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Results for “RNA Interference”

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At least 73 records · Page 4Linked to original sources
PubMed2005

RNA interference targeted to multiple P2X receptor subtypes attenuates zinc-induced calcium entry.

A postulated therapeutic avenue in cystic fibrosis (CF) is activation of Ca(2+)-dependent Cl(-) channels via stimulation of Ca(2+) entry from extracellular solutions independent of CFTR functional status. We have shown that extracellular zinc and ATP induce a sustained increase in cytosolic Ca(2+) in human airway epithelial cells that translates into stimulation of sustained secretory Cl(-) transport in non-CF and CF human and mouse airway epithelial cells, cell monolayers, and nasal mucosa. On the basis of these studies, the Ca(2+) entry channels most likely involved were P2X purinergic receptor channels. In the present study, molecular and biochemical data show coexpression of P2X(4), P2X(5), and P2X(6) subtypes in non-CF (16HBE14o(-)) and CF (IB3-1) human bronchial epithelial cells. Other P2X receptor Ca(2+) entry channel subtypes are expressed rarely or not at all in airway epithelia, epithelial cell models from other CF-relevant tissues, or vascular endothelia. Novel transient lipid transfection-mediated delivery of small interference RNA fragments specific to P2X(4) and P2X(6) (but not P2X(5)) into IB3-1 CF human airway epithelial cells inhibited extracellular zinc- and ATP-induced Ca(2+) entry markedly in fura-2 Ca(2+) measurements and "knocked down" protein by >65%. These data suggest that multiple P2X receptor Ca(2+) entry channel subtypes are expressed in airway epithelia. P2X(4) and P2X(6) may coassemble on the airway surface as targets for possible therapeutics for CF independent of CFTR genotype.

Animals↗
PubMed2006

Inhibition of hepatitis B virus expression and replication by RNA interference in HepG2.2.15.

AIM: To observe the inhibition of hepatitis B virus replication and expression by transfecting vector-based small interference RNA (siRNA) pGenesil-HBV X targeting HBV X gene region into HepG2.2.15 cells. METHODS: pGenesil-HBV X was constructed and transfected into HepG2.2.15 cells via lipofection. HBV antigen secretion was determined 24, 48, and 72 h after transfection by time-resolved immunofluorometric assays (TRFIA). HBV replication was examined by fluorescence quantitative PCR, and the expression of cytoplasmic viral proteins was determined by immunohistochemistry. RESULTS: The secretion of HBsAg and HBeAg into the supernatant was found to be inhibited by 28.5% and 32.2% (P < 0.01), and by 38.67% (P < 0.05) and 42.86% (P < 0.01) at 48 h and 72 h after pGenesil-HBV X transfection, respectively. Immunohistochemical staining for cytoplasmic HBsAg showed a similar decline in HepG2.2.15 cells 48 h after transfection. The number of HBV genomes within culture supernatants was also significantly decreased 48 h and 72 h post-transfection as quantified by fluorescence PCR (P < 0.05). CONCLUSION: In HepG2.2.15 cells, HBV replication and expression is inhibited by vector-based siRNA pGenesil-HBV X targeting the HBV X coding region.

Cell Line, Tumor↗
PubMed2004

[Suppression of c-myc expression by interference RNA in HepG2 hepatocellular carcinoma cells].

OBJECTIVE: To study the inhibitory effect of RNA interference (RNAi) on c-myc expression in hepatocellular carcinoma cell line, HepG2. METHODS: Expression vector of c-myc gene-targeting small interference RNA (siRNA) was constructed (psilencer-c-myc) and transfected into HepG2 cells by lipofectamine, and the unloaded vector was used as control (mock). The expression of c-myc mRNA and protein was identified by quantitive PCR and Western blot. Apoptosis of the transfected cells was examined by flow cytometry and immunofluorescent microscopy. RESULTS: After HepG2 cells were transfected with psilencer-c-myc, the expression of c-myc mRNA and protein was suppressed with an inhibition rate of 67% compared with the mock-transfected cells. Apoptosis was identified in the transfected HepG2 cells. CONCLUSION: The expression of c-myc at transcriptional and translational levels in HepG2 cells transfected with siRNA is markedly inhibited, which may be associated with the induction of apoptosis.

Apoptosis↗
PubMed2004

[Effect of RNA interference on EGF receptor expression of non-small-cell lung cancer A549 cell line].

OBJECTIVE: To investigate changes in biologic properties of non-small-cell lung cancer (NSCLC) A549 cells whose EGF receptor (EGFR) expression was suppressed by short interference RNA (siRNA). METHODS: A549 cells were transfected with synthetic EGFR sequence-specific siRNA by Lipofectamine. EGFR expression was examined by Western blot and flow cytometry. The biological features of the transfected A549 cells were assessed by cell cycle analysis, colony formation and chemosensitivity assay. RESULTS: Sequence-specific siRNAs targeting EGFR significantly down-regulated its expression in A549 cells. Cell growth and colony formation were inhibited by 85.0% and 63.3%, respectively, as compared to the non-sequence-specific siRNA treated cells. Decreased EGFR expression was accompanied by 12.7% increase in A549 cells in G(0)-G(1) phase and 6.6% decrease in S-phase. The EGFR sequence-specific siRNA transfected A549 cells were much more sensitive to the cytotoxic effect of cisplatin with a 77.2% decrease in IC(50) compared to the non-sequence-specific iRNA transfected A540 cells. CONCLUSION: Down regulation of EGFR expression of NSCLC by sequence-specific siRNA may be considered as an additional option in the treatment of EGFR over-expressing cancers, including NSCLC.

Antineoplastic Agents↗
PubMed2005

[Reducing the maize amylopectin content through RNA interference manipulation].

To regulate the biosynthesis of maize starch, gene segment of starch branch enzyme (SBE) in maize was cloned and an expression vector of small interference RNA with the fragment of antisense gene + sense gene (pCJSBE2b, Fig.1) was constructed. The expression vector of pCJSBE2b was transformed into maize inbred lines by using pollen-tube pathway. Results of PCR amplification and Southern blotting indicated that the aimed gene with the fragment of antisense gene + sense gene had integrated into receptor maize genome (Fig.4,5) and inherited steadily in the transgenic plants (Table 1). Northern hybridization analysis showed that the aimed gene transcribed normally in the transgenic plants, which resulted in a reduction of endogenous SBE mRNA (Fig.6). The analysis of the activity of starch branching enzyme showed that the enzyme activity decreased obviously in the transgenic plants and maximum decrease was 85% compared to the control plants (Fig.7a). Although the total starch content of transgenic plants showed no change compared to the control plants but the amylose content increased to 50% (Fig.7b) in transgenic plants about 27% higher than receptor maize plants.

1,4-alpha-Glucan Branching Enzyme↗
PubMed2006

Interference RNA technology in the treatment of CNV.

RNA interference is a new technology that allows the production of therapeutic double-stranded RNA molecules (siRNA) that can specifically prevent the production of a particular gene product in a potent and efficient manner. It accomplishes this task by activating a native cellular defense mechanism, which when triggered results in the prevention of gene product production before the protein is made. Because of this amplification, only small quantities of siRNA are required to produce a silencing of gene production. siRNA designed to reduce production of angiogenic molecules provides potent therapies for ocular neovascularization in patients with age-related macular degeneration or diabetic retinopathy.

Animals↗
PubMed2006

Reversal of MDR1 gene-dependent multidrug resistance using low concentration of endonuclease-prepared small interference RNA.

Multidrug resistance following initial chemotherapy is commonly associated with MDR1 gene encoding for P-glycoprotein (P-gp). RNA interference of MDR1 gene expression was used as a strategy to reverse MDR1-mediated multidrug resistance phenotypes. Here we report that endonuclease-prepared small interfering RNA (esiRNA) at concentrations as low as 10 ng/ml (about 0.7 nM) can decrease MDR1 expression and increase chemosensitivity in the Adriamycin-induced resistant MCF-7/R cells. When MCF-7/R cells were transiently transfected with esiRNA of MDR1 (esiMDR1), the MDR1 mRNA was reduced by about 50%, drug accumulation increased by about 30%, and the IC50 for daunorubicin was reduced from 4.5 to 1.2 microM. These results provide evidence that esiRNA of MDR1 could be an alternative to P-gp inhibitors with the advantage of avoiding non-specific suppression with a lower effective dosage than using a single siRNA duplex, offering a potential therapeutic application of siRNA.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2006

Gene silencing using small interference RNA in mast cells.

Small interfering RNA (siRNA) is a potent and specific method of inducing gene silencing through induction of RNA interference. siRNAs can be allowed for in vitro and in vivo applications. siRNAs have been successfully studied in vitro, but little is known about its efficacy in vivo. We have successfully applied the siRNA technique for silencing glucose transporter 3 in cultured L6 muscle cells. The siRNA technique has been used efficiently to silence RasGRP4 expression in mast cells.

Cell Line↗
PubMed2004

Sense and antisense: therapeutic potential of oligonucleotides and interference RNA in asthma and allergic disorders.

Advances in our understanding of asthma pathogenesis and delineation of the human genome project are yielding novel candidate targets for therapeutic intervention. In parallel with target identification, the past decade has produced novel approaches to normalizing expression genes that are upregulated in disease processes. Single-stranded antisense oligonucleotides and double-stranded short-interfering RNA molecules, which specifically mark target transcripts for degradation, are being investigated for their ability to modulate disease processes. In both cases, the targets are RNA transcripts, and not protein; therefore, all types of molecular gene products can be inhibited, including historically undrugable species such as transcription factors and phosphatases. Various RNA interference strategies have been successfully tested in vitro and in animal models of disease, and data is beginning to accumulate from human clinical trials.EPI-2010, a 21-mer phosphorothioate against the adenosine A1 receptor promoter region,has completed preclinical pharmacology testing and its initial clinical trials. The rationale for EPI-2010 is that overactivity of the adenosine signaling pathway in asthmatic lungs contributes to airway inflammation and hyperresponsiveness. Phase I/IIa clinical trials have shown EPI-2010 to be safe and well-tolerated, with modest indications of efficacy in patients with mild asthma.

Asthma↗
PubMed2006

Functional characterization of Src-interacting Na/K-ATPase using RNA interference assay.

We have shown that the Na/K-ATPase and Src form a signaling receptor complex. Here we determined how alterations in the amount and properties of the Na/K-ATPase affect basal Src activity and ouabain-induced signal transduction. Several alpha1 subunit knockdown cell lines were generated by transfecting LLC-PK1 cells with a vector expressing alpha1-specific small interference RNA. Although the alpha1 knockdown resulted in significant decreases in Na/K-ATPase activity, it increased the basal Src activity and tyrosine phosphorylation of focal adhesion kinase, a Src effector. Concomitantly it also abolished ouabain-induced activation of Src and ERK1/2. When the knockdown cells were rescued by a rat alpha1, both Na/K-ATPase activity and the basal Src activity were restored. In addition, ouabain was able to stimulate Src and ERK1/2 in the rescued cells at a much higher concentration, consistent with the established differences in ouabain sensitivity between pig and rat alpha1. Finally both fluorescence resonance energy transfer analysis and co-immunoprecipitation assay indicated that the pumping-null rat alpha1 (D371E) mutant could also bind Src. Expression of this mutant restored the basal Src activity and focal adhesion kinase tyrosine phosphorylation. Taken together, the new findings suggest that LLC-PK1 cells contain a pool of Src-interacting Na/K-ATPase that not only regulates Src activity but also serves as a receptor for ouabain to activate protein kinases.

Animals↗
PubMed2005

Dicing and slicing: the core machinery of the RNA interference pathway.

RNA interference (RNAi) is broadly defined as a gene silencing pathway that is triggered by double-stranded RNA (dsRNA). Many variations have been described on this theme. The dsRNA trigger can be supplied exogenously, as an experimental tool, or can derive from the genome in the form of microRNAs. Gene silencing can be the result of nucleolytic degradation of the mRNA, or by translational suppression. At the heart of the pathway are two ribonuclease machines. The ribonuclease III enzyme Dicer initiates the RNAi pathway by generating the active short interfering RNA trigger. Silencing is effected by the RNA-induced silencing complex and its RNaseH core enzyme Argonaute. This review describes the discovery of these machines and discusses future lines of work on this amazing biochemical pathway.

Animals↗
PubMed2006

The virion-associated incoming HIV-1 RNA genome is not targeted by RNA interference.

BACKGROUND: RNA interference (RNAi) has proven to be a powerful tool to suppress gene expression and can be used as a therapeutic strategy against human pathogenic viruses such as human immunodeficiency virus type 1 (HIV-1). Theoretically, RNAi-mediated inhibition can occur at two points in the replication cycle, upon viral entry before reverse transcription of the RNA genome, and on the newly transcribed viral RNA transcripts. There have been conflicting results on whether RNAi can target the RNA genome of infecting HIV-1 particles. We have addressed this issue with HIV-1-based lentiviral vectors. RESULTS: We determined the transduction efficiency of a lentiviral vector, as measured by GFP expressing cells, which reflects the number of successful integration events in a cell line stably expressing shNef. We did not observe a difference in the transduction efficiency comparing lentiviral vectors with or without the Nef target sequence in their genome. The results were similar with particles pseudotyped with either the VSV-G or HIV-1 envelope. Additionally, no reduced transduction efficiencies were observed with multiple other shRNAs targeting the vector genome or with synthetic siNef when transiently transfected prior to transduction. CONCLUSION: Our findings indicate that the incoming HIV-1 RNA genome is not targeted by RNAi, probably due to inaccessibility to the RNAi machinery. Thus, therapeutic RNAi strategies aimed at preventing proviral integration should be targeting cellular receptors or co-factors involved in pre-integration events.

Cloning, Molecular↗
PubMed2005

Sequence-specific suppression of mdr1a/1b expression in mice via RNA interference.

PURPOSE: RNA interference (RNAi) is a powerful tool for silencing gene expression posttranscriptionally. The purpose of this study was to examine whether in vivo RNAi can be induced against endogenous mdr1a/1b in adult mice and to assess the feasibility of generating P-glycoprotein (P-gp) knockdown mice based on RNAi by a very simple intravenous injection of synthetic small interfering RNA (siRNAs) or siRNA-expressing plasmid DNAs. METHODS: The targeted sequences for silencing mdr1a specifically or mdr1a/1b simultaneously were examined in an in vitro study using a mouse colon carcinoma cell line, colon26 cells, in culture. Mice were repeatedly treated with intravenous synthetic siRNAs or siRNA-expressing plasmid DNAs in naked form administered via a large-volume and high-speed injection, i.e., the hydrodynamics-based procedure. The amount of targeted mRNA and P-gp in the liver were determined by real-time polymerase chain reaction and Western blot analysis, respectively. RESULTS: Among several targeted sequences, two and one optimized sequences were selected for mdr1a and mdr1a/1b, respectively, in the in vitro study. Following administration of synthetic siRNAs or siRNA-expressing plasmid DNAs directed against mdr1a, the mRNA level in the liver was significantly reduced to approximately 50-60% of that in control mice. Furthermore, a slight reduction was observed at the protein level. Similar results were obtained in the experiments using siRNA-expressing pDNA directed against mdr1a/1b. CONCLUSIONS: Our results demonstrate that sequence-specific suppression of mdr1 gene expression is possible at the mRNA level as well as the protein level in mice following intravenous delivery of siRNA effectors.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2006

The interplay between virus infection and the cellular RNA interference machinery.

RNA interference (RNAi) plays a pivotal role in the regulation of gene expression to control cell development and differentiation. In plants, insects and nematodes RNAi also functions as an innate defence response against viruses. Similarly, there is accumulating evidence that RNAi functions as an antiviral defence mechanism in mammalian cells. Viruses have evolved highly sophisticated mechanisms for interacting with the host cell machinery, and recent evidence indicates that this also involves RNAi pathways. The cellular RNAi machinery can inhibit virus replication, but viruses may also exploit the RNAi machinery for their own replication. In addition, viruses can encode proteins or RNA molecules that suppress existing RNAi pathways or trigger the silencing of specific host genes. Besides the natural interplay between RNAi and viruses, induced RNAi provides an attractive therapy approach for the fight against human pathogenic viruses. Here, we summarize the latest news on virus-RNAi interactions and RNAi based antiviral therapy.

Animals↗
PubMed2002

Inhibition of retroviral pathogenesis by RNA interference.

BACKGROUND: RNA interference (RNAi) is a newly discovered cellular defense system that is known to suppress replication of genomic parasites in model organisms. It has been widely conjectured that RNAi may also serve as an antiviral system in vertebrates. RESULTS: Retroviral infection could be initiated by electroporation of cloned Rous sarcoma virus (RSV) proviral DNA into the developing chick neural tube. Coelectroporation of proviral DNA and short double-stranded RNAs matching sequences of avain retroviruses, which were designed to induce RNAi against RSV, inhibited viral replication. Replication of RSV after electroporation resulted in disruption of embryonic development and early death, but this, too, could be suppressed by RNAi against the RSV genome. RNAi could also inhibit the growth of RSV and HIV in cell culture. Analysis of the step of the retroviral life cycle that is inhibited by RNAi revealed that it primarily prevented accumulation of the viral RNAs synthesized late during infection. RNA genomes introduced in viral particles early during infection were less sensitive. CONCLUSIONS: RNAi can block retroviral infection in vertebrates. The tissue electroporation method described here should allow RNAi to be used widely to study gene function and control of infection in vertebrate animals.

Animals↗
PubMed2006

Prevalence of off-target effects in Drosophila RNA interference screens.

RNA interference (RNAi) in both plants and animals is mediated by small RNAs of approximately 21-23 nucleotides in length for regulation of target gene expression at multiple levels through partial sequence complementarities. Combined with widespread genome sequencing, experimental use of RNAi has the potential to interrogate systematically all genes in a given organism with respect to a particular function. However, owing to a tolerance for mismatches and gaps in base-pairing with targets, small RNAs could have up to hundreds of potential target sequences in a genome, and some small RNAs in mammalian systems have been shown to affect the levels of many messenger RNAs besides their intended targets. The use of long double-stranded RNAs (dsRNAs) in Drosophila, where Dicer-mediated processing produces small RNAs inside cells, has been thought to reduce the probability of such 'off-target effects' (OTEs). Here we show, however, that OTEs mediated by short homology stretches within long dsRNAs are prevalent in Drosophila. We have performed a genome-wide RNAi screen for novel components of Wingless (Wg) signal transduction in Drosophila S2R + cells, and found few, if any, legitimate candidates. Rather, many of the top candidates exert their effects on Wg response through OTEs on known pathway components or through promiscuous OTEs produced by tandem trinucleotide repeats present in many dsRNAs and genes. Genes containing such repeats are over-represented in candidate lists from published screens, suggesting that they represent a common class of false positives. Our results suggest simple measures to improve the reliability of genome-wide RNAi screens in Drosophila and other organisms.

Animals↗
PubMed2005

[Progress of RNA interference mechanism].

RNA interference (RNAi) is a phenomenon that the double-stranded RNA (dsRNA) intermediates the degradation of complementary mRNA found in many organisms. This is a specifically mechanism involved in kinds of proteins to complete the interference function. Structure of siRNA affects which strand will be assembled into RISC. Another role of siRNA is directing RITS complex to bind with homologue chromosome, and then induces heterochromatinization. Although systemic silence induced by dsRNA is observed in Caenorhabditis elegans and plants, this progress is probably transmembrane protein-dependent, and mostly, the systemic silencing is controlled by multi-factors.

Animals↗
PubMed2002

Prevention of HIV-1 infection in human peripheral blood mononuclear cells by specific RNA interference.

The RNA interference (RNAi) phenomenon is a recently observed process in which the introduction of a double-stranded RNA (dsRNA) into a cell causes the specific degradation of a mRNA containing the same sequence. The 21-23 nt guide RNAs, generated by RNase III cleavage from longer dsRNAs, are associated with sequence-specific mRNA degradation. Here, we show that dsRNA specifically suppresses the expression of HIV-1 genes. To study dsRNA-mediated gene interference in HIV-1-infected cells, we have designed six long dsRNAs containing the HIV-1 gag and env genes. HIV-1 replication was totally suppressed in a sequence-specific manner by the dsRNAs in HIV-1-infected cells. Especially, E2 dsRNA containing the major CD4-binding domain sequence of gp120, as the target of the HIV-1 env gene, dramatically inhibited the expression of the HIV-1 p24 antigen in PBMCs for a relatively long time. The dsRNA interference method seems to be a promising new strategy for anti-HIV-1 gene therapeutics.

Animals↗