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Results for “RNA interference”

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At least 793 records · Page 44Linked to original sources

PubMed2004

Suppression of Epstein-Barr virus-encoded latent membrane protein-1 by RNA interference inhibits the metastatic potential of nasopharyngeal carcinoma cells.

Nasopharyngeal carcinoma (NPC) is a highly metastatic tumor characterized by close association with EBV. Of the EBV-encoded products, latent membrane protein-1 (LMP-1) is thought to be the only oncoprotein playing an essential role in cell transformation as well as tumor metastasis. In this study, we tested the effect of suppressing LMP-1 by RNA interference (RNAi) on the proliferative and metastatic potentials of an EBV-positive NPC cell line, C666. We showed that stably suppressing LMP-1 by short hairpin RNA (shRNA) plasmid significantly altered cell motility, substratum adhesion, and transmembrane invasion ability. However, it has little effect on the rate of cell growth and cell cycle control. These results demonstrated the effectiveness of RNAi in suppressing LMP-1, supporting an important role of LMP-1 in NPC metastasis, and suggested a potential application of RNAi-mediated therapeutic strategy for EBV-related NPC.

Cell Adhesion↗

PubMed2006

RNA interference of cytosolic leucine aminopeptidase reduces fecundity in the hard tick, Haemaphysalis longicornis.

Ticks are effective vectors of pathogens because of their blood feeding and high fecundity. This high fecundity is related to the size of the blood meal. Therefore, knowledge of how blood proteins are degraded and converted to proteins, including yolk protein, is important for the development of ways to inhibit the utilization of blood proteins by ticks. RNA interference (RNAi) is becoming a powerful post-transcriptional gene silencing technique that provides insight into gene function. We constructed a double-stranded RNA (dsRNA) based on a previously cloned Haemaphysalis longicornis leucine aminopeptidase (HlLAP) gene to reevaluate the biological role in tick blood digestion. Gene specific transcriptional, translational, and functional disruptions were achieved by the introduction of dsRNA into the ticks. Significantly delayed onset of egg-laying and reduced egg oviposition resulted from the RNAi for the HlLAP gene. These results suggest that HlLAP actually works as a blood digestive enzyme and affects tick fecundity via unknown mechanisms. The reduction of egg oviposition may be caused by a decrease in nutrients, especially free amino acids generated by HlLAP, from the blood meal. This is the first report of an impact on tick reproduction caused by gene silencing of a blood digestion-related molecule.

Animals↗

PubMed2006

Inhibition of HIV-1 replication by RNA interference of p53 expression.

p53 expression and activation have been associated to faster human immunodeficiency virus (HIV) disease progression, most probably by inducing CD4+ T cell death but also through its cooperative effect in the control of viral gene transcription by viral regulatory proteins. Here, we show that RNA interference of p53 in HIV-1 reporter (HeLa P4-R5 MAGI) and lymphoid (SupT1) cell lines blocked HIV-1 and Tat-induced transcription from the HIV-1 promoter and HIV-1 replication in acutely infected cells, suggesting a cooperative role of p53 in HIV-1 transcription. Contrary to SupT1 cells, which encode several mutations on the p53 DNA binding domain, death of HIV-1-induced syncytia was reduced in cocultures of HeLa P4-R5 MAGI with persistently infected HIV-1 cells. To our knowledge, this is the first demonstration of the effect of the loss of function of p53 in HIV-1 replication, which is independent on its classical DNA binding activity. Our results suggest two independent roles for p53 in HIV-1 infection: cooperation in HIV long-terminal repeat transcription and virus-induced cell death.

Cell Death↗

PubMed2004

Intravenous RNA interference gene therapy targeting the human epidermal growth factor receptor prolongs survival in intracranial brain cancer.

PURPOSE: The human epidermal growth factor receptor (EGFR) plays an oncogenic role in solid cancer, including brain cancer. The present study was designed to prolong survival in mice with intracranial human brain cancer with the weekly i.v. injection of nonviral gene therapy causing RNA interference (RNAi) of EGFR gene expression. EXPERIMENTAL DESIGN: Human U87 gliomas were implanted in the brain of adult scid mice, and weekly i.v. gene therapy was started at day 5 after implantation of 500000 cells. An expression plasmid encoding a short hairpin RNA directed at nucleotides 2529-2557 within the human EGFR mRNA was encapsulated in pegylated immunoliposomes. The pegylated immunoliposome was targeted to brain cancer with 2 receptor-specific monoclonal antibodies (MAb), the murine 83-14 MAb to the human insulin receptor and the rat 8D3 MAb to the mouse transferrin receptor. RESULTS: In cultured glioma cells, the delivery of the RNAi expression plasmid resulted in a 95% suppression of EGFR function, based on measurement of thymidine incorporation or intracellular calcium signaling. Weekly i.v. RNAi gene therapy caused reduced tumor expression of immunoreactive EGFR and an 88% increase in survival time of mice with advanced intracranial brain cancer. CONCLUSIONS: Weekly i.v. nonviral RNAi gene therapy directed against the human EGFR is a new therapeutic approach to silencing oncogenic genes in solid cancers. This is enabled with a nonviral gene transfer technology that delivers liposome-encapsulated plasmid DNA across cellular barriers with receptor-specific targeting ligands.

Animals↗

PubMed2005

Correction of cellular phenotypes of Hutchinson-Gilford Progeria cells by RNA interference.

The great majority of cases of the Hutchinson-Gilford progeroid syndrome (HGPS) ("Progeria of Childhood'') are caused by a single nucleotide mutation (1824 C->T) in the LMNA gene which encodes lamin A and C, nuclear intermediate filaments that are important components of the nuclear lamina. The resultant mutant protein (Delta50 lamin A) is thought to act in a dominant fashion. We exploited RNA interference technology to suppress Delta50 lamin A expression, with the long range goal of intervening in the pathogenesis of the coronary artery atherosclerosis that typically leads to the death of HGPS patients. Short hairpin RNA (shRNA) constructs were designed to target the mutated pre-spliced or mature LMNA mRNAs, and were expressed in HGPS fibroblasts carrying the 1824 C->T mutations using lentiviruses. One of the shRNAs targeted to the mutated mRNA reduced the expression levels of Delta50 lamin A to 26% or lower. The reduced expression was associated with amelioration of abnormal nuclear morphology, improvement of proliferative potential, and reduction in the numbers of senescent cells. These findings provide a rationale for potential gene therapy.

Cell Culture Techniques↗

PubMedDate not supplied

Targeted delivery of antisense oligodeoxynucleotide and small interference RNA into lung cancer cells.

Selective gene inhibition by antisense oligodeoxynucleotide (AS-ODN) or by small interference RNA (siRNA) therapeutics promises the treatment of diseases that cannot be cured by conventional drugs. However, antisense therapy is hindered due to poor stability in physiological fluids and limited intracellular uptake. To address these problems, a ligand targeted and sterically stabilized nanoparticle formulation has been developed in our lab. Human lung cancer cells often overexpress the sigma receptor and, thus, can be targeted with a specific ligand such as anisamide. AS-ODN or siRNA against human survivin was mixed with a carrier DNA, calf thymus DNA, before complexing with protamine, a highly positively charged peptide. The resulting particles were coated with cationic liposomes consisting of DOTAP and cholesterol (1:1, molar ratio) to obtain LPD (liposome-polycation-DNA) nanoparticles. Ligand targeting and steric stabilization were then introduced by incubating preformed LPD nanoparticles with DSPE-PEG-anisamide, a PEGylated ligand lipid developed earlier in our lab, by the postinsertion method. Nontargeted nanoparticles coated with DSPE-PEG were also prepared as a control. Antisense activities of nanoparticles were determined by survivin mRNA down-regulation, survivin protein down-regulation, ability to trigger apoptosis in tumor cells, tumor cell growth inhibition, and chemosensitization of the treated tumor cells to anticancer drugs. We found that tumor cell delivery and antisense activity of PEGylated nanoparticles were sequence dependent and rely on the presence of anisamide ligand. The uptake of oligonucleotide in targeted, PEGylated nanoparticles could be competed by excess free ligand. Our results suggest that the ligand targeted and sterically stabilized nanoparticles can provide a selective delivery of AS-ODN and siRNA into lung cancer cells for therapy.

Cell Line, Tumor↗

PubMed2003

Short interfering RNA (siRNA)-mediated RNA interference (RNAi) in human cells.

Transient gene silencing in mammalian cells can be mediated by double stranded RNA (dsRNAs) molecules of approximately 20-25 nucleotides termed short interfering (siRNAs). Naturally occurring siRNAs in lower eukaryotes have characteristic structural elements; however, little is known about what features are critical for an exogenous siRNA to mediate RNAi in mammals. We have recently determined some of the critical parameters that influence the efficiency of siRNA-mediated RNAi in mammalian cells and have been considering the use of RNAi as a functional genomics tool, particularly for high throughput analysis, and the potential use of RNAi as a therapeutic tool.

Humans↗

PubMed2006

Functional and selective RNA interference in developing axons and growth cones.

Developing axons and growth cones contain "local" mRNAs that are translated in response to various extracellular signaling molecules and have roles in several processes during axonal development, including axonal pathfinding, orientation of axons in chemotactic gradients, and in the regulation of neurotransmitter release. The molecular mechanisms that regulate mRNA translation within axons and growth cones are unknown. Here we show that proteins involved in RNA interference (RNAi), including argonaute-3 and argonaute-4, Dicer, and the fragile X mental retardation protein, are found in developing axons and growth cones. These proteins assemble into functional RNA-induced silencing complexes as transfection of small interfering RNAs selectively into distal axons results in distal axon-specific mRNA knock-down, without reducing transcript levels in proximal axons or associated diffusion of small interfering RNA into proximal axons or cell bodies. RhoA mRNA is localized to axons and growth cones, and intra-axonal translation of RhoA is required for growth cone collapse elicited by Semaphorin 3A (Sema3A), an axonal guidance cue. Selective knock-down of axonal RhoA mRNA abolishes Sema3A-dependent growth cone collapse. Our results demonstrate functional and potent RNAi in axons and identify an approach to spatially regulate mRNA transcripts at a subcellular level in neurons.

Animals↗

PubMed2003

Induction of G0/G1 cell cycle arrest in ovarian carcinoma cells by the anti-inflammatory drug NS-398, but not by COX-2-specific RNA interference.

Cyclooxygenases, particularly COX-2, play an important role in tumor development and progression. We have previously shown that COX-2 expression is an independent prognostic factor in human ovarian carcinoma. In this study, we investigated the effects of the inhibition of COX isoforms by the NSAID NS-398 as well as by COX-isoform-specific RNA interference (RNAi) in the human ovarian carcinoma cell lines OVCAR-3 and SKOV-3. OVCAR-3 cells showed a constitutive expression of COX-1 and an induction of high levels of COX-2 and PGE(2) after stimulation with interleukin-1beta. In contrast, SKOV-3 cells were negative for both COX isoforms. In OVCAR-3 cells, PGE(2) production was inhibited by NS-398 in concentrations of 1 microM and by a COX-2-specific silencing RNA (siRNA), while a COX-1-specific siRNA did not have an effect. This suggests that COX-2 is the major source of PGE(2) in this cell line. To dissociate COX-2-specific and non-COX-2-specific effects on cell proliferation, a proliferation assay was performed after incubation of cells with NS-398 and COX siRNAs. NS-398 induced an inhibition of cell proliferation at concentrations of 50-500 microM, which are above the concentrations needed for the inhibition of PGE(2) production. This inhibitory effect was present in the COX-positive cell line OVCAR-3 as well as in the COX-negative cell line SKOV-3 and could not be reverted by addition of exogenous PGE(2). Neither COX-1- nor COX-2-specific siRNAs had an effect on cell proliferation of OVCAR-3 cells. Cell cycle analysis showed an increased accumulation of cells in the G0/G1 phase after treatment with NS-398, but not with COX siRNAs. These experiments suggest that NS-398 reduced cell proliferation in ovarian carcinoma cells by induction of G0/G1 cell cycle arrest independent of COX-2 inhibition. Our study shows that specific inhibition of COX isoforms by RNAi could be used to dissociate effects of NSAIDs. Furthermore, our results suggest that cell cycle arrest is one of the primary mechanisms responsible for the antiproliferative effects of NS-398 on ovarian carcinoma cells.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed2004

Inhibition of cathepsin B and MMP-9 gene expression in glioblastoma cell line via RNA interference reduces tumor cell invasion, tumor growth and angiogenesis.

Extracellular proteases have been shown to cooperatively influence matrix degradation and tumor cell invasion through proteolytic cascades, with individual proteases having distinct roles in tumor growth, invasion, migration and angiogenesis. Matrix metalloproteases (MMP)-9 and cathepsin B have been shown to participate in the processes of tumor growth, vascularization and invasion of gliomas. In the present study, we used a cytomegalovirus promoter-driven DNA template approach to induce hairpin RNA (hpRNA)-triggered RNA interference (RNAi) to block MMP-9 and cathepsin B gene expression with a single construct. Transfection of a plasmid vector-expressing double-stranded RNA (dsRNA) for MMP-9 and cathepsin B significantly inhibited MMP-9 and cathepsin B expression and reduced the invasive behavior of SNB19, glioblastoma cell line in Matrigel and spheroid invasion models. Downregulation of MMP-9 and cathepsin B using RNAi in SNB19 cells reduced cell-cell interaction of human microvascular endothelial cells, resulting in the disruption of capillary network formation in both in vitro and in vivo models. Direct intratumoral injections of plasmid DNA expressing hpRNA for MMP-9 and cathepsin B significantly inhibited established glioma tumor growth and invasion in intracranial tumors in vivo. Further intraperitoneal (i.p.) injections of plasmid DNA expressing hpRNA for MMP-9 and cathepsin B completely regressed pre-established tumors for a long time (4 months) without any indication of these tumor cells. For the first time, these observations demonstrate that the simultaneous RNAi-mediated targeting of MMP-9 and cathepsin B has potential application for the treatment of human gliomas.

Animals↗

PubMed2003

RNA interference for HIF-1alpha inhibits its downstream signalling and affects cellular proliferation.

Transcription factor HIF-1 is a key determinant of oxygen-dependent gene regulation. Suppression of HIF-1alpha is important for exploring HIF-1-dependent processes and for interfering with hypoxia-induced pathophysiological events. This study applied RNA-interference targeting HIF-1alpha to the human lung A549 cell line. Transfection of HIF-1alpha-siRNA reduced HIF-1alpha synthesis as measured on mRNA and protein level by realtime RT-PCR, Western blot, and immuncytochemistry. A time kinetic for hypoxic stabilization of HIF-1alpha protein and its inhibition by HIF-1alpha-siRNA is included. Hypoxic induction of HIF-1-controlled target genes as heme oxygenase I (HO-1), phosphoglycerate kinase (PGK), and vascular endothelial growth factor (VEGF) was markedly attenuated by HIF-1alpha-siRNA treatment. Correspondingly, gene activation via hypoxia-responsive-element, as shown by reporter gene assay, was inhibited by HIF-1alpha-siRNA. Moreover, this approach was found to suppress the shift from from S-phase to G1-phase observed in A549 cells in response to hypoxia, supporting a role of HIF-1alpha in oxygen-dependent cell cycle regulation.

Cell Division↗

PubMed2005

Heterotopia formation in rat but not mouse neocortex after RNA interference knockdown of DCX.

Subcortical band heterotopia (SBH) or double cortex is associated with significant impairments in neocortical function including mental retardation and epilepsy. Mutant alleles of DCX in humans typically cause SBH in females and lissencephaly in males, whereas Dcx null mutations in mice neither disrupt neocortical neuronal migration nor cause SBH formation. In utero RNA interference (RNAi) of Dcx in rats, in contrast, creates an animal model of SBH. Possible explanations for the discrepancies in results following loss of Dcx function include species differences and/or differences between RNAi knockdown and genetic deletion. We have carried out a series of in utero RNAi experiments to investigate possible species differences between rat and mouse to determine the molecular specificity of RNAi against Dcx and to identify the cellular constituents of SBH in the rat model. In utero RNAi in the rat consistently leads to both the formation of SBH and laminar displacement of transfected cells in normotopic cortex, whereas the same treatment in mouse fails to induce SBH but does create laminar displacement. Induction of SBH and impaired radial migration following RNAi against Dcx is rescued by overexpression of Dcx. Thus, both disruptions induced by RNAi are specific to interference of Dcx. SBHs contain transfected pyramidal cells as well as nontransfected cell types, including neocortical interneurons and glia. Together these results indicate that there is a species difference between rat and mouse with respect to RNAi-induced SBH formation and that SBH formation involves the recruitment of several unaltered cell types.

Animals↗

PubMed2005

RNA interference improves motor and neuropathological abnormalities in a Huntington's disease mouse model.

Huntington's disease (HD) is a fatal, dominant neurogenetic disorder. HD results from polyglutamine repeat expansion (CAG codon, Q) in exon 1 of HD, conferring a toxic gain of function on the protein huntingtin (htt). Currently, no preventative treatment exists for HD. RNA interference (RNAi) has emerged as a potential therapeutic tool for treating dominant diseases by directly reducing disease gene expression. Here, we show that RNAi directed against mutant human htt reduced htt mRNA and protein expression in cell culture and in HD mouse brain. Importantly, htt gene silencing improved behavioral and neuropathological abnormalities associated with HD. Our data provide support for the further development of RNAi for HD therapy.

Animals↗

PubMed2004

RNA interference targeting Stat3 inhibits growth and induces apoptosis of human prostate cancer cells.

Stat3, a member of the signal transduction and activation of transcription (STAT) family, is a key signal transduction protein that mediates signaling by cytokines, peptide growth factors, and oncoproteins and is constitutively activated in numerous cancers including prostate. Previous studies demonstrated that constitutively activated Stat3 plays an important role in the development and progression of prostate cancer by promoting cell proliferation and protecting against apoptosis. The present study was designed to investigate the potential use of RNA interference to block Stat3 expression and activation and the effect on the growth of human prostate cancer cells. We identified a small interfering RNA (siRNA) specific for Stat3 and demonstrate that blockade of Stat3 activation by the Stat3 siRNA suppresses the growth of human prostate cancer cells and Stat3-mediated gene expression and induces apoptotic cell death. The Stat3 siRNA does not inhibit the proliferation nor induces apoptosis of Stat3-inactive human prostate cancer cells. In addition, the Stat3 siRNA inhibits the levels of androgen-regulated prostate specific antigen (PSA) expression in prostate cancer cells. These results demonstrate that targeting Stat3 signaling using siRNA technique may serve as a novel therapeutic strategy for treatment of prostate cancer expressing constitutively activated Stat3.

Acute-Phase Proteins↗

PubMed2006

In vivo RNA interference demonstrates a role for Nramp1 in modifying susceptibility to type 1 diabetes.

Type 1 diabetes is an autoimmune disease influenced by multiple genetic loci. Although more than 20 insulin-dependent diabetes (Idd) loci have been implicated in the nonobese diabetic (NOD) mouse model, few causal gene variants have been identified. Here we show that RNA interference (RNAi) can be used to probe candidate genes in this disease model. Slc11a1 encodes a phagosomal ion transporter, Nramp1, that affects resistance to intracellular pathogens and influences antigen presentation. This gene is the strongest candidate among the 42 genes in the Idd5.2 region; a naturally occurring mutation in the protective Idd5.2 haplotype results in loss of function of the Nramp1 protein. Using lentiviral transgenesis, we generated NOD mice in which Slc11a1 is silenced by RNAi. Silencing reduced the frequency of type 1 diabetes, mimicking the protective Idd5.2 region. Our results demonstrate a role for Slc11a1 in modifying susceptibility to type 1 diabetes and illustrate that RNAi can be used to study causal genes in a mammalian model organism.

Animals↗

PubMed2003

Targeting oncogenic fusion genes in leukemias and lymphomas by RNA interference.

Leukemias and lymphomas are often characterized by non-random chromosomal translocations that, at the molecular level, induce the activation of specific oncogenes or create novel chimeric genes. They have frequently been regarded as optimal targets for gene-silencing approaches because of the large body of evidence that these single abnormalities directly initiate and maintain the malignant process. Herein, we discuss RNA interference (RNAi)-based approaches for targeting the fusion sites of chromosomal translocations as a future treatment option in leukemias and lymphomas.

Animals↗

PubMed2005

Assaying the Drosophila negative feedback loop with RNA interference in S2 cells.

Transcriptional negative feedback loops play a critical role in the molecular oscillations of circadian genes and contribute to robust behavioral rhythms. In one key Drosophila loop, CLOCK and CYCLE (CLK/CYC) positively regulate transcription of period (per). The period protein (PER) then represses this transcriptional activation, giving rise to the molecular oscillations of per RNA and protein. There is evidence that links molecular oscillations with behavioral rhythms, suggesting that PER also regulates the expression of downstream genes, ultimately resulting in proper behavior rhythmicity. Phosphorylation of PER has also been shown to be critical for rhythms. Doubletime (DBT) and casein kinase II (CKII) have been implicated in the phosphorylation of PER, which affects its stability as well as nuclear localization. We investigated the role of these kinases on PER transcriptional repression using the Drosophila S2 cell line in combination with RNA interference (RNAi) to knock down specific gene expression. This article describes the methods used to study PER repression activity in the S2 cell system as well as to exploit RNAi in this system. We also include protocols for immunocytochemistry and the application of leptomycin to differentiate direct effects on repression from indirect effects on subcellular localization. Finally, we discuss the generation of stable cell lines in the S2 cell system; these will be useful for experiments requiring homogeneous cell populations.

ARNTL Transcription Factors↗

PubMed2003

A functional genomic screen for cardiogenic genes using RNA interference in developing Drosophila embryos.

Identifying genetic components is an essential step toward understanding complex developmental processes. The primitive heart of the fruit fly, the dorsal vessel, which is a hemolymph-pumping organ, has provided a unique model system to identify cardiogenic genes and to further our understanding of the molecular mechanisms of cardiogenesis. Using RNA interference in developing Drosophila embryos, we performed a genomewide search for cardiogenic genes. Through analyses of the >5800 genes that cover approximately 40% of all predicted Drosophila genes, we identified a variety of genes encoding transcription factors and cell signaling proteins required for different steps during heart development. Analysis of mutant heart phenotypes and identified genes suggests that the Drosophila heart tube is segmentally patterned, like axial patterning, but assembled with regional modules. One of the identified genes, simjang, was further characterized. In the simjang mutant embryo, we found that within each segment a subset of cardial cells is missing. Interestingly, the simjang gene encodes a protein that is a component of the chromatin remodeling complex recruited by methyl-CpG-DNA binding proteins, suggesting that epigenetic information is crucial for specifying cardiac precursors. Together, these studies not only identify key regulators but also reveal mechanisms underlying heart development.

Animals↗