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Results for “Small interfering RNA”

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At least 163 records · Page 9Linked to original sources

PubMed2003

Inhibition of breast and ovarian tumor growth through multiple signaling pathways by using retrovirus-mediated small interfering RNA against Her-2/neu gene expression.

The Her-2/neu oncogene is overexpressed in approximately 30% of breast and ovarian cancer cases and often indicates a poor prognosis. Therapeutic agents against Her-2/neu have been intensively sought over the past decade. Here we show that small interfering RNA (siRNA) can silence the expression of Her-2/neu in models of human breast or ovarian cancer through retrovirus-mediated transfer of an siRNA against Her-2/neu. Cells infected with retrovirus expressing anti-Her-2/neu siRNA exhibit slower proliferation, increased apoptosis, increased G0/G1 arrest, and decreased tumor growth. Changes in cell cycle-associated factors included decreased levels of phosphatidylinositol 3-kinase, pAkt, and cyclin D1 and increased levels of p27 and phosphorylated retinoblastoma protein. Knockdown of Her-2/neu expression by siRNA is also associated with increased expression of the anti-angiogenic factor thrombospondin-1 and decreased expression of the pro-angiogenic vascular endothelial growth factor, suggesting that Her-2/neu stimulates tumor growth at least in part by regulating angiogenesis. siRNA-mediated gene silencing of Her-2/neu and increasing the expression of thrombospondin-1 may be a useful therapeutic strategy for Her-2/neu-over-expressing breast or ovarian cancer.

Apoptosis↗

PubMed2004

Inhibition of glucocorticoid-induced apoptosis by targeting the major splice variants of BIM mRNA with small interfering RNA and short hairpin RNA.

Glucocorticoids (GCs) induce apoptosis in lymphocytes and are effective agents for the treatment of leukemia. The activated glucocorticoid receptor initiates a transcriptional program leading to caspase activation and cell death, but the critical signaling intermediates in GC-induced apoptosis remain largely undefined. We have observed that GC induction of the three major protein products of the Bcl-2 relative Bim (BimEL, BimS, and BimL) correlates with GC sensitivity in a panel of human precursor B-cell (pre-B) acute lymphoblastic leukemia (ALL) cell lines. To test the hypothesis that Bim facilitates GC-induced apoptosis, we reduced BIM mRNA levels and Bim protein levels by RNA interference in highly GC-sensitive pre-B ALL cells. Reducing Bim proteins by either electroporation of synthetic small interfering RNA (siRNA) duplexes or lentivirus-mediated stable expression of short hairpin RNA inhibited the activation of caspase-3 and increased cell viability following GC exposure. We also observed that the extent of GC resistance correlated with siRNA silencing potency. siRNA duplexes that reduced only BimEL or BimEL and BimL (but not BimS) exhibited less GC resistance than a potent siRNA that silenced all three major isoforms, implying that induction of all three Bim proteins contributes to cell death. Finally, the modulation of GC-induced apoptosis caused by Bim silencing was independent of Bcl-2 expression levels, negating the hypothesis that the ratio of Bim to Bcl-2 regulates apoptosis. These results offer evidence that the induction of Bim by GC is a required event for the complete apoptotic response in pre-B ALL cells.

Acetylcysteine↗

PubMed2006

Therapeutic RNA interference of malignant melanoma by electrotransfer of small interfering RNA targeting Mitf.

Microphthalmia-associated transcription factor (Mitf) is critically involved in melanin synthesis as well as differentiation of cells of the melanocytic lineage. Some earlier studies suggested that Mitf is also essential in the survival of melanoma cells, but this notion remains controversial. We synthesized short interfering RNA (siRNA) duplexes corresponding to the mitf sequence and transfected them into B16 melanoma. Lipid-mediated transfection in vitro of Mitf-specific siRNA resulted in specific downregulation of Mitf and of the tyrosinase that is a transcriptional target of Mitf. This treatment also remarkably reduced the viability of melanoma cells by inducing apoptosis. To examine the potential feasibility of RNAi therapy against melanoma, B16 cells were subcutaneously injected into syngenic mice and siRNA was transfected into the pre-established tumor by means of electroporation. The Mitf-specific siRNA drastically reduced outgrowth of subcutaneous melanoma, while nonspecific siRNA failed to affect tumor progression. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-based analysis of tumor specimens demonstrated that the tumor cells transfected with Mitf-siRNA effectively underwent apoptosis in vivo. The present results indicate that Mitf plays important roles in melanoma survival. Intratumor electrotransfer of Mitf-specific siRNA may provide a powerful strategy for therapeutic intervention of malignant melanoma.

Animals↗

PubMed2006

Fiber molecular model of atelocollagen-small interfering RNA (siRNA) complex.

Previously we represented molecular model of collagen triple helix-DNA double helix complex [G.M. Mrevlishvili, D.V. Svintradze, Int. J. Macromol. 36 (2005) 324-326; G.M. Mrevlishvili, D.V. Svintradze, Int. J. Macromol. 35 (2005) 243-245]. We also proved that during the complex formation hydration of triple helix destroys and forms new water bridges maintaining the complex nano-structure. In this paper we demonstrate that small interfering RNA binds to atelocollagen directly to form siRNA-atelocollagen fiber complex.

Animals↗

PubMed2005

Small interfering RNA and gene expression analysis using a multiplex branched DNA assay without RNA purification.

The authors have developed a novel multiplex detection system that quantitatively measures the expression level of 11 messenger RNAs (mRNAs) directly from cell lysates or tissue homogenates without RNA purification. The system incorporates branched DNA (bDNA) technology from Bayer and a multiplex bead array platform from Luminex. In this study, a 21-nt synthetic small interfering RNA (siRNA; specifically designed to knockdown interleukin-8 [IL-8] expression) was delivered into HeLa cells. Using the multiplex bDNA assay, gene expression levels were measured simultaneously from cell lysates for 11 genes. After treating the HeLa cells for 20 h with phorbol myristate acetate (PMA), IL-8 mRNA levels were induced by almost 50-fold; transfection with 30 nM IL-8-specific siRNA reduced the PMA-induced IL-8 mRNA by 80%. In addition, PMA induced mRNA expression in IL-1alpha (3-fold) and IL-6 (4-fold); however, the IL-8 siRNA did not affect the expression of either of these 2 cytokine genes, indicating that the siRNA was selective for IL-8 mRNA expression. Three housekeeping genes' expression levels were measured under all conditions tested. The multiplex bDNA assay provides a powerful tool for quantitative multiplex gene expression analysis directly from cell lysates, which could be extremely valuable for conservation of rare or difficult-to-obtain samples.

Cytokines↗

PubMed2006

Effective gene suppression using small interfering RNA in hard-to-transfect human T cells.

RNA interference (RNAi) is an evolutionarily conserved cellular defense mechanism that protects cells from hostile genes and regulates the function of normal genes during growth and development. In this study, we established proof of principle of small interfering RNA (siRNA) silencing in hard-to-transfect human T cell lines and primary human CD4 T cells. We used public and in-house programs to design four siRNAs each for GFP, for our novel cellular gene HALP, and for their corresponding scrambled siRNA controls. We generated siRNA expression cassettes (SECs) by PCR and directly transfected the PCR products into T cells using amaxa Nucleofector technology. The most effective SECs were selected and cloned into a TA cloning vector and titered with their respective controls to increase transfection efficiency. Flow cytometry and fluorescence microscopy analyses were performed for GFP siRNAs, and Northern blot analysis was done to assess the HALP silencing effect. These experiments demonstrate that SECs are an excellent screening tool to identify siRNA sequences effective in silencing expression of genes of interest. The vector expressing the most effective siRNA robustly inhibited GFP expression (up to 92%) in the context of co-transfection in human T cell lines and primary CD4 T cells. The optimized siRNA for our endogenous cellular gene HALP also silenced its target RNA expression by more than 90%. These studies demonstrate that the combination of SEC, siRNA expression vectors and Nucleofector technology can be successfully applied to hard-to-transfect human T cell lines and primary T cells to effectively silence genes.

Asparaginase↗

PubMed2002

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.

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.

Catalysis↗

PubMed2005

Intravesical administration of small interfering RNA targeting PLK-1 successfully prevents the growth of bladder cancer.

The mainstay in the management of invasive bladder cancer continues to be radical cystectomy. With regard to improvement of quality of life, however, therapies that preserve the bladder are desirable. We investigated the use of intravesical PLK-1 small interfering RNA (siRNA) against bladder cancer. Patients with bladder cancers expressing high levels of PLK-1 have a poor prognosis compared with patients with low expression. Using siRNA/cationic liposomes, the expression of endogenous PLK-1 could be suppressed in bladder cancer cells in a time- and dose-dependent manner. As a consequence, PLK-1 functions were disrupted. Inhibition of bipolar spindle formation, accumulation of cyclin B1, reduced cell proliferation, and induction of apoptosis were observed. In order to determine the efficacy of the siRNA/liposomes in vivo, we established an orthotopic mouse model using a LUC-labeled bladder cancer cell line, UM-UC-3(LUC). PLK-1 siRNA was successfully transfected into the cells, reduced PLK-1 expression, and prevented the growth of bladder cancer in this mouse model. This is the first demonstration, to our knowledge, of inhibition of cancer growth in the murine bladder by intravesical siRNA/cationic liposomes. We believe intravesical siRNA instillation against bladder cancer will be useful as a therapeutic tool.

Administration, Intravesical↗

PubMed2005

Suppression of DNA methyltransferase 1 levels in head and neck squamous carcinoma cells using small interfering RNA results in growth inhibition and increase in Cdk inhibitor p21.

The ectopic expression of DNA methyltransferase 1 (DNMT 1) can transforms mammalian cells, and the inhibition of DNMT1 activity reverses that phenotypic transformation. Therefore, DNMT1 is considered to be an excellent target for therapeutic intervention. Previously, inhibition of DNMT1 was accomplished by using an antagonist or by antisense oligonucleotides. In this study, we examined the ability of the novel approach using small interfering RNA (siRNA) to disrupt the expression of DNMT1 in human non-small cell lung carcinoma A549 cells and the consequences of such an intervention. Transfection of DNMT1 siRNA decreased DNMT1 protein levels specifically and effectively. This decrease was accompanied by suppression of cell proliferation and colony-forming ability. The mechanism of this inhibition may be related to the increased levels of the cyclin dependent kinase inhibitor p21. These results suggest that the siRNA approach can be used to disrupt effectively DNMT1 activity and cancer cell growth.

Carcinoma, Squamous Cell↗

PubMed2005

CXCR4 knockdown by small interfering RNA abrogates breast tumor growth in vivo.

Breast cancer cells express the chemokine receptor CXCR4 and frequently metastasize to organs with an abundant source of the CXCR4 ligand, stromal cell-derived factor 1 (SDF-1). The chemokine receptor CXCR4 plays an active role in the metastasis of breast cancer. Here, we show that a small interfering RNA (siRNA) against CXCR4 effectively downregulates CXCR4 expression in human MDA-MB-231 breast cancer cells, leading to significant decrease in breast cancer cell invasion and adhesion. It was further found that tumor cells lacking CXCR4 expression proliferated at a much slower rate than control cells in vitro. Surprisingly, tumor cells lacking CXCR4 expression failed to grow in SCID mice in repeated experiments, providing the first direct evidence for an essential role of CXCR4 in breast cancer growth in vivo. This finding suggests an expanded role for the CXCR4 molecule in tumor cell growth in vivo, in addition to its role in breast cancer metastasis. This study implies the CXCR4 molecule as a potential target to control breast tumor growth as well as metastasis.

Animals↗

PubMed2004

[Reversing multidrug resistance in breast cancer cell line MCF-7/ADR by small interfering RNA].

BACKGROUND & OBJECTIVE: Multidrug resistance of tumor cells often leads to failure of chemotherapy. The over-expression of P-glycoprotein (P-gp), encoded by multidrug resistance 1 (mdr1) gene, plays an important role in multidrug resistance of breast cancer. This study was to explore the feasibility of silencing mdr1 gene by small interfering RNA (siRNA) in drug resistant breast cancer cell line MCF-7/ADR. METHODS: The siRNA oligonucleotides strand designed previously was inserted into pSilencer3.1-H1 Hygro vector, the plasmid was transformed into E.coli. After amplification, the plasmid was purified, and sequenced to determine whether the ligation between siRNA insert and the vector was correct, then transfected into MCF-7/ADR cells, and relevant sensitive MCF-7 cells. MCF-7/ADR cells were screened by hygromycin, surviving cells were cultured. The positive rate of P-gp was detected by flow cytometry, and positive rate of mdr1 gene was detected by real-time relatively quantitative polymerase chain reaction (PCR). Adriamycin (ADM) resistant experiment was performed on MCF-7/ADR cells with siRNA. RESULTS: Positive rate of P-gp in MCF-7/ADR cells was decreased from 99.8% (before siRNA transfection) to 12.3% (after siRNA transfection). Real- time PCR revealed that the threshold cycle value of MCF-7/ADR cells increased from 25.22 to 30.64 after transfected with siRNA. The IC(50) of ADM for MCF-7/ADR cells transfected with siRNA was 0.51 micromol/L, while that for MCF-7/ADR cells without transfection was 17.88 micromol/L. CONCLUSION: siRNA can silence mdr1 gene in MCF-7/ADR cells, may become a new, effective medical technique.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2006

Comparative studies of suppression of malignant cancer cell phenotype by antisense oligo DNA and small interfering RNA.

One of the distinguishing features of malignant tumor cells is the ability to proliferate in an anchorage-independent manner; methods that effectively suppress this phenotype may be applicable to the therapeutic inhibition of the malignancy of cancers. Interfering RNA is a potentially powerful tool for cancer therapy because of its specificity of target selection and remarkably high efficiency in target mRNA suppression. We studied the use of two knockdown strategies, antisense oligo DNA (AS-ODN) and small interfering RNA (siRNA), and showed how the anchorage-independent proliferation of malignant cells could be blocked efficiently. Anchorage-independent proliferation of rat fibroblasts transformed with v-src was suppressed with only a single 1-microM dose of AS-ODN; similar suppression using siRNA required treatment with 1 nM siRNA every 12 h. With our experimental system, the molecular stability of AS-ODN allowed the use of a simple treatment regimen to control the amount of the target molecule, providing that the treatment dose was sufficiently high. In comparison, siRNA treatment was effective at lower doses, but more frequent treatment was necessary to achieve the same suppression of proliferation.

Animals↗

PubMed2006

Antitumor activity of an oncolytic adenovirus-delivered oncogene small interfering RNA.

Despite successes in animal models, cancer gene therapy with small interfering RNAs (siRNA) is hindered by the lack of an optimal delivery platform. We examined the applicability of the replication-competent, oncolytic adenovirus, ONYX-411, to deliver a mutant K-ras siRNA transgene to human cancer cells. Proof-of-principle studies showed an additive tumor growth-inhibitory response through siRNA-mediated K-ras knockdown and ONYX-411-mediated cancer cell lysis. A novel construct, termed Internavec (for interfering RNA vector), was generated by cloning a K-ras(v12)-specific siRNA(ras-4) hairpin construct under the control of the human H1 promoter into the deleted E3b region of ONYX-411. Internavec acquired an increase in potency of approximately 10-fold in human cancer cells expressing the relevant K-ras(v12) mutation (H79, H441, and SW480), as defined by a reduction in the effective dose needed to achieve 50% growth inhibition (ED(50)). Internavec remained attenuated in nonmalignant epithelial cells. Daily intratumoral injections of Internavec (five daily injections of 1 x 10(8) plaque-forming units) significantly reduced the growth of s.c. H79 pancreatic cancer xenografts in nu/nu mice by 85.5%, including complete growth suppression in three of five mice. Parental ONYX-411 or ONYX-411-siRNA(GFP) was markedly less effective (47.8% growth reduction, P = 0.03; and 44.1% growth reduction, P = 0.03, respectively). siRNA(ras) transgene activity contributed to cell cycle blockage, increased apoptosis, and marked down-regulation of Ras signaling-related gene expression (AKT2, GSK3 beta, E2F2, and MAP4K5). These findings indicate that Internavec can generate a two-pronged attack on tumor cells through oncogene knockdown and viral oncolysis, resulting in a significantly enhanced antitumor outcome.

Adenocarcinoma↗

PubMed2005

Inhibition of Hepatitis B virus gene expression by single and dual small interfering RNA treatment.

RNA interference (RNAi) has been successfully applied in suppression of Hepatitis B virus (HBV) replication. To circumvent the problem that mutation in HBV genome may result in resistance when siRNA is further developed as an anti-viral drug, in this study, we established a dual small interfering RNA (siRNA) expression system, which could simultaneously express two different siRNA molecules that can specifically target two genes. To test the effectiveness of this system, we applied this new approach to express simultaneously two different 21-bp hairpin siRNA duplexes that specifically attack the HBs and HBx genes of HBV, respectively, in Bel-7402 and HepG2.2.15 cells. Results indicated that dual siRNA could simultaneously inhibit the expression of HBs and HBx gene by 83.7% and 87.5%, respectively, based on luciferase assays. In addition, dual siRNA molecules were able to significantly reduce the amount of HBV core associated DNA, which is considered as an intracellular replicative intermediate, and the viral DNA in culture supernatant. Therefore, this dual siRNA system provides a more powerful tool for the study of gene function and implicates a potential application in the treatment of viral infection.

Cell Line, Tumor↗

PubMed2005

Small interfering RNA targeting the PINK1 induces apoptosis in dopaminergic cells SH-SY5Y.

PTEN-induced kinase 1 (PINK1) is a recently identified gene, mutations of which cause levodopa-responsive parkinsonism. An over-expression of wild-type PINK1 protects neurons from stress-induced mitochondrial dysfunction and apoptosis. We studied the effects of PINK1 suppression using small interfering RNA (siRNA), which can inhibit PINK1 mRNA expression up to 87%, and decrease PINK1 protein up to 80% in human dopaminergic cell line SH-SY5Y. Incubation with PINK1 siRNA decreased SH-SY5Y cell viability and significantly increased MPP(+) or rotenone-induced cytotoxicity. Our results indicate that reduction in PINK1 expression can trigger apoptotic process that can be exacerbated by the presence of MPP(+) or rotenone. These findings support the hypothesis that PINK1 participates in the protection of dopaminergic neurons.

Amino Acid Sequence↗

PubMed2007

Genome-wide sreening by using small-interfering RNA expression libraries.

RNA interference (RNAi) is an evolutionarily conserved phenomenon in which gene expression is silenced by double-stranded RNA (dsRNA) in a sequence-specific manner. This technology has the potential to affect all aspects of target discovery and validation. With the completion of the human genome, it is now possible to design small-interfering RNA (siRNA) libraries targeting every human gene. Specific siRNAs, libraries containing a pathway, gene family, or gene set of interest, are expected to unsecure new targets in pathways of therapeutic interest. Here, we highlight the potential of siRNA screens for target identification by using cell-based assays.

Apoptosis↗

PubMed2006

Successful silencing of plasminogen activator inhibitor-1 in human vascular endothelial cells using small interfering RNA.

Clinical as well as experimental evidence suggests that vascular overexpression of plasminogen activator inhibitor (PAI)-1, the primary physiological inhibitor of both urokinase and tissue-type plasminogen activator, may be involved in the pathophysiology of atherosclerosis and cardiovascular disease. We investigated the feasibility, efficacy and functional effects of PAI-1 gene silencing in human vascular endothelial cells using small interfering RNA. Double-stranded 21 bp-RNA molecules targeted at sequences within the human PAI-1 gene were constructed. Successful siRNA transfection of HUVEC was confirmed using fluorescence microscopy and flow cytometry. One of five candidate siRNA sequences reduced PAI-1 mRNA and protein in a concentration- and time-dependent manner. Suppression of PAI-1 mRNA was detected up to 72 hours after transfection. Moreover, siRNA treatment reduced the activity of PAI-1 released from HUVEC, and prevented the oxLDL- or LPS-induced upregulation of PAI-1 secretion. Importantly, siRNA treatment did not affect the expression of other endothelial-cell markers. Moreover, downregulation of PAI-1 significantly enhanced the ability of endothelial cells to adhere to vitronectin, and this effect could be reversed upon addition of recombinant PAI-1. SiRNA-mediated reduction of PAI-1 expression may be a promising strategy for dissecting the effects of PAI-1 on vascular homeostasis.

Base Sequence↗

PubMed2005

[Regulatory effect of small interfering RNA targeting multidrug resistant protein 1 on chemosensitivity of human multiforme glioblastoma cell line BT325].

BACKGROUND & OBJECTIVE: Multidrug resistance is a major reason of failure of chemotherapy for glioma. Overexpression of P-glycoprotein (P-gp), encoded by multidrug resistant protein 1 (MDR1) gene, is one of the key factors. This study was to explore the regulatory effect of small interfering RNA (siRNA) targeting MDR1 on chemosensitivity of human multiforme glioblastoma cell line BT325. METHODS: MDR1 siRNAs containing sequences of 3,051-3,069 (MDR1 A group), 502-520 (MDR1 B group), and 1,534-1,552 (MDR1 C group) were designed, and transfected into BT325 cells. Positive clones were screened with puromycin. The expression of MDR1 was measured by reverse transcription-polymerase chain reaction (RT-PCR); the expression of P-gp was detected by immunohistochemistry and flow cytometry (FCM). Drug sensitivity assay was performed in the transfected cells. RESULTS: BT325 cells proliferated exponentially after MDR1 siRNA transfection. After transfection of MDR1 siRNAs, the expression of MDR1 mRNA was significantly lower in MDR1 A, B, and C groups than in control group (0.18+/-0.05, 0.30+/-0.09, and 0.36+/-0.13 vs. 0.76+/-0.06, P<0.001); the positive rate of P-gp was decreased from 85.73% to 1.44%; the 50% inhibitory concentrations (IC(50)) of doxorubicin and vincristine to BT325 cells were decreased markedly; the G0/G1 phase proportions of MDR1 A, B, and C groups were increased by 13.55%, 14.35%, and 1.46% of control, respectively (P<0.05). CONCLUSION: MDR1 siRNA may modulate multidrug resistance through down-regulating the expression of MDR1 gene, enhancing the chemosensitivity of glioma, and inducing cell apoptosis.

ATP Binding Cassette Transporter, Subfamily B, Mem↗