Small-interfering-RNA expression in cells based on an efficiently constructed dumbbell-shaped DNA.
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DNA↗
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Results for “Small interfering RNA”
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Chemoresistance: impact of nuclear factor (NF)-kappaB inhibition by small interfering RNA. Commentary re J. Guo et al., Enhanced chemosensitivity to irinotecan by RNA interference-mediated down-regulation of the NF-kappaB p65 subunit. Clin Cancer Res 2004;10:3333-3341.
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Animals↗
Re: Intravitreal injection of vascular endothelial growth factor small interfering RNA inhibits growth and leakage in a nonhuman primate, laser-induced model of choroidal neovascularization.
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Animals↗
Inhibition of ABCB1 (MDR1) and ABCB4 (MDR3) expression by small interfering RNA and reversal of paclitaxel resistance in human ovarian cancer cells.
Ovarian cancer is currently the most lethal gynecologic malignancy in developed countries, and paclitaxel is a cornerstone in the treatment of this malignancy. Unfortunately, the efficacy of paclitaxel is limited by the development of drug resistance. Clinical paclitaxel resistance is often associated with ABCB1 (MDR1) overexpression, and in vitro paclitaxel resistance typically demonstrates overexpression of the ABCB1 gene. In this study, we demonstrate that paclitaxel-resistant cell lines overexpress both ABCB1 and ABCB4 (MDR3). To evaluate the role of these transporters in paclitaxel-resistant ovarian cancer cells, small interference RNAs (siRNAs) were used to target ABCB1 and ABCB4 RNA in the paclitaxel-resistant SKOV-3TR and OVCAR8TR ovarian cancer cell lines. Treatment of these lines with either chemically synthesized siRNAs or transfection with specific vectors that express targeted siRNAs demonstrated decreased mRNA and protein levels of ABCB1 or ABCB4. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays of siRNA-treated cells demonstrated 7- to 12.4-fold reduction of paclitaxel resistance in the lines treated with the synthesized siRNA of ABCB1 and 4.7- to 7.3-fold reduction of paclitaxel resistance in the cell lines transfected with siRNA of ABCB1 expressing vectors. ABCB4 siRNA-treated cell lines showed minor reduction in paclitaxel resistance. These results indicate that siRNA targeted to ABCB1 can sensitize paclitaxel-resistant ovarian cancer cells in vitro and suggest that siRNA treatment may represent a new approach for the treatment of ABCB1-mediated drug resistance.
ATP Binding Cassette Transporter, Subfamily B↗
Knockdown of Ki-67 by small interfering RNA leads to inhibition of proliferation and induction of apoptosis in human renal carcinoma cells.
To investigate the effect of small-interfering RNA (siRNA) targeted against Ki-67, which is an attractive molecular target for cancer therapy, on inhibiting Ki-67 expression and cell proliferation in human renal carcinoma cells (HRCCs), siRNAs were used to inhibit the expression of Ki-67 in HRCCs. Ki-67 mRNA levels were detected by RT-PCR and in situ hybridization analysis. Ki-67 protein levels were detected by Western blot and immunocytochemistry analysis. TUNEL assay was used to measure the apoptosis of carcinoma cells. Results of RT-PCR and in situ hybridization demonstrated reduction of Ki-67 mRNA expression in Ki-67 siRNAs treated 786-0 cells. Similar reduction in Ki-67 protein measured by Western blot and immunocytochemistry was observed in cells transfected with Ki-67 siRNA. Ki-67-siRNA treatment of HRCCs resulted in specific inhibition of proliferation and increased apoptotic cell death. From these findings we conclude that inhibition of Ki-67 expression by siRNA may be a reasonable approach in renal cancer therapy.
Apoptosis↗
Bcl-XL small interfering RNA enhances sensitivity of Hepg2 hepatocellular carcinoma cells to 5-fluorouracil and hydroxycamptothecin.
Changes in drug sensitivity in Bcl-XL small interfering RNA (siRNA) transfected Hepg2 hepatocellular carcinoma cells were investigated in this study. Bcl-XL siRNA and negative siRNA expression vector were constructed and stably transfected into Hepg2 cells. Reverse transcription (RT)-PCR, western blot and immunofluorescence were used to detect the target gene expression at mRNA and protein levels. Drug sensitivity of the cells to 5-fluorouracil (5-FU) and hydroxycamptothecin (HCPT) were evaluated with MTT. The Bcl-XL mRNA and protein expression levels in Bcl-XL siRNA transfectants were reduced compared with negative siRNA transfectants or mock cells. MTT results showed that Bcl-XL siRNA transfected cells have a higher cell inhibition rate than negative vector transfected cells or untreated cells after treatment with 13, 130, 1300 and 13,000 mg/L of 5-FU. Bcl-XL siRNA transfected cells also showed increased drug-sensitivity compared with negative vector transfected cells or untreated cells after treatment with 0.18, 0.36, 0.72 and 1.44 mg/L HCPT. Flow cytometry (FCM) results demonstrated that the sub-G1 population increased in the Bcl-XL siRNA group, compared with the negative siRNA group and untreated control group, after the addition of 5-FU (1300 mg/L) and HCPT (0.72 mg/L). siRNA targeting Bcl-XL gene can specifically down-regulate Bcl-XL expression in Hepg2 cells, and can increase spontaneous cell apoptosis and sensitize cells to 5-FU or HCPT.
Antimetabolites, Antineoplastic↗
Distinct mechanisms determine transposon inheritance and methylation via small interfering RNA and histone modification.
Heritable, but reversible, changes in transposable element activity were first observed in maize by Barbara McClintock in the 1950s. More recently, transposon silencing has been associated with DNA methylation, histone H3 lysine-9 methylation (H3mK9), and RNA interference (RNAi). Using a genetic approach, we have investigated the role of these modifications in the epigenetic regulation and inheritance of six Arabidopsis transposons. Silencing of most of the transposons is relieved in DNA methyltransferase (met1), chromatin remodeling ATPase (ddm1), and histone modification (sil1) mutants. In contrast, only a small subset of the transposons require the H3mK9 methyltransferase KRYPTONITE, the RNAi gene ARGONAUTE1, and the CXG methyltransferase CHROMOMETHYLASE3. In crosses to wild-type plants, epigenetic inheritance of active transposons varied from mutant to mutant, indicating these genes differ in their ability to silence transposons. According to their pattern of transposon regulation, the mutants can be divided into two groups, which suggests that there are distinct, but interacting, complexes or pathways involved in transposon silencing. Furthermore, different transposons tend to be susceptible to different forms of epigenetic regulation.
Adenosine Triphosphatases↗
Recent developments in the application of plasmid DNA-based vectors and small interfering RNA therapeutics for cancer.
Increased understanding of the molecular pathological mechanisms of cancer, the advent of novel molecular tools such as synthetic small interfering RNA (siRNA) or plasmid DNA-based vectors (pDNA), and technology for the in vivo delivery of such biomolecular therapeutics have provided an encouraging perspective for cancer therapy. Numerous pDNAs and siRNAs have been tested in preclinical cancer models, and these first approaches have reached clinical evaluation. The therapeutic effector mechanisms include interference with neoangiogenesis, blockage of cell division, promotion of apoptosis and sensitization to chemotherapy, delivery of cytotoxic genes, and activation of anticancer immune responses. Physical methods have been developed for highly effective regional delivery. A series of innovative "smart" formulations directs the current development toward safe and effective systemic tumor-targeted delivery of pDNA and siRNA.
Genetic Therapy↗
[Effect of small interfering RNA targeting connective tissue growth factor on the proliferation of rat primary hepatic stellate cells].
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Animals↗
DICER-LIKE 4 functions in trans-acting small interfering RNA biogenesis and vegetative phase change in Arabidopsis thaliana.
Arabidopsis thaliana contains four DICER-LIKE (DCL) genes with specialized functions in small RNA biogenesis for RNA interference-related processes. A mutant with defects in DCL4 was identified and analyzed for microRNA- and endogenous, small interfering RNA (siRNA)-related functions. The dcl4-2 mutant contained normal or near-normal levels of microRNAs (21 nt) and heterochromatin-associated siRNAs (24 nt). In contrast, this mutant lacked each of three families of 21-nt trans-acting siRNAs (ta-siRNAs) and possessed elevated levels of ta-siRNA target transcripts. The dcl4-2 mutant resembled an rna-dependent RNA polymerase 6 mutant in that both mutants lacked ta-siRNAs and displayed heterochronic defects in which vegetative phase change was accelerated. Double mutant analyses with dcl2-1, dcl3-1, and dcl4-2 alleles revealed hierarchical redundancy among DCL activities, leading to alternative processing of ta-siRNA precursors in the absence of DCL4. These data support the concept that plants have specialized and compartmentalized DCL functions for biogenesis of distinct small RNA classes.
Arabidopsis↗
Induction of cell apoptosis in non-small cell lung cancer cells by cyclin A1 small interfering RNA.
Cyclin A1 and cyclin B1 are overexpressed in various tumors but are present at low levels in normal tissues. Cyclin A1 is restricted to germ cells undergoing meiosis. In order to explore the possibility of using cyclin A1 and cyclin B1 as anticancer targets, we knocked them down in two lung cancer cell lines, H157 and H596, using siRNA. As with cyclin A1 siRNA in lung cancer cell lines, cyclin B1, Cdc2 and CDK2 were all significantly downregulated. The S phase fraction increased significantly, and they eventually underwent apoptosis by way of downregulated intrinsic apoptotic pathways and modulators with upregulated extrinsic apoptotic pathways. Our study suggests that cyclin A1 might be a promising anticancer target specific to lung cancer.
Apoptosis↗
A plasmid-based system for expressing small interfering RNA libraries in mammalian cells.
BACKGROUND: RNA interference (RNAi) is an evolutionarily conserved process that functions to inhibit gene expression. The use of RNAi in mammals as a tool to study gene function has rapidly developed in the last couple of years since the discovery that the function-inhibiting units of RNAi are short 21-25 nt double-stranded RNAs (siRNAs) derived from their longer template. The use of siRNAs allows for gene-specific knock-down without induction of the non-specific interferon response in mammalian cells. Multiple systems have been developed to introduce siRNAs into mammals. One of the most appealing of these techniques is the use of vectors containing polymerase III promoters to drive expression of hairpin siRNAs. However, there are multiple limitations to using hairpin siRNA vectors including the observation that some are unstable in bacteria and are difficult to sequence. RESULTS: To circumvent the limitation of hairpin siRNA vectors we have developed a convergent opposing siRNA expression system called pHippy. We have generated pHippy vectors or expression cassettes that knock down the expression of both reporter and endogenous genes. As a proof of principle that pHippy can be used to generate random siRNA libraries, we generated a small siRNA library against PGL3 luciferase and demonstrated that we could recover functional siRNAs that knock down PGL3 luciferase. CONCLUSIONS: siRNA is a powerful tool to study gene function. We have developed a new vector with opposing convergent promoters for the expression of siRNAs, which can be used to knock down endogenous genes in a high throughput manner or to perform functional screening with random or cDNA-derived siRNA libraries.
Animals↗
Inhibition of bcr-abl gene expression by small interfering RNA sensitizes for imatinib mesylate (STI571).
Bcr-Abl proteins are effective inducers of the leukemic phenotype in chronic myeloid leukemia (CML) and distinct variants of acute lymphoblastic leukemia (ALL). Targeting bcr-abl by treatment with the selective tyrosine kinase inhibitor imatinib has proved to be highly efficient for controlling leukemic growth. However, it is unclear whether imatinib is sufficient to eradicate the disease because of primary or secondary resistance of leukemic cells. Therefore, targeting Bcr-Abl with an alternative approach is of great interest. We demonstrate that RNA interference (RNAi) with a breakpoint-specific short-interfering RNA (siRNA) is capable of decreasing Bcr-Abl protein expression and of antagonizing Bcr-Abl-induced biochemical activities. RNAi selectively inhibited Bcr-Abl-dependent cell growth. Furthermore, bcr-abl-homologous siRNA increased sensitivity to imatinib in Bcr-Abl-overexpressing cells and in a cell line expressing the imatinib-resistant Bcr-Abl kinase domain mutation His396Pro, thereby antagonizing 2 of the major mechanisms of resistance to imatinib.
Antineoplastic Agents↗
Small interfering RNA and gene silencing in transgenic mice and rats.
After short duplexes of synthetic 21-23 nt RNAs (siRNA) were reported to be effective in silencing specific genes, a vector-based approach for siRNAs was demonstrated in mammalian cultured cell lines. However, the effect of RNA interference (RNAi) on various differentiated cells in live animals remains unknown. In this report, we demonstrate that transgenically supplied siRNA can silence ubiquitously expressed enhanced green fluorescent protein in every part of the mouse and rat body. These results suggest that transgenic RNAi could function as an alternative method of gene silencing by applying homologous recombination to embryonic stem (ES) cells, and should be successful even in species where ES cell lines remain unestablished.
Animals↗
Modulation of human dUTPase using small interfering RNA.
Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) is responsible for maintaining low intracellular levels of dUTP, thus preventing the incorporation of dUTP into DNA. A 21 bp double-stranded RNA molecule (siRNAdUT3) targeted against motif 3 of human dUTPase resulted in a time- and dose-dependent decrease in dUTPase activity in transfected cells. dUTPase activity was reduced approximately 95+/-5% in all cell lines tested 48 h after transfection with 2 microg siRNAdUT3 and it was maintained at this decreased level for at least 72 h. Down-regulation of dUTPase resulted in a significant increase in intracellular dUTP and a decreased proliferation of the transfected cells. Therefore, we conclude that dUTPase activity/expression can be down-regulated using siRNA specifically targeted to dUTPase mRNA and that this approach can be used to elucidate the role of dUTPase in DNA metabolism, as well as, to determine whether dUTPase is a valid target for drug development.
Cell Line, Tumor↗
Gene silencing of CENP-E by small interfering RNA in HeLa cells leads to missegregation of chromosomes after a mitotic delay.
Centromeric protein-E (CENP-E) is a kinesin-like motor protein required for chromosome congression at prometaphase. Functional perturbation of CENP-E by various methods results in a consistent phenotype, i.e., unaligned chromosomes during mitosis. One unresolved question from previous studies is whether cells complete mitosis or sustain mitotic arrest in the presence of unaligned chromosomes. Using RNA interference and video-microscopy, we analyzed the dynamic process of mitotic progression of HeLa(H2B)-GFP cells lacking CENP-E. Our results demonstrate that these cells initiated anaphase after a delayed mitotic progression due to the presence of unaligned chromosomes. In some dividing cells, unaligned chromosomes are present during anaphase, causing nondisjunction of some sister chromatids producing aneuploid daughter cells. Unlike in Xenopus extract, the loss of CENP-E in HeLa cells does not impair gross checkpoint activation because cells were arrested in mitosis in response to microtubule-interfering agents. However, the lack of CENP-E at kinetochores reduced the hyperphosphorylation of BubR1 checkpoint protein during mitosis, which may explain the loss of sensitivity of a cell to a few unaligned chromosomes in the absence of CENP-E. We also found that presynchronization with nocodazole sensitizes cells to the depletion of CENP-E, leading to more unaligned chromosomes, longer arrest, and cell death.
Calcium-Binding Proteins↗
Suppression of human Mnk1 by small interfering RNA increases the eukaryotic initiation factor 4F activity in HEK293T cells.
Short-interfering RNAs (siRNAs) have proved to be a useful tool in studying gene function in plants, invertebrates and mammalian systems. Herein, we report the use of siRNAs for targeting the human MAP kinase-interacting kinase Mnk1 gene. This study demonstrates the efficacy of the designed siRNA in silencing Mnk1 in the human cell line HEK293T and shows that Mnk1 suppression decreases eukaryotic initiation factor 4E phosphorylation without causing any change in global protein synthesis rate and cell proliferation. Interestingly, suppression of Mnk1 results in a significant increase in eukaryotic initiation factor 4F complex formation after 72 h of transfection.
Animals↗
Downregulation of xIAP expression by small interfering RNA inhibits cellular viability and increases chemosensitivity to methotrexate in human hepatoma cell line HepG2.
The aim of this study was to investigate whether downregulating the expression of xIAP by RNAi (RNA interference) technology can induce the apoptosis of HepG2 cells, inhibit cellular viability and increase chemosensitivity of cancer cells. HepG2 cells were transfected with U6 promoter plasmids coding for short interfering RNAs (siRNAs) targeting xIAP. RT-PCR and western blot analysis were used to assess the mRNA and protein levels of xIAP expression. T he suppression efficiency o f xIAPby RNAi was evaluated using the MTT assay for cellular viability and Annexin V-PI binding assay for apoptosis. These results showed that siRNAs reduced cellular viability and increased cellular apoptosis. Moreover, downregulation of xIAP expression enhanced the chemosensitivity of HepG2 cells to methotrexate. These results suggest that the downregulation of xIAP by RNAi could potentially be used as a therapeutic strategy for human hepatocellular carcinoma.
Antimetabolites, Antineoplastic↗