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Results for “RNA Interference”

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At least 487 records · Page 27Linked to original sources

PubMed2005

[Knockdown of bcl-xL expression with RNA interference induces nasopharyngeal carcinoma cells apoptosis].

OBJECTIVE: To study the inhibition of the expression of bcl-xL gene induced by RNA interference in CNE-2Z cell line in addition to the inhibition of its proliferation and apoptotic induction. METHODS: Small interfering RNAs targeting bcl-xL gene were synthesized by using web design software provided by Amnion and the silencer short interfering RNA (siRNA) construction kit; fluorescein-labeled siRNAs were done by FAM-silencer siRNA labeling kit; siRNAs were transfected into CNE-2Z cells by using lipofectamine 2000 reagent; siRNA transfection efficiencies were analyzed by fluorescent microscopy; down-regulation of bcl-xL was detected by RT-PCR; thiazolyl blue (MTT) assay was used to assess the cell growth; apoptosis of CNE-2Z cells was analyzed by flow cytometry. RESULTS: Green fluorescence in the cells was seen clearly in FAM-labeled siRNA transfected group under the fluorescent microscope while none in the untransfected group. Different down-regulations of bcl-xL mRNA expression were found in the transfected groups. The expression of bcl-xL mRNA decreased by 10% - 70% in the siRNAs transfected CNE-2Z by RT-PCR scan analysis. The inhibitory rate of cell proliferation depended on time and concentrations to some extent. Different cell apoptosis could be induced by different concentrations of siRNA4. CONCLUSIONS: The synthesized siRNAs in vitro were able to down-regulate the expression of bcl-xL There were different capabilities of the specific siRNAs down-regulation. The transient transfected bcl-xL siRNA4 could effectively inhibit the growth of the cancer cells and induce theirs apoptosis. It was suggested that the siRNA technique provide not only an extremely powerful tool for the functional analysis of genome but also a new method for anti-nasopharyngeal carcinoma gene therapy.

Apoptosis↗

PubMed2006

[Construction of RNA interference expression vectors of human neuropathy target esterase and its inhibition for expression of NTE in mammalian cells].

OBJECTIVE: To construct the RNA interference expression vector for expression of human neuropathy target esterase (NTE) gene in mammalian cells. METHODS: Spe I and Xho I-digested insert from pSUPER, which comprised H1 RNA polymerase III promoter and the multiple cloning sites, were cloned into the compatible in the pcDNA3.1 (+) to generate pSUPER/neo that could express small interfering RNA in mammalian cells. The annealed oligos targeting the expression of NTE were ligated into pSUPER/neo vector digested with Bgl II and Hind III to generate pSUPER/neo-NTE, which was transfected into COS7 and SH-SY5Y cells. The inhibitory effect of the expression of NTE was detected by western blot analysis and the enzyme activity assay. RESULTS: pSUPER/neo-NTE could stably express double-stranded RNA of NTE. The expression of pSUPER/neo-NTE in COS7 and SH-SY5Y cells could efficiently inhibit the activity of NTE in the mammalian cells. CONCLUSION: Stable eukaryotic expression vector of double-stranded RNA of NTE, pSUPER/neo-NTE, has been constructed successfully with promoter substitution strategy.

Animals↗

PubMed2005

Tobamovirus-resistant tobacco generated by RNA interference directed against host genes.

Two homologous Nicotiana tabacum genes NtTOM1 and NtTOM3 have been identified. These genes encode polypeptides with amino acid sequence similarity to Arabidopsis thaliana TOM1 and TOM3, which function in parallel to support tobamovirus multiplication. Simultaneous RNA interference against NtTOM1 and NtTOM3 in N. tabacum resulted in nearly complete inhibition of the multiplication of Tomato mosaic virus and other tobamoviruses, but did not affect plant growth or the ability of Cucumber mosaic virus to multiply. As TOM1 and TOM3 homologues are present in a variety of plant species, their inhibition via RNA interference should constitute a useful method for generating tobamovirus-resistant plants.

Amino Acid Sequence↗

PubMed2002

The tomato RNA-directed RNA polymerase has no effect on gene silencing by RNA interference in transgenic mice.

Double-stranded RNA (dsRNA) has been shown to interfere with the function of specific genes in various invertebrate species. The application of dsRNA interference (RNAi) in vertebrates (zebrafish and mouse) is still limited to embryos and it is not clear whether the method is generally applicable. Using a transgenic mouse model we investigated whether a stably inherited dsRNA introduced as a transgene can interfere with the expression of a specific target gene in erythroid tissue during development. In our globin gene system we do not observe any specific RNA interference. We, therefore, also introduced another gene that may be involved in a mechanism of post transcriptional gene silencing (PTGS), namely RNA-dependent RNA polymerase (RdRP) that was proposed to be involved in producing RNAs that trigger PTGS in plants. However, even though the tomato RdRP is catalytically active in erythroid tissue, no RNAi was observed.

Animals↗

PubMed2006

[Screening of aryl hydrocarbon receptor gene specific RNA interference fragment by quantitative competitive RT-PCR].

OBJECTIVE: To evaluate and screen the specific RNAi fragments which can effectively inhibit Aryl hydrocarbon receptor(AHR) gene mRNA expression in human bronchial epithelial cell line (16HBE). METHODS: AHR mRNA of 16HBE cells transfected 4 different AHR gene interfere sites were determined quantitatively with the quantitative competitive RT-PCR by using self-prepared internal standard as competitive templates, and the RNA interfere effect wasevaluated. RESULTS: AHR mRNA average expression per 40ng total RNA of 16HBE cells transfected 4 different AHR gene interfere fragments were 5.65fg, 14.78fg, 3.14fg and 0.68fg respectively, the average rates of inhibition were 61.6%, -0.5%, 78.6% and 95.4% respectively. CONCLUSION: AHR gene specific effective RNA interfere sequence ware screened by quantitative competitive RT-PCR which could accurately quantify gene mRNA level, and offered condition for studying the gene function of AHR.

Binding, Competitive↗

PubMed2006

Inhibition of rho GTPases by RNA interference.

Selective down-modulation or silencing of individual members of the Rho-GTPase family is now practical using RNA interference. Transfection of mammalian cells with an individual siRNA duplex or siRNA pools can suppress expression of a specific isoform to understand its function. By adjusting the dose of siRNA, intermediate levels of suppression can be attained to test the biological role of different levels of a GTPase such as Rac. Nevertheless, there are significant potential pitfalls, including "off-target" effects of the siRNA on other genes. Besides demonstrating successful, noncytotoxic suppression of protein and activity levels of a specific GTPase, controls are essential to establish specificity. In this chapter, we provide methods for selective knockdown of expression by siRNA and confirmation of the effectiveness of Rho GTPase silencing, as well as descriptions and some examples of controls for specificity that include evaluations of dose-response, negative and positive controls, GTPase specificity, confirmation by using more than one siRNA for the same gene, rescue by a mutated siRNA-resistant cDNA encoding the target gene, and complementary supporting evidence. Selective silencing of specific Rho family GTPases should provide increasing insight into the regulatory and functional roles of each isoform in a wide variety of biological processes.

Cell Line↗

PubMed2003

Maintenance of heterochromatin by RNA interference of tandem repeats.

Tandem repeats are prone to epigenetic silencing regulated by RNA interference. This may be because siRNAs from tandem array transcripts are regenerated by RNA-dependent RNA polymerase (RdRP) and Dicer, but siRNAs from single-copy sequences are exhausted by sequential use of downstream primers by RdRP. This could account for the formation of heterochromatin from tandem repeats.

Heterochromatin↗

PubMed2003

A functional genomic analysis of cell morphology using RNA interference.

BACKGROUND: The diversity of metazoan cell shapes is influenced by the dynamic cytoskeletal network. With the advent of RNA-interference (RNAi) technology, it is now possible to screen systematically for genes controlling specific cell-biological processes, including those required to generate distinct morphologies. RESULTS: We adapted existing RNAi technology in Drosophila cell culture for use in high-throughput screens to enable a comprehensive genetic dissection of cell morphogenesis. To identify genes responsible for the characteristic shape of two morphologically distinct cell lines, we performed RNAi screens in each line with a set of double-stranded RNAs (dsRNAs) targeting 994 predicted cell shape regulators. Using automated fluorescence microscopy to visualize actin filaments, microtubules and DNA, we detected morphological phenotypes for 160 genes, one-third of which have not been previously characterized in vivo. Genes with similar phenotypes corresponded to known components of pathways controlling cytoskeletal organization and cell shape, leading us to propose similar functions for previously uncharacterized genes. Furthermore, we were able to uncover genes acting within a specific pathway using a co-RNAi screen to identify dsRNA suppressors of a cell shape change induced by Pten dsRNA. CONCLUSIONS: Using RNAi, we identified genes that influence cytoskeletal organization and morphology in two distinct cell types. Some genes exhibited similar RNAi phenotypes in both cell types, while others appeared to have cell-type-specific functions, in part reflecting the different mechanisms used to generate a round or a flat cell morphology.

Animals↗

PubMed2006

RNA interference: unraveling a mystery.

Andrew Fire and Craig Mello have won the Nobel Prize in Medicine or Physiology for their discovery of RNA interference. Mary K. Montgomery, then a postdoc in the Fire laboratory, participated in some of the key experiments.

Animals↗

PubMed2004

Stable and complete overcoming of MDR1/P-glycoprotein-mediated multidrug resistance in human gastric carcinoma cells by RNA interference.

Multidrug resistance (MDR) is the major cause of failure of effective chemotherapeutic treatment of disseminated neoplasms. The "classical" MDR phenotype of human malignancies is mediated by drug extrusion by the adenosine triphosphate binding cassette (ABC)-transporter P-glycoprotein (MDR1/P-gp). For stable reversal of "classical" MDR by RNA interference (RNAi) technology, an H1-RNA gene promoter-driven expression vector encoding anti-MDR1/P-gp short hairpin RNA (shRNA) molecules was constructed. By introduction of anti-MDR1/P-gp shRNA expression vectors into the extremely high drug-resistant human gastric carcinoma cell line EPG85-257RDB, the MDR phenotype was completely reversed. The reversal of MDR was accompanied by a complete suppression of MDR1/P-gp expression on mRNA and protein level, and by a considerable increased intracellular anthracyline accumulation in the anti-MDR1/P-gp shRNA-treated cells. The data indicate that stable shRNA-mediated RNAi can be tremendously effective in reversing MDR1/P-gp-mediated MDR and is therefore a promising strategy for overcoming MDR by gene therapeutic applications.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2006

RNA interference: natural, experimental, and clinical roles in cancer biology.

The old idea of using antisense RNA to block messenger RNA has recently led to powerful new techniques for knocking down expression of individual protein-coding genes. The simplicity and general applicability of these new methods for RNA interference (RNAi) have turned them into fundamental tools in molecular and cellular biology, with more than 5000 publications using them during the few years since they were developed. These experimental methods are now known to exploit fundamental cellular processes that regulate differentiation via genomically encoded RNAi sequences known as microRNAs (miRNAs); changes in endogenous microRNA regulation have now been implicated in oncogenesis. Clinical trials based on local delivery of interfering RNA have already begun. More general methods for safe and effective delivery of interfering RNA to intact organisms are being developed, which could open the way to widespread clinical applications. Because RNAi can provide selective knockdown of almost any protein, it may soon provide an approach to individualized cancer therapy.

Genetic Techniques↗

PubMed2006

Suppression of glucosylceramide synthase by RNA interference reverses multidrug resistance in human breast cancer cells.

Glucosylceramide synthase (GCS), the enzyme that converts ceramide to glucosylceramide, induce multidrug resistance (MDR) in cancer cells. Recently, RNA interference (RNAi) is a powerful strategy for gene therapy by introducing double-stranded RNA and leading to the sequence-specific destruction. We have designed two different short hairpin RNAs (shRNAs) targeting GCS and introduced them into adriamycin- resistant human breast cancer cells (MCF-7/AdrR cells) to inhibit GCS expression. The results demonstrated that the shRNAs targeting GCS decreased GCS mRNA, abolished GCS protein levels and restored the sensitivity of MCF-7/AdrR cells to several antineoplastic drugs. This study revealed that this approach can reverse MDR effectively and it may be applicable to cancer patients as a specific means to restore the sensitivity to chemotherapy.

Antineoplastic Agents↗

PubMed2005

Improvement of the fatty acid composition of an oil-producing filamentous fungus, Mortierella alpina 1S-4, through RNA interference with delta12-desaturase gene expression.

An oleaginous fungus, Mortierella alpina 1S-4, is used commercially for arachidonic acid production. Delta12-Desaturase, which desaturates oleic acid (18:1n-9) to linoleic acid (18:2n-6), is a key enzyme in the arachidonic acid biosynthetic pathway. To determine if RNA interference (RNAi) by double-stranded RNA occurs in M. alpina 1S-4, we silenced the Delta12-desaturase gene. The silenced strains accumulate 18:2n-9, 20:2n-9, and Mead acid (20:3n-9), which are not detected in either the control strain or wild type strain 1S-4. The fatty acid composition of stable transformants was similar to that of Delta12-desaturation-defective mutants previously identified. Thus, RNAi occurs in M. alpina and could be used to alter the types and relative amounts of fatty acids produced by commercial strains of this fungus without mutagenesis or other permanent changes in the genetic background of the producing strains.

Arachidonic Acid↗

PubMed2006

Gene silencing analyses against amyloid precursor protein (APP) gene family by RNA interference.

Amyloid precursor protein (APP) and amyloid precursor-like proteins 1 and 2 (APLP1 and APLP2) are members of a large gene family. Although APP is known to be the source of the beta-amyloid peptides involved in the development of Alzheimer's disease, the normal functions of APP, APLP1 and APLP2 in cells are poorly understood. In this study, we carried out gene silencing analysis by means of RNA interference with synthetic small interfering RNA duplexes targeting the App, Aplp1 and Aplp2 genes in Neuro2a (N2a) cells, a mouse neuroblastoma cell line. The results demonstrated that cell viability and neurite outgrowth of N2a cells undergoing knockdown of Aplp1 were significantly reduced, compared with N2a cells undergoing knockdown of either App or Aplp2.

Amyloid beta-Protein Precursor↗

PubMed2006

Gene silencing of virus replication by RNA interference.

Small interfering RNAs (siRNAs) are as effective as long double-stranded RNAs (dsRNAs) at targeting and silencing genes by RNA interference (RNAi). siRNAs are widely used for assessing gene function in cultured mammalian cells or early developing vertebrate embryos. They are also promising reagents for developing gene-specific therapeutics. The specific inhibition of viral replication is particularly well suited to RNAi, as several stages of the viral life cycle and many viral and cellular genes can be targeted. The future success of this approach will depend on the recent advances in siRNA-based clinical trials.

Animals↗

PubMed2002

[RNA interference for mammalian cells].

Knock-out of mammalian genes is technically troublesome and time-consuming compared to those of lower animals. RNA interference (RNAi) is a strategy of sequence-specific post-translational gene silencing, and it has been successfully applied for disruption of gene transcripts in C. elegans. Gene silencing by RNAi had not been accomplished in mammalian cells until recently, because bystander activation of DNA-dependent protein kinase (PKR) coincides to disturb gene silencing by long double-stranded RNA (dsRNA), resulting in non-specific repression of translation of many proteins. A breakthrough has come recently with a report suggesting that 20- or 21-bp duplex RNA with 2 bp 3' overhang are made by DICER protein that cleaves long dsRNA during RNAi reactions in vivo and the resultant short dsRNAs mediate RNAi. Those short dsRNAs, namely, small interference RNA (siRNA), barely activate PKR. Using synthesized 21 bp siRNA, T. Tuschl's group has challenged to establish an artificial RNAi method suitable for mammalian cells. Their report was favorable in that siRNA specifically suppressed targeted gene translation in mammalian cells during culture without activation of PKR. Recently K. Taira's group developed the vector-based siRNA expression system by which RNAi is feasible in mammalian cells. Almost all genes can be targeted by RNAi. RNAi methods require minimal time and labor; therefore, mammalian gene knockdown by RNAi will become popular in the near future.

Gene Silencing↗

PubMed2006

RNA interference effector proteins localize to mobile cytoplasmic puncta in Schizosaccharomyces pombe.

Ago1, Dcr1 and Rdp1 are the core components of the RNA interference (RNAi) apparatus in the fission yeast Schizosaccharomyces pombe. They function in distinct gene-silencing pathways that direct homology-dependent degradation of mRNA and modification of chromatin. In addition, Ago1 and Dcr1 regulate enactment of Cdc2-dependent cell cycle checkpoints. The ability of the RNAi apparatus to perform multiple roles in these divergent pathways is sure to require dynamic localization of Ago1, Dcr1 and/or Rdp1. Although limited information is available, comprehensive studies regarding the relative localizations of Ago1, Dcr1 and Rdp1 are lacking. To this end, we employed live-cell imaging and immunoelectron microscopy to study the intracellular localizations of these proteins. In contrast to previous reports, our study results indicate that the bulk of Ago1 and Dcr1 form stable complexes and are associated with large, mobile, highly dynamic cytoplasmic elements. The majority of Rdp1 is localized to the nucleus, but a pool of Rdp1 is associated with the same cytoplasmic structures. The movements of these structures were dependent upon ATP and intact microtubules. Recruitment of the RNAi core proteins to these structures was not dependent upon siRNAs. Together, our data indicate that the enzymes required for the initiation and effector phases of RNA-dependent gene silencing are concentrated in a common intracellular location, an arrangement that would be expected to result in highly efficient post-transcriptional gene silencing.

Base Sequence↗

PubMed2006

Therapeutic potential of RNA interference against cancer.

One of the most dramatic events of the past 5 years in the field of molecular biology has been the discovery of RNA interference (RNAi). Although RNAi is an evolutionarily conserved phenomenon for sequence-specific gene silencing in mammalian cells, exogenous small interfering RNA (siRNA) and vector-based short hairpin RNA (shRNA) can also invoke RNAi responses. Both are now not only experimental tools for analyzing gene function but are expected to be excellent avenues for drug target discovery and the emerging class of gene medicine for targeting incurable diseases such as cancer. The success of cancer therapeutic use of RNAi relies on the development of safe and efficacious delivery systems that introduce siRNA and shRNA expression vectors into target tumor cells. For their delivery, a variety of strategies have been used, most of them based on traditional gene therapy delivery systems. In this review, we present siRNA delivery method strategies and discuss the potential of RNAi-based gene therapy in cancer treatment.

Animals↗