Intrahepatic gene silencing by RNA interference.
Explore the source record for details and available documents.
Comment↗
SEARCH · PubMed Health
Results for “RNA Interference”
Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
A Hoechst H33258 agarose plate assay for the estimation of nanogram DNA levels without RNA interference: applications in PCR and in estimations of plasmid and cytoplasmic DNA.
Explore the source record for details and available documents.
Bisbenzimidazole↗
Suppression of human brain tumor with interference RNA specific for tenascin-C.
Glioblastoma multiforme (GBM) accounts for approximately 12-15% of intracranial neoplasms. The GBM remains refractory to therapy because of tumor heterogeneity, local invasion, and non-uniform vascular permeability to drugs. Patients with GBM have the median survival of approximately 8-10 months, and for those cases where tumor recurs, the average time of tumor progression after therapy is only eight weeks. A combination of different treatment modes as surgery and chemo- or/and radiotherapy extend survival only for a short time, if any. Recently, tenascin-C (TN-C) as a dominant epitope in glioblastoma has been discovered. It is transiently expressed during organogenesis, absent or much reduced in most fully developed organs, but reappears under pathological conditions such as infection, inflammation, or tumorigenesis. It was found that the intensity of TN-C staining correlates with the tumor grade and that the strongest staining indicates poor prognosis.
Adult↗
Suppression of chemokine receptor expression by RNA interference allows for inhibition of HIV-1 replication, by Martínez et al.
Explore the source record for details and available documents.
Comment↗
[Effective inhibition of EB virus-encoded latent membrane protein-1 by siRNA in EB virus (+) nasopharyngeal carcinoma cell].
OBJECTIVE: To evaluate the application of RNA interference in the study of latent membrane protein-1 (LMP-1) in EB virus-positive nasopharyngeal carcinoma (NPC) cell line C611. Observe the effects of LMP-1 silencing on NPC cell growth. METHODS: Four synthesized small interference RNA (siRNA) were transfected into NPC cell using Oligofectamine reagent. LMP-1 mRNA level was determined by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Flow cytometry and MTT assay were employed to analyze the effects on cell cycle and proliferation. RESULTS: The most effective sequence found out among the 4 candidates. Single dose of this siRNA caused nearly 90% loss of LMP-1 mRNA in C611 cell. The specific inhibition could last for 96 hours if re-transfection was preformed. LMP-1 siRNA treatment resulted in cell cycle arrest at G0-G1 stage in C611, accompany with a reduction of cell proliferation by 32.9%. While EBV-negative NPC cells appeared unaffected. CONCLUSIONS: These results provided solid testimony that EBV-encoded latent membrane protein-1 was vulnerable to RNA interference and, selective inhibition of LMP-1 had anti-proliferation effect on NPC cell. RNA interference could be a powerful tool in further investigations of LMP-1 and a novel therapeutic strategy for EBV-related NPC patients.
Cell Line, Tumor↗
Pharmacological and small interference RNA-mediated inhibition of breast cancer-associated fatty acid synthase (oncogenic antigen-519) synergistically enhances Taxol (paclitaxel)-induced cytotoxicity.
The relationship between breast cancer-associated fatty acid synthase (FAS; oncogenic antigen-519) and chemotherapy-induced cell damage has not been studied. We examined the ability of C75, a synthetic slow-binding inhibitor of FAS activity, to modulate the cytotoxic activity of the microtubule-interfering agent Taxol (paclitaxel) in SK-Br3, MDA-MB-231, MCF-7 and multidrug-resistant MDR-1 (P-Glycoprotein)-overexpressing MCF-7/AdrR breast cancer cells. When the combination of C75 with Taxol in either concurrent (C75 + Taxol 24 hr) or sequential (C75 24 hr --> Taxol 24 hr) schedules were tested for synergism, addition or antagonism using the isobologram and the median-effect plot analyses, co-exposure of C75 and Taxol mostly demonstrated synergistic effects, whereas sequential exposure to C75 followed by Taxol mainly showed additive or antagonistic interactions. Because the nature of the cytotoxic interactions was definitely schedule-dependent in MCF-7 cells, we next evaluated the effects of C75 on Taxol-induced apoptosis as well as Taxol-activated cell death and cell survival-signaling pathways in this breast cancer cell model. An ELISA for histone-associated DNA fragments demonstrated that C75 and Taxol co-exposure caused a synergistic enhancement of apoptotic cell death, whereas C75 pre-treatment did not enhance the apoptosis-inducing activity of Taxol. Co-exposure to C75 and Taxol induced a remarkable nuclear accumulation of activated p38 mitogen-activated protein kinase (p38 MAPK), which was accompanied by a synergistic nuclear accumulation of the p53 tumor-suppressor protein that was phosphorylated at Ser46, a p38 MAPK-regulated pro-apoptotic modification of p53. As single agents, FAS blocker C75 and Taxol induced a significant stimulation of the proliferation and cell survival mitogen-activated protein kinase extracellular signal-regulated kinase (ERK1/ERK2 MAPK) activity, whereas, in combination, they interfered with ERK1/ERK2 activation. Moreover, the combined treatment of C75 and Taxol inactivated the anti-apoptotic AKT (protein kinase B) kinase more than either agent alone, as evidenced by a synergistic down-regulation of AKT phosphorylation at its activating site Ser(473) without affecting AKT protein levels. To rule out a role for non-FAS C75-mediated effects, we finally used the potent and highly sequence-specific mechanism of RNA interference (RNAi) to block FAS-dependent signaling. Importantly, SK-Br3 and multi-drug resistant MCF-7/AdrR cells transiently transfected with sequence-specific double-stranded RNA oligonucleotides targeting FAS gene demonstrated hypersensitivity to Taxol-induced apoptotic cell death. Our findings establish for the first time that FAS blockade augments the cytotoxicity of anti-mitotic drug Taxol against breast cancer cells and that this chemosensitizing effect is schedule-dependent. We suggest that the alternate activation of both the pro-apoptotic p38 MAPK-p53 signaling and the cytoprotective MEK1/2 --> ERK1/2 cascade, as well as the inactivation of the anti-apoptotic AKT activity may explain, at least in part, the sequence-dependent enhancement of Taxol-induced cytotoxicity and apoptosis that follows inhibition of FAS activity in breast cancer cells. If chemically stable FAS inhibitors demonstrate systemic anticancer effects of FAS inhibition in vivo, these findings may render FAS as a valuable molecular target to enhance the efficacy of taxanes-based chemotherapy in human breast cancer.
Antineoplastic Agents, Phytogenic↗
siRNA for gene silencing: a route to drug target discovery.
The identification of RNA interference in mammalian cells, mediated via both virally-derived short interference RNA (siRNA) and endogenously produced microRNA, has revolutionised our understanding of the translational control of gene expression. Indeed, since its initial discovery, siRNA has been rapidly deployed for the elucidation of gene function and the identification of potential drug targets, a process often known as target discovery. In this review, we briefly discuss the mechanism of RNA interference and then critically examine the use of siRNA in target discovery, with a particular emphasis upon issues such as efficacy, selectivity, delivery and application in high-throughput studies.
Animals↗
[Knocking down rat Mecp2 expression by RNAi].
OBJECTIVE: To find the valid siRNA (small interference RNA) sequence to knock down rat Mecp2 expression for the analysis of Mecp2 function by RNA interference (RNAi). METHODS: A plasmid (pMecp2-RFP) expressing rat Mecp2 and marker gene red fluorescent protein (RFP) as a fusion gene was constracted. We first selected a candidate valid sequence by cotransfecting the pMecp2-RFP with 4 candidate siRNAs targeting rat Mecp2 to HEK293T cells respectively, and then identified the RNAi efficiency of the candidate valid siRNA by detecting its effect on suppressing the expression of endogenous rat Mecp2 in PC12 cells. RESULTS: siRNA sequence (5'-GCUGUGAAGGAAUCUUCUA-3') targeting rat Mecp2 mRNA 918-936nt was the valid sequence to knock down rat Mecp2 expression by RNA interference. As the target sequence was located in exon 4 of rat Mecp2 mRNA, we assumed that it could suppress the expression of Mecp2alpha and Mecp2beta at the same time. And as the target sequence was located in the coding region of rat Mecp2, we assumed that it could suppress the expression of both 1.9 kb and 10 kb of rat Mecp2 transcript. CONCLUSION: This study has laid the foundation for constructing rat Mecp2 knocked-down neuronal cell model to study the gene function of Mecp2 in brain development.
Animals↗
Different effects of enhanced and reduced expression of pub gene on the formation of embryoid bodies by cultured embryonic mouse stem cell.
The effects of pub gene on proliferation and initial stages of differentiation of embryonic mouse stem cells were studied in vitro. To this end we used enhanced expression of human pub gene (hpub) and suppression of expression of mouse endogenous pub gene with RNA-interference in embryonic stem cells. Proliferative activity of genetically modified polyclonal lines of the embryonic stem cells transfected with plasmids carrying expressing hpub gene or plasmids generating small interference RNA to this gene did not differ from that of the control cells. Inhibition of expression of endogenous pub gene in embryonic stem cells using small interference RNA 2-fold decreased the formation of embryoid bodies, at the same time additional expression of exogenous hpub gene almost 2-fold increased their number in comparison with the control. It was hypothesized that pub gene participates in early stages of differentiation of embryonic stem cells leading to the formation of embryoid bodies.
Analysis of Variance↗
RNAi: nature abhors a double-strand.
In organisms as diverse as nematodes, trypanosomes, plants, and fungi, double-stranded RNA triggers the destruction of homologous mRNAs, a phenomenon known as RNA interference. RNA interference begins with the transformation of the double-stranded RNA into small RNAs that then guide a protein nuclease to destroy their mRNA targets.
Animals↗
Transient RNA silencing of scoulerine 9-O-methyltransferase expression by double stranded RNA in Coptis japonica protoplasts.
RNAi (RNA interference, RNA silencing) is a powerful tool for functional genomics, but the construction of an RNAi vector(s) and the establishment of stable transformants are time-consuming and laborious. Here we report the transient RNAi of endogenous biosynthetic genes involved in isoquinoline alkaloid biosynthesis in Coptis japonica protoplasts. Double stranded (ds) RNA fragments of various lengths prepared from several different positions of the coding sequence of scoulerine 9-O-methyltransferase (SMT) were introduced into C. japonica protoplasts by polyethylene glycol-mediated transformation, and their effects were monitored by reverse transcription-polymerase chain reaction. Substantial silencing of SMT gene expression was obtained by the introduction of these SMT dsRNAs. A significant reduction in SMT protein levels was also observed. The potentials of this transient RNAi system to evaluate the functions of biosynthetic genes in Coptis alkaloid research are discussed.
Berberine↗
[Specific inhibition of hTERT gene expression by short interfering RNAs in gastric cancer SGC7901 cell].
OBJECTIVE: Activation of hTERT, the human telomerase catalytic subunit, has been implicated as the critical event in triggering telomerase activity of cancer cells. In present research, we investigated whether RNA interference (RNAi) induced by small interference RNA (siRNA) could suppress human telomerase catalytic unit (hTERT) gene expression in gastric SGC7901 cells. METHODS: As a pilot study, we utilized green fluorescent protein (GFP) plasmid pCX-GFP (5 510 bp) as a reporter system and generated constructs SHi-pU6-GFP expressing small hairpin RNA (shRNA) specific for green fluorescence protein (GFP) in K562 and SGC7901 cell respectively. Furthermore, we constructed pU6-hTERT-siRNAs carried hairpin siRNA for hTERT gene and transfected in SGC7901 by using Lipofectamine trade mark 2000. The expression of hTERT gene was detected by reverse transcription polymerase chain reaction (RT-PCR) and fluorescence quantitative polymerase chain reaction (FQ-PCR) assay. RESULTS: Our pilot study showed the short hairpin RNA (shRNA) expression vector driven by the murine U6 small nuclear RNA promoter can specifically induce potent gene knockdown effect (i.e., inhibit GFP expression specifically) when transfected transiently into SGC7901 cell. The constructed pU6-hTERT-siRNAs carried hairpin siRNA for hTERT gene was proved to be the same as designed by restriction endonuclease analysis. pU6-hTERT-siRNAs were successfully transferred into SGC7901 cell and their stable expression were obtained. The expression of hTERT gene were specific inhibited by pU6-hTERT-siRNAs in SGC7901 cell. CONCLUSIONS: Short hairpin RNAs (shRNAs) could induce sequence-specific hTERT gene silencing in SGC7901 cell. Our results prove the feasibility of the U6 promoter-driven shRNA expression technique to be used to cancer gene therapy.
Catalytic Domain↗
[Inhibitory effect of siRNA targeting HIF-1alpha on differentiation of peripheral blood endothelial progenitor cells].
BACKGROUND & OBJECTIVE: Transcription factor hypoxia inducible factor-1 alpha (HIF-1alpha) is a key determinant of oxygen-dependent gene regulation in angiogenesis. Suppression of HIF-1alpha is important for exploring HIF-1-dependent processes and hypoxia-induced pathophysiologic events. This study applied RNA interference targeting HIF-1alpha to human peripheral blood endothelial progenitor cells (EPCs) for therapeutic antineovascularization in vitro. METHODS: Small interference RNA (siRNA) targeting HIF-1alpha was constructed and transfected into human EPCs. Transfection efficiency of siRNA-HIF-1alpha was measured by reverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry, and flow cytometry. The content of endothelial nitric oxide synthase (eNOS) was measured by ELISA. The morphology of EPCs/endothelial cells (ECs) cultured in normoxia or hypoxia was observed after siRNA-HIF-1alpha transfection. RESULTS: SiRNA-HIF-1alpha was successfully constructed with the transfection efficiency of about 20%. After transfected for 6 h, HIF-1alpha mRNA was detected in EPCs cultured in normoxia or hypoxia with suppression; the expression of vascular endothelial growth factor (VEGF) was significantly attenuated (P < 0.05); the expression of HIF-1beta had no obvious change (P > 0.05). Stable expression of HIF-1alpha protein was induced by hypoxia, and was inhibited by siRNA-HIF-1alpha (P < 0.05). When cultured in 1% or 21% oxygen pressure for 3 days, CD31+ EPCs were inhibited more efficiently by siRNA-HIF-1alpha than by pEGFP (P < 0.05). The content of eNOS was significantly attenuated by siRNA-HIF-1alpha in time- and VEGF concentration-dependent manners (P < 0.01). EPCs differentiation and proliferation were induced by hypoxic culture, and suppressed by siRNA-HIF-1alpha. CONCLUSIONS: The constitutive or hypoxia-induced expression of HIF-1alpha in EPCs is sufficient to affect the expression of downstream target genes. SiRNA targeting HIF-1alpha could inhibit the expression of the genes which promote neovascularization, and suppress the differentiation of EPC to EC.
Aryl Hydrocarbon Receptor Nuclear Translocator↗
[Effect of ST6Gal I siRNA-mediated gene silencing on the adhesion and invasion of SW480 cells].
AIM: To study the effect of synthesized ST6Gal I specific siRNA on the adhesion and invasiveness of human colon carcinoma cell line SW480 with over expression of ST6Gal I. METHODS: A double strand small interference RNA (siRNA) targeting ST6Gal I was designed and synthesized, and then transfected into SW480 cells by lipofectmine 2000. SW480 cells were cultured and divided into 4 groups: blank control group, liposome control group, non-specific siRNA group and ST6Gal I siRNA group. The expression of ST6Gal I mRNA was examined by RT-PCR and the amount of alpha-2, 6-sialylation on the SW480 cell surface was detected by flow cytometry. The adhesion and invasion of SW480 cells to extracellular matrix (ECM) were analyzed by using CytoMatrix kit and cell invasion assay kit, respectively. RESULTS: After SW480 cells were transfected for 48 hours, the expression of ST6Gal I mRNA in ST6Gal I siRNA group was significantly decreased compared with that in the blank control group, liposome control group, and non-specific siRNA group (P<0.05). After SW480 cells were transfected for 72 hours, the amount of alpha-2, 6-sialylation on cell surface, the adhesion and invasion of the cells in ST6Gal I siRNA group were markedly lower than those in the other 3 groups (P<0.05). CONCLUSION: The chemically synthesized specific siRNA targeting ST6Gal I can effectively inhibit the expression of ST6Gal I and reduce cell adhesion and invasion to ECM in SW480 cells. Our research is important for further study of anti-tumor treatment with RNA interference.
Cell Adhesion↗
The gene-silencing effect of siRNA in cationic lipoplexes is enhanced by incorporating pDNA in the complex.
Efficient delivery is a key issue in translating interference RNA technology into a feasible therapy. The efficiency of carrier systems used for this technology is commonly tested by co-transfection, i.e. simultaneous transfection with an exogenous gene and with the siRNA. Two approaches can be distinguished: (1) with the two transfectants in the same carrier complex (siRNA/pDNA/carrier) and (2) with the two transfectants in different carrier complexes (pDNA/carrier and siRNA/carrier). The process to prepare the nucleic acid(s)-carrier complexes and the transfection procedure may affect the effectiveness of the gene-silencing process. In this study, two preparation methods were compared, namely the co-preparation of an siRNA/pDNA/liposome lipoplex (Method I) and the separate preparation of an siRNA/liposome lipoplex and a pDNA/liposome lipoplex (Method II). siRNA in the lipoplex produced by Method I showed a stronger gene-silencing effect than that in the lipoplexes prepared by Method II. There was no significant difference between the two methods in the amount of siRNA delivered to cells. Cellular entry and intracellular trafficking of siRNA/pDNA/liposome lipoplex is likely to differ from those of the separate lipoplexes. When in Method II non-transcriptional pDNA was included in the complex with siRNA, the gene-silencing effect was significantly enhanced. If and to what extent the experimental design is suitable to quantify RNA interference remains to be demonstrated.
Animals↗
High glucose upregulates connective tissue growth factor expression in human vascular smooth muscle cells.
BACKGROUND: Connective tissue growth factor (CTGF) is a potent profibrotic factor, which is implicated in fibroblast proliferation, angiogenesis and extracellular matrix (ECM) synthesis. It is a downstream mediator of some of the effects of transforming growth factor beta (TGFbeta) and is potentially induced by hyperglycemia in human renal mesangial cells. However, whether high glucose could induce the CTGF expression in vascular smooth muscle cells (VSMCs) remains unknown. Therefore, this study was designed to test whether high glucose could regulate CTGF expression in human VSMC. The effect of modulating CTGF expression on VSMC proliferation and migration was further investigated. RESULTS: Expression of CTGF mRNA was up-regulated as early as 6 hours in cultured human VSMCs after exposed to high glucose condition, followed by ECM components (collagen type I and fibronectin) accumulation. The upregulation of CTGF mRNA appears to be TGFbeta-dependent since anti-TGFbeta antibody blocks the effect of high glucose on CTGF gene expression. A small interference RNA (siRNA) targeting CTGF mRNA (CTGF-siRNA) effectively suppressed CTGF up-regulation stimulated by high glucose up to 79% inhibition. As a consequence of decreased expression of CTGF gene, the deposition of ECM proteins in the VSMC was also declined. Moreover, CTGF-siRNA expressing vector partially inhibited the high glucose-induced VSMC proliferation and migration. CONCLUSION: Our data suggest that in the development of macrovascular complications in diabetes, CTGF might be an important factor involved in the patho-physiological responses to high glucose in human VSMCs. In addition, the modulatory effects of CTGF-siRNA during this process suggest that specific targeting CTGF by RNA interference could be useful in preventing intimal hyperplasia in diabetic macrovascular complications.
Cell Movement↗
HIV Nef-mediated CD4 down-regulation is adaptor protein complex 2 dependent.
Nef is a crucial viral protein for HIV to replicate at high titers and in the development of AIDS. One Nef function is down-regulating CD4 from the cell surface, which correlates with Nef-enhanced viral pathogenicity. Nef down-regulates CD4 by linking CD4 to clathrin-coated pits. However, the mechanistic connection between the C-terminal dileucine motif of Nef and the component(s) of the clathrin-coated pits has not been pinpointed. In this report we used two AP-2 complex-specific inhibitors: a dominant negative mutant of Eps15 (Eps15DIII) that binds to the alpha subunit of AP-2 complex and a small interference RNA that is specific for the mu2 subunit of AP-2 complex. We show that both HIV Nef- and SIV Nef-mediated CD4 down-regulations were profoundly blocked by the synergistic effect of Eps15DIII and RNA interference of AP-2 expression. The results demonstrate that HIV/SIV Nef-mediated CD4 down-regulation is AP-2 dependent. We also show that the PMA-induced CD4 down-regulation was blocked by these two inhibitors. Therefore, PMA-induced CD4 down-regulation is also AP-2 dependent. The results demonstrate that, like the tyrosine sorting motif-dependent endocytosis (for which the transferrin receptor and the epidermal growth factor receptor are the two prototypes), dileucine sorting motif-dependent endocytosis of Nef and CD4 are also AP-2 dependent.
Adaptor Protein Complex 2↗