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Results for “Small interfering RNA”

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At least 73 records · Page 4Linked to original sources
PubMed2002

RNAi in human cells: basic structural and functional features of small interfering RNA.

We investigated the mechanism of RNA interference (RNAi) in human cells. Here we demonstrate that the status of the 5' hydroxyl terminus of the antisense strand of a siRNA determines RNAi activity, while a 3' terminus block is tolerated in vivo. 5' hydroxyl termini of antisense strands isolated from human cells were phosphorylated, and 3' end biotin groups were not efficiently removed. We found no requirement for a perfect A-form helix in siRNA for interference effects, but an A-form structure was required for antisense-target RNA duplexes. Strikingly, crosslinking of the siRNA duplex by psoralen did not completely block RNA interference, indicating that complete unwinding of the siRNA helix is not necessary for RNAi activity in vivo. These results suggest that RNA amplification by RNA-dependent RNA polymerase is not essential for RNAi in human cells.

Animals↗
PubMed2006

Induction of CML28-specific cytotoxic T cell responses using co-transfected dendritic cells with CML28 DNA vaccine and SOCS1 small interfering RNA expression vector.

CML28 is an attractive target for antigen-specific immunotherapy. SOCS1 represents an inhibitory control mechanism for DC antigen presentation and the magnitude of adaptive immunity. In this study, we evaluated the potential for inducing CML28-specific cytotoxic T lymphocytes (CTL) responses by dendritic cells (DCs)-based vaccination. We constructed a CML28 DNA vaccine and a SOCS1 siRNA vector and then cotransfect monocyte-derived DCs. Flow cytometry analysis showed gene silencing of SOCS1 resulted in higher expressions of costimulative moleculars in DCs. Mixed lymphocyte reaction (MLR) indicated downregulation of SOCS1 stronger capability to stimulate proliferation of responder cell in DCs. The CTL assay revealed transfected DCs effectively induced autologous CML28-specific CTL responses and the lytic activities induced by SOCS1-silenced DCs were significantly higher compared with those induced by SOCS1-expressing DCs. These results in our study indicates gene silencing of SOCS1 remarkably enhanced the cytotoxicity efficiency of CML28 DNA vaccine in DCs.

Antigens, Neoplasm↗
PubMed2004

Effect of inhibition of EBV-encoded latent membrane protein-1 by small interfering RNA on EBV-positive nasopharyngeal carcinoma cell growth.

OBJECTIVE: To evaluate the feasibility of using small interfering RNA (siRNA) for selective inhibiting latent membrane protein-1 (LMP1) expression in Epstein-Barr virus (EBV)-positive nasopharyngeal carcinoma (NPC) cell line C611, and observe the effects of LMP1 gene silencing on the NPC cell growth. METHODS: Four synthesized double-strand siRNA were respectively transfected into NPC cells, using Oligofectamine reagent. The subsequent changes in LMP1 mRNA level were determined by semi-quantitative reverse transcriptase (RT)-PCR. Flow cytometry and MTT assay were employed to examine the alterations in cell cycle and cell proliferation. RESULTS: The most effective sequence was identified among the 4 candidate siRNAs, and its single dose caused nearly 90% loss of LMP1 mRNA in C611 cells, with sustained specific inhibition for 96 h following a re-transfection. LMP1 siRNA treatment resulted in cell cycle arrest at G(0)-G(1) phase, accompanied by a reduction of cell proliferation by 33%, whereas EBV-negative NPC cells appeared unaffected. CONCLUSIONS: EBV-encoded LMP-1 is vulnerable to RNA interference and selective inhibition of LMP1 suppresses the proliferation of EBV-positive NPC cells, a finding that sheds light on the possible use of RNA interference in further investigations of LMP1 and for therapeutic purposes of EBV-related NPC.

Apoptosis↗
PubMed2006

Knockdown of survivin expression by small interfering RNA suppresses proliferation of two human cancer cell lines.

OBJECTIVE: To construct an expression vector of small interfering RNA (siRNA) against survivin and observe its effects on survivin expression and proliferation of human pancreatic cancer cell line PC-2 and breast cancer cell line MCF-7. METHODS: Constructed an expression vector of siRNA against survivin and transfected it into PC-2 and MCF-7 cells using lipofectamine 2000. The expression of survivin was detected by semi-quantitive RT-PCR and immunohistochemistry, and its effects on proliferation of PC-2 and MCF-7 cells were detected by MTT assay. RESULTS: The introduction of sequence-specific siRNA could efficiently suppress survivin expression at both mRNA and protein levels in the two cancer cell lines. In PC-2 cells, the expression inhibition rates were 81.25% at mRNA level and 74.24% at protein level. In MCF-7 cells, the expression inhibition rates were 64.91% at mRNA level and 79.72% at protein level. The proliferation of PC-2 and MCF-7 cells was also suppressed, and 24 and 48 hours after the cells were reseeded, the proliferation inhibition rates of PC-2 cells were 28.00% and 33.38%, and that of MCF-7 cells were 31.58% and 33.02%, respectively. CONCLUSIONS: The expression vector of siRNA against survivin can block survivin expression in PC-2 and MCF-7 cells efficiently and specifically. Down regulation of survivin expression can suppress proliferation of PC-2 and MCF-7 cells. Survivin RNAi may have potential value in gene therapy of human cancers.

Base Sequence↗
PubMed2003

Use of adeno-associated viral vector for delivery of small interfering RNA.

Post-transcriptional gene silencing by small interfering RNAs (siRNAs) is rapidly becoming a powerful tool for genetic analysis of mammalian cells. Delivery of siRNA into mammalian cells is usually achieved via the transfection of double-stranded oligonucleotides or plasmids encoding RNA polymerase III promoter-driven small hairpin RNA. Recently, retroviral vectors have been used for siRNA delivery, which overcome the problem of poor transfection efficiency seen with the plasmid-based systems. However, retroviral vectors have several limitations, such as the need for active cell division for gene transduction, oncogenic potential, low titers and gene silencing. In this report, we have adapted a commercially available adenoassociated virus (AAV) vector for siRNA delivery into mammalian cells. We demonstrate the ability of this modified vector to deliver efficiently siRNA into HeLa S3 cells and downregulate p53 and caspase 8 expression. Our results suggest that AAV-based vectors are efficient vectors for the delivery of siRNA into mammalian cells. Based on the known ability of these vectors to infect both dividing and nondividing cells, their use as a therapeutic tool for the delivery of siRNA deserves further study.

Caspase 8↗
PubMed2006

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

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

Apoptosis↗
PubMed2004

Silencing of SARS-CoV spike gene by small interfering RNA in HEK 293T cells.

Two candidate small interfering RNAs (siRNAs) corresponding to severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike gene were designed and in vitro transcribed to explore the possibility of silencing SARS-CoV S gene. The plasmid pEGFP-optS, which contains the codon-optimized SARS-CoV S gene and expresses spike-EGFP fusion protein (S-EGFP) as silencing target and expressing reporter, was transfected with siRNAs into HEK 293T cells. At various time points of posttransfection, the levels of S-EGFP expression and amounts of spike mRNA transcript were detected by fluorescence microscopy, flow cytometry, Western blot, and real-time quantitative PCR, respectively. The results showed that the cells transfected with pEGFP-optS expressed S-EGFP fusion protein at a higher level compared with those transfected with pEGFP-S, which contains wildtype SARS-CoV spike gene sequence. The green fluorescence, mean fluorescence intensity, and SARS-CoV S RNA transcripts were found significantly reduced, and the expression of SARS-CoV S glycoprotein was strongly inhibited in those cells co-transfected with either EGFP- or S-specific siRNAs. Our findings demonstrated that the S-specific siRNAs used in this study were able to specifically and effectively inhibit SARS-CoV S glycoprotein expression in cultured cells through blocking the accumulation of S mRNA, which may provide an approach for studies on the functions of SARS-CoV S gene and development of novel prophylactic or therapeutic agents for SARS-CoV.

Cell Line↗
PubMed2005

Electro-transfer of small interfering RNA ameliorated arthritis in rats.

RNA interference provides the powerful means of sequence-specific gene silencing. Particularly, small interfering RNA (siRNA) duplexes may be potentially useful for therapeutic molecular targeting of human diseases, although novel delivery systems should be devised to achieve efficient and organ-specific transduction of siRNA. In the present study, we demonstrated that electro-transfer of a siRNA-polyamine complex made efficient and specific gene knockdown possible in the articular synovium. Targeted suppression of the tumor necrosis factor-alpha gene through this procedure significantly ameliorated collagen-induced arthritis in rats. Our results suggest the potential feasibility of therapeutic intervention with RNA medicines for treatment of rheumatoid and other locomotor diseases.

Animals↗
PubMed2003

Immune modulation by silencing IL-12 production in dendritic cells using small interfering RNA.

RNA interference is a mechanism of posttranscriptional gene silencing that functions in most eukaryotic cells, including human and mouse. Specific gene silencing is mediated by short strands of duplex RNA of approximately 21 nt in length (termed small interfering RNA or siRNA) that target the cognate mRNA sequence for degradation. We demonstrate here that RNAi can be used for immune modulation by targeting dendritic cell (DC) gene expression. Transfection of DC with siRNA specific for the IL-12 p35 gene resulted in potent suppression of gene expression and blockade of bioactive IL-12 p70 production without affecting unrelated genes or cellular viability. Inhibition of IL-12 was associated with increased IL-10 production, which endowed the DC with the ability to stimulate production of Th2 cytokines from allogenic T cells in vitro. Furthermore, siRNA-silenced DC lacking IL-12 production were poor allostimulators in MLR. IL-12-silenced and KLH-pulsed DC polarized the immune response toward a Th2 cytokine profile in an Ag-specific manner. These data are the first to demonstrate that RNA interference is a potent and specific tool for modulating DC-mediated immune responses.

Adjuvants, Immunologic↗
PubMed2005

Delivery of small interfering RNA to mammalian cells in culture by using cationic lipid/polymer-based transfection reagents.

RNA interference (RNAi) has become a powerful tool for the knockdown of target gene expression and subsequent phenotypic analysis of gene function in mammalian cells in culture. Critical to the success of any small inhibitory RNA (siRNA)-mediated RNAi knockdown in mammalian cells is the efficient delivery of the siRNA to those cells. This chapter describes the use of popular cationic lipid?polymer-based transfection reagents for in vitro siRNA delivery and includes a general protocol with special emphasis on key transfection parameters important to the success of siRNA delivery.

Animals↗
PubMed2006

[Inhibitory effects of small interfering RNA specific to protein kinase CK2a on the growth of laryngeal carcinoma cells].

OBJECTIVE: To assess the effect of small interfering RNA (siRNA) specific to protein kinase CK2a on proliferation and apoptosis of Hep-2 cell line. METHODS: siRNA expression plasmid psiRNA-hH1neo-CK2 specific to protein kinase CK2a and non-specific siRNA expression plasmid psiRNA-hH1neo-cont were constructed respectively, and then were transfected into Hep-2 cells by lipofectamine methods. Protein kinase CK2a mRNA and protein of the transfected cells were detected by reverse transcription polymerase chain reaction (RT-PCR) and Western Blot, respectively. Proliferation and apoptosis of the transfected cells were observed by methyl thiazolyl tetrazolium (MTT) method and flow cytometry (FCM), respectively. RESULTS: Protein kinase CK2a mRNA and protein expressions were significantly decreased in the cells transfected with psiRNA-hH1neo-CK2 (P < 0.05). The Hep-2 cells grew slowly after transfected with psiRNA-hH1neo-CK2(P < 0.05). Obvious subdiploid peaks were found in the cells transfected with psiRNA-hH1neo-CK2 (P < 0.05). CONCLUSIONS: siRNA expression plasmid specific to protein kinase CK2a suppressed the protein kinase CK2a expression and the proliferation of Hep-2, and induced apoptosis of Hep-2 cells.

Carcinoma↗
PubMed2006

Expression of vector-based small interfering RNA against West Nile virus effectively inhibits virus replication.

RNA interference is one of the effective emerging anti-viral strategies to inhibit virus infection in cells. In this study, a small interfering RNA expressing vector (pSilencer-NS5) targeting the NS5 gene of West Nile virus (WNV) was employed to target and destroy WNV transcripts. Real-time PCR revealed drastic reduction in WNV RNA transcripts in pSilencer-NS5-transfected Vero cells. The virus infectious titre was also significantly reduced by 90% as determined by plaque assays. The resulting decrease in virus replication was shown to be specific since both scrambled and nucleotide(s) mismatch siRNA against WNV NS5 gene did not have any effect on WNV productive yields. Furthermore, Western immunoblot analysis on the expression of viral NS5 and envelope (E) proteins showed significant down-regulation on the expression of viral NS5 and envelope (E) proteins in virus-infected cells that were pre-transfected with pSilencer-NS5. These data clearly supported the notion that the expression of vector-based siRNA against WNV NS5 gene is able to exert its silencing effect on WNV-infected cells without inducing cytotoxicity, hence holding promise in therapeutic treatment of this important emerging infectious disease.

Animals↗
PubMed2006

Nuclear factor-kB signaling pathway constitutively activated in esophageal squamous cell carcinoma cell lines and inhibition of growth of cells by small interfering RNA.

Although constitutive nuclear factor (NF)-kappaB activation has been reported in many human tumors, the role of the NF-kappaB pathway in esophageal squamous cell carcinoma (ESCC) has not been known. In this study, NF-kappaB pathway in two ESCC cell lines was investigated using immunocytochemistry, Western blot and reverse transcription-polymerase chain reaction. The activation of NF-kappaB DNA binding was determined by electrophoretic mobility-shift assay. RNA interference was used to specifically inhibit the expression of p65. Growth of cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The results showed that p50, p65, IkappaBalpha p-IkappaBalpha and IkappaB kinase beta were expressed and mainly localized in the cytoplasm. Reverse transcription-polymerase chain reaction results showed the constitutive expressions of p50, p65 and IkappaBalpha mRNA in the two ESCC cell lines. Furthermore, the nuclear extracts revealed that p50 and p65 translocated to the nucleus had DNA-binding activity. Finally, small interfering RNA of p65 decreased the expression of p65, and the viability of cells transfected with p65 small interfering RNA was significantly suppressed at the same concentration of 5-fluorouracil (P < 0.05) compared to untransfected cells. The results of this study showed that there was the constitutively activated NF-kB signaling pathway in the ESCC cell lines. RNA interference targeting at p65 increased the sensitivity of the ESCC cell lines to 5-fluorouracil, suggesting that NF-kappaB might be a good target for cancer treatment.

Carcinoma, Squamous Cell↗
PubMed2006

Small interfering RNA expression vector targeting hypoxia-inducible factor 1 alpha inhibits tumor growth in hepatobiliary and pancreatic cancers.

Hepatobiliary and pancreatic carcinomas are hypovascular tumors that can proliferate under hypoxic conditions. Recent reports have demonstrated that hypoxia-inducible factor 1 alpha (HIF1alpha) plays an important role in the survival of these cancers. Given these findings, the inhibition of the HIF1alpha pathway might prove to be a powerful tool in the treatment of these cancers. To inhibit HIF1alpha expression, we used small interference RNA (siRNA) expression vectors in this study. The transient transfection of siRNA expression vectors significantly reduced both HIF1alpha mRNA levels (13% of control) and protein levels (41% of control) and significantly inhibited the growth of cancer cell lines (P<0.05). VEGF, Glut1, and aldorase A expressions were also significantly reduced by transfection with these vectors (P<0.05), and we found that these vectors induced apoptosis but not cell cycle arrest. In a subcutaneous tumor model using nude mice, transfected MIA PaCa-2 cells, stably expressing siRNAs, barely formed tumors compared to control (P<0.05). This study thus demonstrates the usefulness of siRNA expression vector in targeting HIF1alpha and points to a potential clinical role in the treatment of pancreatic and hepatobiliary carcinomas.

Animals↗
PubMed2007

Decoy receptor-2 small interfering RNA (siRNA) strategy employing three different siRNA constructs in combination defeats adenovirus-transferred tumor necrosis factor-related apoptosis-inducing ligand resistance in lung cancer cells.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in cancer cells but not in normal cells. However, studies have indicated that more than half of human tumors exhibit TRAIL resistance. Although the mechanism of TRAIL resistance is not understood, it represents a barrier to any TRAIL-mediated gene therapy approach. In addition, no correlation between TRAIL receptor (TRAIL-R) expression profile and TRAIL resistance has been demonstrated in cancer cells. In this study, three different lung cancer cell lines and three different primary cell cultures established from patients with lung cancer (two patients with squamous cell lung carcinoma and one with adenocarcinoma) were screened for sensitivity to adenoviral delivery of TRAIL. Whereas TRAIL-resistant primary lung cell cultures and the A549 lung cancer cell line exhibited high levels of surface decoy receptor-2 (DcR2/TRAIL-R4) expression, TRAIL-sensitive lung cancer cell lines (HBE and H411) failed to express it. A DcR2 short interfering RNA (siRNA) approach involving three different siRNA constructs in combination downregulated DcR2/TRAIL-R4 expression and sensitized lung cancer cells to TRAIL-induced apoptosis. Immunohistochemical staining of samples from 10 patients with lung carcinoma suggested that high-level DcR2/TRAIL-R4 expression is a common phenotype observed in patients with non-small cell lung carcinoma.

Adenocarcinoma↗