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Results for “RNA interference”

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At least 757 records · Page 42Linked to original sources

PubMed2004

Lentivirus-mediated RNA interference of DC-SIGN expression inhibits human immunodeficiency virus transmission from dendritic cells to T cells.

In the early events of human immunodeficiency virus type 1 (HIV-1) infection, immature dendritic cells (DCs) expressing the DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) receptor capture small amounts of HIV-1 on mucosal surfaces and spread viral infection to CD4(+) T cells in lymph nodes (22, 34, 45). RNA interference has emerged as a powerful tool to gain insight into gene function. For this purpose, lentiviral vectors that express short hairpin RNA (shRNA) for the delivery of small interfering RNA (siRNA) into mammalian cells represent a powerful tool to achieve stable gene silencing. In order to interfere with DC-SIGN function, we developed shRNA-expressing lentiviral vectors capable of conditionally suppressing DC-SIGN expression. Selectivity of inhibition of human DC-SIGN and L-SIGN and chimpanzee and rhesus macaque DC-SIGN was obtained by using distinct siRNAs. Suppression of DC-SIGN expression inhibited the attachment of the gp120 envelope glycoprotein of HIV-1 to DC-SIGN transfectants, as well as transfer of HIV-1 to target cells in trans. Furthermore, shRNA-expressing lentiviral vectors were capable of efficiently suppressing DC-SIGN expression in primary human DCs. DC-SIGN-negative DCs were unable to enhance transfer of HIV-1 infectivity to T cells in trans, demonstrating an essential role for the DC-SIGN receptor in transferring infectious viral particles from DCs to T cells. The present system should have broad applications for studying the function of DC-SIGN in the pathogenesis of HIV as well as other pathogens also recognized by this receptor.

Amino Acid Sequence↗

PubMed2002

RNA interference reveals a requirement for myocyte enhancer factor 2A in activity-dependent neuronal survival.

RNA interference (RNAi) provides a powerful method of gene silencing in eukaryotic cells, including proliferating mammalian cells. However, the utility of RNAi as a method of gene knock-down in primary postmitotic mammalian neurons remained unknown. Here, we asked if RNAi might be utilized to allow the assessment of the biological function of a specific gene in the nervous system. We employed a U6 promoter-driven DNA template approach to induce hairpin RNA-triggered RNAi to characterize the role of the transcription factor myocyte enhancer factor 2A (MEF2A) in the neuronal activity-dependent survival of granule neurons of the developing rat cerebellum. We found that the expression of MEF2A hairpin RNAs leads to the efficient and specific inhibition of endogenous MEF2A protein expression in primary cerebellar granule neurons. We also found that RNAi of MEF2A reduces significantly MEF2 response element-mediated transcription in granule neurons and inhibits activity-dependent granule neuron survival. Taken together, our RNAi experiments have revealed that MEF2A plays a critical role in activity-dependent neuronal survival. In addition, our findings indicate that RNAi does operate in postmitotic mammalian neurons and thus offers a rapid genetic method of studying gene function in the development and function of the mammalian nervous system.

Animals↗

PubMed2003

In vivo analysis of the RNA interference mechanism in Trypanosoma brucei.

Flagellate protozoa of the family Trypanosomatidae, which includes various members of the genera Leishmania and Trypanosoma, are model systems for unicellular pathogens to study fundamentally important biological phenomena. Recently, ablation of gene expression by RNA interference (RNAi) has become the method of choice to study gene function in Trypanosoma brucei, an early divergent eukaryote that infects humans and animals. As has been shown in multicellular organisms, the RNAi mechanism in T. brucei involves processing of double-stranded RNA 24- to 26-nt RNAs, termed small interfering RNAs (siRNAs), which guide degradation of the target mRNA. In this article, we describe some of the methods we employ for the analysis of the RNAi mechanism in T. brucei with particular emphasis on detection, cloning, and fractionation of siRNAs and siRNA complexes.

Animals↗

PubMed2004

Flower color modulations of Torenia hybrida by downregulation of chalcone synthase genes with RNA interference.

Suppression of biosynthetic genes involved in flower color formation is an important approach for obtaining target flower colors. Here we report that flower color of the garden plant Torenia hybrida was successfully modulated by RNA interference (RNAi) against a gene of chalcone synthase (CHS), a key enzyme for anthocyanin and flavonoid biosynthesis. By using each of the coding region and the 3'-untranslated region of the CHS mRNA as an RNAi target, exhaustive and gene-specific gene silencing were successfully induced, and the original blue flower color was modulated to white and pale colors, respectively. Our results indicate that RNAi is quite useful for modulations of flower colors of commercially important garden plants.

Acyltransferases↗

PubMed2005

Knockdown of STAT3 expression by RNA interference inhibits the induction of breast tumors in immunocompetent mice.

Constitutively activated STAT3 is involved in the formation of multiple types of tumors including breast cancer. We examined the effects of Stat3 protein knockdown by RNA interference using a dicistronic lentivirus small hairpin (shRNA) delivery system on the growth of mammary tumors in BALB/c mice induced by the 4T1 cell line. A single exposure of 4T1 cells to shRNA/STAT3 lentivirus transduced 75% of the cells with green fluorescent protein (GFP) within 96 hours. In cells selected for GFP expression, neither Stat3 protein nor phosphotyrosine Stat3 was detected. Tumor formation induced by injecting 4T1 cells into the mammary fat pad was blocked by expression of the shRNA for STAT3 whereas all mice injected with 4T1 cells expressing only GFP efficiently formed tumors. c-Myc expression was reduced 75% in cells expressing greatly reduced levels of Stat3 compared with the GFP control. Of interest, the level of activated Src, which is known to activate Stat3, was virtually eliminated but the level of the Src protein itself remained the same. Importantly, expression of Twist protein, a metastatic regulator, was eliminated in STAT3 knockdown cells. Invasion activity of STAT3 knockdown cells was strongly inhibited. However, the proliferation rate of cells in Stat3 knockdown cells was similar to that of the GFP control; the cell cycle was also not affected. We conclude from these studies that activated Stat3 protein plays a critical role in the induction of breast tumors induced by 4T1 cells by enhancing the expression of several important genes including c-Myc and the metastatic regulator Twist. These studies suggest that stable expression of small interfering RNA for STAT3 has potential as a therapeutic strategy for breast cancer.

Animals↗

PubMed2003

Modulation of the classical multidrug resistance (MDR) phenotype by RNA interference (RNAi).

For reversal of MDR1 gene-dependent multidrug resistance (MDR), two small interfering RNA (siRNA) constructs were designed to inhibit MDR1 expression by RNA interference. SiRNA duplexes were used to treat human pancreatic carcinoma (EPP85-181RDB) and gastric carcinoma (EPG85-257RDB) cells. In both cellular systems, siRNAs could specifically inhibit MDR1 expression up to 91% at the mRNA and protein levels. Resistance against daunorubicin was decreased to 89% (EPP85-181RDB) or 58% (EPG85-257RDB). The data indicate that this approach may be applicable to cancer patients as a specific means to reverse tumors with a P-glycoprotein-dependent MDR phenotype back to a drug-sensitive one.

Carcinoma↗

PubMed2005

[Inhibitory effect of RNA interference on chronic myeloid leukemia bcr/abl oncogene expression].

OBJECTIVE: To investigate the inhibitory effect of RNA interference on chronic myeloid leukemia (CML) bcr/abl oncogene expression. METHODS: The small interference RNAs (siRNAs) were synthesized in vitro. K562 cells stably expressing bcr/abl gene were transfected with the siRNA by electroporation, both the non-transfected cells and non-specific siRNAs transfected cells were taken as controls. The enhanced green fluorescent protein (EGFP) plasmid was used as positive control and the transfection efficiency was detected by flow cytometry. Inhibitory effect of siRNAs was demonstrated by real-time quantitative RT-PCR and Western blots. Cell proliferation was measured by MTT assay and apoptosis by Annexin V-FITC assay. RESULTS: The transfection efficiency was about 70%. The synthesized siRNAs inhibited CML bcr/abl oncogene expression at both mRNA and protein levels. siRNAs could inhibit K562 cell proliferation to 47% and 56% at 24 h and 48 h after transfection, respectively, and induce cell apoptosis from 1.00% in control group to 15.05% and 19.4% at 24 h and 48 h respectively. CONCLUSION: At the cell level, inhibition of CML bcr/abl oncogene expression by chemically synthesized siRNAs provides the new method for anti-leukemia study.

Apoptosis↗

PubMed2005

Inhibiting the growth of malignant melanoma by blocking the expression of vascular endothelial growth factor using an RNA interference approach.

BACKGROUND: Vascular endothelial growth factor (VEGF) is overexpressed in malignant melanoma (MM). OBJECTIVES: To develop an RNA interference approach that specifically targets VEGF by constructing a eukaryotic expression plasmid containing short interfering RNA (siRNA), and to evaluate the effects of this vector on the proliferation and apoptosis of MM in vitro and in vivo. METHODS: pU-VEGF-siRNA plasmid was transfected into MM cell line A375 and colorectal carcinoma cell line Lovo by electroporation. Expression of VEGF mRNA and protein in A375 and Lovo cells after gene transfer was detected by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Proliferation of pU-VEGF-siRNA-transfected A375 and Lovo cells and control cells was observed by cell counting through the microscope. The proliferation of human umbilical vein endothelial cells (ECV-304) cultured in medium containing supernatants of transfected and control A375 cells was measured by the cell counting method. Flow cytometry (FCM) was used to analyse the apoptosis of transfected and control groups. In a mouse model, tumorigenicity and tumour growth of transfected cells were studied in vivo. VEGF expression and microvessel density (MVD) in tumour tissue were measured by immunohistochemistry. Apoptosis in tumours was detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling. RESULTS: Expression of VEGF mRNA and protein in pU-VEGF-siRNA-transfected A375 and Lovo cells was significantly decreased on days 3, 10, 17 and 24 post-transfection, compared with controls. The greatest suppression occurred on days 3 and 10 post-transfection. The proliferation of transfected A375 cells and ECV-304 cocultured with supernatants of transfected A375 cells was inhibited. FCM analysis showed that a hypodiploidy peak was found only in A375 cells transfected by pU-VEGF-siRNA. After subcutaneous inoculation with pU-VEGF-siRNA-transfected A375 cells, tumour growth in mice was inhibited, VEGF expression and MVD were decreased, and tumour apoptosis was increased significantly, in comparison with mice inoculated with untransfected A375 cells. CONCLUSIONS: The delivery of siRNA directed against VEGF was shown not only to give efficient and specific downregulation of the expression of VEGF, inhibit proliferation of A375 and ECV-304 cells and induce apoptosis of A375 cells in vitro, but also to suppress growth of MM in vivo. These results suggest that a strategy based on siRNA targeting of VEGF may build the foundation to the clinical management of MM.

Animals↗

PubMed2004

Monitoring the effect of gene silencing by RNA interference in human CD34+ cells injected into newborn RAG2-/- gammac-/- mice: functional inactivation of p53 in developing T cells.

Tumor suppressor p53 plays an important role in regulating cell cycle progression and apoptosis. Here we applied RNA interference to study the role of p53 in human hematopoietic development in vivo. An siRNA construct specifically targeting the human tumor-suppressor gene p53 was introduced into human CD34(+) progenitor cells by lentivirus-mediated gene transfer, which resulted in more than 95% knockdown of p53. We adapted the human-SCID mouse model to optimize the development of hematopoietic cells, particularly of T cells. This was achieved by the intraperitoneal injection of CD34(+) precursor cells into newborn Rag2(-/-) gammac(-/-) mice that lack T, B, and NK cells. Robust development of T cells was observed in these mice, with peripheral T-cell repopulation 8 weeks after injection of the precursor cells. Other lymphocyte and myeloid subsets also developed in these mice. Injecting p53 siRNA-transduced CD34(+) cells resulted in stable expression and down-modulation of p53 in the mature T-cell offspring. Inactivating p53 did not affect the development of CD34(+) cells into various mature leukocyte subsets, including T cells, but it conferred resistance to gamma-irradiation and other p53-dependent apoptotic stimuli to the T cells.

Animals↗

PubMed2004

RNA interference of Trypanosoma brucei topoisomerase IB: both subunits are essential.

Type IB topoisomerases are enzymes essential for the orderly synthesis of nucleic acids and are the molecular target for antitumor camptothecins. In dozens of organisms, including eukaryotes, bacteria, and viruses, this enzyme is monomeric. However, we previously found that topoisomerase IB in trypanosomes is a heteromultimer, comprised of two distinct subunits encoded by separate genes. A large 90 kDa subunit contains the DNA binding domain and a small 36 kDa subunit contains the catalytic domain. In this study we use RNA interference to silence each of the subunits separately. For each subunit, tetracycline-induced expression of double-stranded RNA results in drastic reduction of cognate mRNA and protein. For the large subunit, nucleic acid biosynthesis (as monitored by the incorporation of radiolabeled precursors into DNA and RNA) is halved by 39 h, and cell growth halts by 72 h, after induction. The steady state level of both nuclear and mitochondrial mRNAs is reduced. Virtually identical results are obtained by silencing the small subunit. Interestingly, although interference is specific at the level of mRNA, silencing of one subunit leads to a profound reduction in the level of protein for both subunits, suggesting that survival, or perhaps synthesis, of each subunit depends upon the presence of the other. These findings underscore the essential nature of type IB topoisomerase activity in Trypanosoma brucei and its suitability as a target for rational drug design.

Animals↗

PubMed2006

Delivery methods for RNA interference in C. elegans.

This chapter describes four methods for delivery of double-stranded RNA to Caenorhabditis elegans (injection, feeding, soaking, and in vivo delivery), and suggests schemes that should facilitate detection of specific gene silencing.

Animals↗

PubMed2006

[Effect of silencing hypoxia-inducible factor-1 alpha by RNA interference on human breast carcinoma cell line].

OBJECTIVE: To investigate the effect of short hairpin RNA (shRNA) targeting hypoxia-inducible factor-1 alpha (HIF-1 alpha) on the human breast carcinoma MCF-7 cell line. METHODS: The hypoxia environment was achieved by treating cells with cobalt chloride. The shRNA eukaryotic expression vector targeting HIF-1 alpha was constructed, and transfected into MCF-7 cells through lipofectamine 2000. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to study the expression of vascular endothelial growth factor (VEGF). The mRNA and protein level of HIF-1 alpha were detected by real-time PCR and Western blot. Sub-G1 apoptotic population analysis, Annexin V/PI binding assay, and DNA ladder analysis were applied to investigate the cell apoptosis. The cell cycle was detected by flow cytometry. RESULTS: The mRNA and protein level of HIF-1 alpha increased after exposure of MCF-7 cells to hypoxia (P < 0.01). However, apoptosis was lower in hypoxia compared with normoxia (P < 0.05). The HIF-1 level of MCF-7 transfected with HIF-1 alpha shRNA decreased approximately 91.63% (P < 0.01). When the cells were treated with or without apoptosis inducer Ara-C, the apoptosis of MCF-7 cells transfected with HIF-1 alpha shRNA increased by 1.75 times (P < 0.01) and 61. 31 times (P < 0.01), respectively. The expression of VEGF in MCF-7 cells transfected with HIF-1 alpha shRNA decreased 66.8% compared with untransfected cells (P < 0.05). Cell cycle progression was inhibited when the MCF-7 cells were transfected with HIF-1 alpha shRNA. CONCLUSIONS: HIF-1 alpha plays an anti-apoptotic role in human breast carcinoma MCF-7 cell line. The shRNA we designed targeting HIF-1 alpha in MCF-7 can promote cell apoptosis, inhibit the expression of VEGF, and delay cell cycle progression.

Apoptosis↗

PubMed2005

Downregulation of Apaf-1 and caspase-3 by RNA interference in human glioma cells: consequences for erucylphosphocholine-induced apoptosis.

Erucylphosphocholine (ErPC) exerts strong anticancer activity in vivo and in vitroand induces apoptosis even in chemoresistant glioma cell lines. We investigated the contribution of Apaf-1 and caspase-3 to the apoptotic response to ErPC using RNA interference (RNAi) in human glioblastoma cells. We could demonstrate that human glioma cell lines are susceptible to RNAi. Apaf-1 and caspase-3 are amenable to specific small interfering RNA (siRNA)-induced degradation resulting in a reduction of protein levels to 8-33% (Apaf-1) and to 30-50% (caspase-3). Transfection of siRNA directed to Apaf-1 and caspase-3 specifically reduced caspase-3 processing induced by ErPC treatment and yielded a reduction in cells that undergo ErPC-induced apoptosis to 17-33% (Apaf-1) and to 38-50% (caspase-3). The caspase-3 siRNA experiments were corroborated in caspase-3-deficient and -reconstituted MCF-7 breast cancer cells. Survival assays and morphological observations revealed that caspase-3 reconstitution significantly sensitized MCF-7 cells to ErPC. Exploring the caspase cascade responsible for ErPC-induced apoptosis MCF-7 cells provided evidence that caspase-3 is required for the activation of caspases-2, -6 and -8 and also participates in a feedback amplification loop. Our results provide evidence that Apaf-1 and caspase-3 are major determinants of ErPC-induced apoptosis and the possible use of ErPC in a clinical setting is discussed.

Apoptosis↗

PubMed2005

Gene therapy for human small-cell lung carcinoma by inactivation of Skp-2 with virally mediated RNA interference.

Increase of Skp-2, which is involved in the degradation of cell cycle regulators including p27Kip1, p21 and c-myc, is one of the important mechanisms for dysregulation of cell cycles in various cancers. We applied RNA interference (RNAi) for Skp-2 by using HIV-lentiviral or adenoviral vectors for a human small-cell lung carcinoma cell line with increased Skp-2 to evaluate RNAi strategy for cancer gene therapy. HIV-lentivirus-mediated RNAi for Skp-2 resulted in efficient inhibition of the in vitro cell growth of cancer cells with increased Skp-2 through the increase of p27Kip1 and p21, but no significant effect on the growth of cells without high Skp-2 expression. Furthermore, intratumoral administration of adenovirus siRNA vector for Skp-2 efficiently inhibited growth of established subcutaneous tumor on NOD/SCID mice. These results indicate that the Skp-2 RNAi may be a useful strategy for gene therapy of cancers with high Skp-2 expression.

Adenoviridae↗

PubMed2004

Gene interactions in the DNA damage-response pathway identified by genome-wide RNA-interference analysis of synthetic lethality.

Here, we describe a systematic search for synthetic gene interactions in a multicellular organism, the nematode Caenorhabditis elegans. We established a high-throughput method to determine synthetic gene interactions by genome-wide RNA interference and identified genes that are required to protect the germ line against DNA double-strand breaks. Besides known DNA-repair proteins such as the C. elegans orthologs of TopBP1, RPA2, and RAD51, eight genes previously unassociated with a double-strand-break response were identified. Knockdown of these genes increased sensitivity to ionizing radiation and camptothecin and resulted in increased chromosomal nondisjunction. All genes have human orthologs that may play a role in human carcinogenesis.

Animals↗

PubMed2006

Modeling CTLA4-linked autoimmunity with RNA interference in mice.

The CTLA4 gene is important for T lymphocyte-mediated immunoregulation and has been associated with several autoimmune diseases, in particular, type 1 diabetes. To model the impact of natural genetic variants of CTLA4, we constructed RNA interference (RNAi) "knockdown" mice through lentiviral transgenesis. Variegation of expression was observed in founders but proved surmountable because it reflected parental imprinting, with derepression by transmission from male lentigenics. Unlike the indiscriminate multiorgan autoimmune phenotype of the corresponding knockout mice, Ctla4 knockdown animals had a disease primarily focused on the pancreas, with rapid progression to diabetes. As with the human disease, the knockdown phenotype was tempered by genetic-modifier loci. RNAi should be more pertinent than gene ablation in modeling disease pathogenesis linked to a gene-dosage variation.

Animals↗

PubMed2003

Transgenic RNA interference in ES cell-derived embryos recapitulates a genetic null phenotype.

Gene targeting via homologous recombination in murine embryonic stem (ES) cells has been the method of choice for deciphering mammalian gene function in vivo. Despite improvements in this technology, it still remains a laborious method. Recent advances in RNA interference (RNAi) technology have provided a rapid loss-of-function method for assessing gene function in a number of organisms. Studies in mammalian cell lines have shown that introduction of small interfering RNA (siRNA) molecules mediates effective RNA silencing. Plasmid-based systems using RNA polymerase III (RNA pol III) promoters to drive short hairpin RNA (shRNA) molecules were established to stably produce siRNA. Here we report the generation of knockdown ES cell lines with transgenic shRNA. Because of the dominant nature of the knockdown, embryonic phenotypes could be directly assessed in embryos completely derived from ES cells by the tetraploid aggregation method. Such embryos, in which endogenous p120-Ras GTPase-activating protein (RasGAP), encoded by Rasa1 (also known as RasGAP), was silenced, had the same phenotype as did the previously reported Rasa1 null mutation.

Animals↗

PubMed2003

Transgene-induced RNA interference: a strategy for overcoming gene redundancy in polyploids to generate loss-of-function mutations.

Gene redundancy in polyploid species complicates genetic analyses by making the generation of recessive, loss-of-function alleles impractical. We show that this problem can be circumvented using RNA interference (RNAi) to achieve dominant loss of function of targeted genes. Arabidopsis suecica is an allotetraploid (amphidiploid) hybrid of A. thaliana and A. arenosa. We demonstrate that A. suecica can be genetically transformed using the floral dip method for Agrobacterium-mediated transformation. Transgenes segregate as in a diploid, indicating that chromosome pairing occurs exclusively (or almost so) among homologs and not among homeologs. Expressing a double-stranded (ds) RNA corresponding to the A. thaliana gene, decrease in DNA methylation 1 (DDM1) caused the elimination of DDM1 mRNAs and the loss of methylation at both A. thaliana- and A. arenosa-derived centromere repeats. These results indicate that a single RNAi-inducing transgene can dominantly repress multiple orthologs.

Arabidopsis↗