. Both centromeric siRNAs and the heterochromatin component Chp1 are components of the RITS (RNA-induced initiation of transcriptional gene silencing) complex, and the association of RITS with centromeres is linked to Dicer activity. In turn, centromeric binding of RITS promotes Clr4-mediated methylation of histone H3 lysine 9 (K9), recruitment of Swi6, and formation of heterochromatin. Similar to centromeres, the mating type locus (Mat) is coated in K9-methylated histone H3 and is bound by Swi6. Here we report that Chp1 associates with the mating type locus and telomeres and that Chp1 localization to heterochromatin depends on its chromodomain and the C-terminal domain of the protein. Another protein component of the RITS complex, Tas3, also binds to Mat and telomeres. Tas3 interacts with Chp1 through the C-terminal domain of Chp1, and this interaction is necessary for Tas3 stability. Interestingly, in cells lacking the Argonaute (Ago1) protein component of the RITS complex, or lacking Dicer (and hence siRNAs), Chp1 and Tas3 can still bind to noncentromeric loci, although their association with centromeres is lost. Thus, Chp1 and Tas3 exist as an Ago1-independent subcomplex that associates with noncentromeric heterochromatin independently of the RNAi pathway.

Argonaute Proteins↗

PubMed2006

Depletion of SMN by RNA interference in HeLa cells induces defects in Cajal body formation.

Neuronal degeneration in spinal muscular atrophy (SMA) is caused by reduced expression of the survival of motor neuron (SMN) protein. The SMN protein is ubiquitously expressed and is present both in the cytoplasm and in the nucleus where it localizes in Cajal bodies. The SMN complex plays an essential role for the biogenesis of spliceosomal U-snRNPs. In this article, we have used an RNA interference approach in order to analyse the effects of SMN depletion on snRNP assembly in HeLa cells. Although snRNP profiles are not perturbed in SMN-depleted cells, we found that SMN depletion gives rise to cytoplasmic accumulation of a GFP-SmB reporter protein. We also demonstrate that the SMN protein depletion induces defects in Cajal body formation with coilin being localized in multiple nuclear foci and in nucleolus instead of canonical Cajal bodies. Interestingly, the coilin containing foci do not contain snRNPs but appear to co-localize with U85 scaRNA. Because Cajal bodies represent the location in which snRNPs undergo 2'-O-methylation and pseudouridylation, our results raise the possibility that SMN depletion might give rise to a defect in the snRNA modification process.

Autoantigens↗