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Results for “RNA Interference”

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At least 163 records · Page 9Linked to original sources
PubMed2004

[Effects of RNA interference on epidermal growth factor receptor expression in SPC-A-1 cells].

OBJECTIVE: To investigate whether RNA interference (RNAi) induced by small interference RNA (siRNA) could suppress epidermal growth factor receptor (EGFR) expression in non-small-cell lung carcinoma (NSCLC) cells. METHODS: SPC-A-1 cells were transfected using chemically synthesized double stranded RNA (dsRNA) formulated with Lipofectamine 2000. The EGFR numbers were determined by both Western blot and flow cytometry. The antiproliferative effects of dsRNA-EGFR were assessed using cell counts and colony assay. Cell cycle analysis was carried out via flow cytometry. The chemosensitivity of transfected cells to cisplatin was determined by MTT. RESULTS: Sequence specific siRNAs targeting EGFR down-regulated EGFR expression significantly. Compared with the control group, dsRNA-EGFR reduced the cell numbers by 78.3% and decreased the colonies by 66.8%. Cell cycle analysis showed that dsRNA-EGFR induced accumulation of cells in G0-G1 phase by 17.48% with a significant decrease in the percentage of cells in S-phase by 19.20% relative to the control. Based on the value of IC50 obtained by Origin 6.0 software, we concluded that dsRNA-EGFR increased the sensitivity of SPC-A-1 to cisplatin by seven-fold. CONCLUSIONS: Sequence specific siRNAs targeting EGFR was capable of suppressing EGFR expression, and therefore, significantly inhibiting cellular proliferation and inducing cell cycle arrest. The finding from chemosensitivity assay further revealed that dsRNA-EGFR was associated with an addictive or synergistic effect on tumor growth inhibition when combined with cisplatin. The successful application of dsRNA-EGFR for inhibition of proliferation in EGFR overexpressing cells extends the list of available therapeutic modalities in the treatment of human cancer.

Antineoplastic Agents↗
PubMedDate not supplied

[Great potential of small RNAs: RNA interference and microRNA].

Double-stranded RNA (dsRNA) induces a sequence-specific silencing in eukaryotic cells. This silencing process beggins when long dsRNA is cleaved to 21 to 26 long small RNA by means of the RNAse III-type enzyme Dicer. These small dsRNA are included into silencing effector complexes, that are targeted to complementary sequences. Small RNA dependent gene silencing can be achieved by distinct mechanisms based depending mainly on the nature of target sequences and on the proteins present in the effector complex. The route of interference RNA (RNAi) begins when Dicer yields small interference RNA (siRNA) that bind to complementary mRNA for its degradation, forming the RISC complex. siRNA are naturally formed from transposons and dsRNA viruses during its replication, as well as from other bidirectional transcribed repetitive sequences. Some of the enzymes thar are part of the RNAi machinery, including Dicer, are encoded by multigene families in many species, that also play a role in other mechanisms of RND-dependent gene silencing. MicroRNA's (miRNA) are other small RNA's that can induce gene silencing at the mRNA level. These are formed in a general manner when Dicer process hairpin structures resulting from the transcription of non-coding sequences from plant and animal genomes. miRNA's are integrated into a RISC-like complex, after which, depending on their degree of complementarity with target mRNA, can either repress translation or induce mRNA degradation. miRNA-dependent silencing is essential for the development of multicellular organisms. Artificial RNAi induction by means of siRNA or miRNA is being used as a tool to inactivate gene expression in culture cells and in living organisms. This review focuses on the progress in the understanding of the mechanisms involved in gene regulation by RNA in animals and details some current efforts to apply theses phenomena as a tool in research and in the therapeutic of human diseases.

Humans↗
PubMed2005

RNA interference in vivo: toward synthetic small inhibitory RNA-based therapeutics.

Small interfering RNA (siRNA) mediated inhibition of gene expression has rapidly become a major tool for in vitro analysis of protein function. In vivo gene silencing by siRNAs will play an important role for target validation and is the first step towards the development of siRNA-based therapeutics. This chapter reviews the early and intriguing successes in using siRNAs for in vivo gene silencing. The impact of chemical modification on siRNA efficacy in vitro and the potential for employing such modifications to alter the pharmacokinetic properties of siRNAs is also summarized. A protocol describing siRNA-based gene silencing in tumor models can serve as guide for the design of individual in vivo RNA interference experiments.

Base Sequence↗
PubMed2003

Efficiency and specificity of RNA interference generated by intra- and intermolecular double stranded RNA in Trypanosoma brucei.

In many eukaryotes, double-stranded (ds) RNA leads to specific degradation of RNA of cognate sequence, a process termed RNA interference (RNAi). Here we used the protozoan Trypanosoma brucei as a model to investigate efficiency and specificity of RNAi generated by expression of long dsRNA of PFRA and PFRC genes, which code for flagellar proteins required for cell motility. Consequences of RNAi were monitored at all three levels: target RNA expression, protein expression and phenotype observation, using population or individual cell analysis. Expression of PFRA dsRNA from an inverted repeat was extremely efficient, knocking down PFRA RNA and PFRA protein, and producing a severe paralysis phenotype. Silencing by expression of PFRA dsRNA using a dual facing promoter system was also very efficient, producing a clear phenotype, although low amounts of PFRA RNA and PFRA protein were detected. Expression via the dual facing promoters of PAR2 dsRNA (83% overall identity with PFRA, including nine blocks of >20 nt total identity) did not produce significant reduction of total amounts of PFRA RNA or PFRA protein. However, individual cell analysis by immunofluorescence revealed that 10-60% cells (depending on subclones) exhibited lower PFRA amounts in their flagellum, producing a reduced-motility phenotype.

Animals↗
PubMed2004

RNA interference in crop plants.

RNA interference (RNAi) is a post-transcriptional gene-silencing phenomenon induced by double-stranded RNA. It has been widely used as a knockdown technology to analyze gene function in various organisms. Although RNAi was first discovered in worms, related phenomena such as post-transcriptional gene silencing and coat protein mediated protection from viral infection had been observed in plants prior to this. In plants, RNAi is often achieved through transgenes that produce hairpin RNA. For genetic improvement of crop plants, RNAi has advantages over antisense-mediated gene silencing and co-suppression, in terms of its efficiency and stability. It also offers advantages over mutation-based reverse genetics in its ability to suppress transgene expression in multigene families in a regulated manner.

Animals↗
PubMed2004

RNA interference in protozoan parasites.

RNA interference or RNAi is defined as the mechanism through which gene-specific, double-stranded RNA (dsRNA) triggers degradation of homologous transcripts. Besides providing an invaluable tool to downregulate gene expression in a variety of organisms, it is now evident that RNAi extends its tentacles into both the nucleus and the cytoplasm and is involved in a variety of gene silencing phenomena. Here we review the current status of RNAi in protozoan parasites that cause diseases of considerable medical and veterinary importance throughout Africa, Asia and the Americas. RNAi was first discovered in Trypanosoma brucei, a species of the family Trypanosomatidae, and it rapidly became the method of choice to downregulate gene expression in these organisms. At the same time, mechanistic studies exposed a role for RNAi in the control of retroposon transcript abundance. Whereas RNAi is also present in T. congolense, other members of the same family of organisms, namely T. cruzi and Leishmania major, are RNAi-negative. In apicomplexan parasites, there is experimental evidence for RNAi in Plasmodium, but this is not supported by their genetic make up. In contrast, the genome of Toxoplasma gondii harbours gene candidates with convincing similarity to 'classical' RNAi genes. Thus, as previously shown in fungi, protozoan parasites are genetically heterogeneous as far as the RNAi pathway is concerned. Finally, database mining predicts that Entamoeba histolytica and Giardia intestinalis have an RNAi pathway and the presence of RNAi genes in Giardia supports the view that gene silencing by dsRNA appeared very early during evolution of the eukaryotic lineage.

Animals↗
PubMed2003

Small nucleolar RNA interference induced by antisense or double-stranded RNA in trypanosomatids.

In trypanosomes the C/D- and H/ACA-like small nucleolar RNAs (snoRNAs) are clustered and repeated in the genome. The snoRNAs studied to date are transcribed as polycistronic transcripts by RNA polymerase II and then processed, resulting in mature snoRNAs. In this study we demonstrated that snoRNA genes can be silenced in three trypanosomatid species: Leptomonas collosoma, Leishmania major, and Trypanosoma brucei. Silencing was achieved in L. collosoma and L. major by the expressing of an antisense transcript complementary to the snoRNA gene and was accompanied by the accumulation of small interfering RNA. Silencing eliminated the mature snoRNA but not its precursor and abolished the specific 2'-O-methylation guided by the snoRNA. In T. brucei, silencing was achieved by using the inducible synthesis of double-stranded RNA from T7 opposing promoters. Silencing varied between the different snoRNA genes, which may reflect the accessibility of small interfering RNA to the target RNAs. This study suggests that RNA interference can degrade snoRNAs. This study has further implications in elucidating the function of nucleolar RNAs and specific modifications guided by these RNAs in trypanosomatids and perhaps in other eukaryotes as well.

Animals↗
PubMedDate not supplied

Kinetic and efficacy analysis of RNA interference in stably and transiently expressing cell lines.

RNA interference, particularly through the use of small interfering RNA (siRNA), has become an important laboratory tool for both fundamental and applied investigations. However, it is currently unknown whether siRNA-mediated knockdown of transiently expressed proteins is an acceptable quantitative surrogate for stably expressed proteins. Further, the best means by which to transfect cells with functionally active siRNA are poorly defined, and determination of the best reagent and transfection conditions for a particular cell line is a burdensome prerequisite for RNA interference studies. We therefore established the optimal transfection conditions for six commercial siRNA delivery reagents in three cell lines (HR5-CL11, HeLa, and NIH/3T3) transiently or stably expressing the firefly luciferase gene. The delivery efficiency, knockdown kinetics, and cytotoxicity of the reagents were evaluated. siPORT Amine, X-tremeGENE, and TransIT-siQUEST achieved the best knockdown and consistency of performance among the three cell lines. Delivery efficiency varied and was cell line dependent in some cases. The knockdown kinetics were reagent-dependent, and knockdown was generally more rapid in the stably transfected cells. Cytotoxicity of the reagents was variable. GeneSilencer was the least cytotoxic reagent for all three cell lines, and TransIT-siQUEST was the most cytotoxic to the HeLa and HR5-CL11 cell lines. These comparative results provide an initial basis for reagent selection and experimental design for RNA interference studies in HeLa, NIH/3T3, and their respective derivative cell lines.

Animals↗
PubMed2006

[Influence of survivin gene repression by RNA interference on the radiosensitivity and chemosensitivity to cisplatin of cervical cancer cell HeLa].

OBJECTIVE: To observe the effect of survivin gene repression by RNA interference (RNAi) on the radiosensitivity and the chemosensitivity to cisplatin of cervical cancer cell HeLa. METHODS: The recombined eukaryotic expression plasmid pSilencer2.1-s2 containing human survivin gene small interference RNA (siRNA) was transfected into human cervical cancer cell line HeLa by using lipofectamine 2000. The expression of survivin gene mRNA and its protein was detected by semi-quantitative RT-PCR and western blot respectively. The changes of caspase-3 activity was assessed by kinase activity test. Cell apoptosis was examined by flow cytometry. The changes of cell radiosensitivity was observed by plate clone formation assay. Cell viability was examined by methyl thiazolyl tetrazolium (MTT) assay and 50% inhibitive concentration (IC(50)) of cisplatin was examined also. RESULTS: Compared with the cells which were transfected with negative control plasmid (HeLa-NC), pure plasmid (HeLa-U6 neo) and un-transfected cells (HeLa), the expression level of survivin gene mRNA and protein declined evidently in the cells transfected with pSilencer2.1-s2 plasmid (HeLa-s2), the expression inhibitory rates were (62.8 +/- 0.3)% and (60.1 +/- 0.5)%; the caspase-3 activity was enhanced and A(405) was 1.261 +/- 0.043 (P < 0.05); the apoptotic rate was increased obviously (29.23 +/- 1.41)% (P < 0.05). At the same dose of radiation, clone formation rate declined significantly (P < 0.05); at the same concentration of cisplatin, cell viability declined sharply and the IC(50) of cisplatin was (0.873 +/- 0.021) microg/ml (P < 0.05). CONCLUSION: Survivin gene repression by RNAi can enhance caspase-3 activity, induce cell apoptosis, significantly increase the radiosensitivity and chemosensitivity to cisplatin in human cervical cancer cell line HeLa.

Antineoplastic Agents↗
PubMed2006

[Inhibitory effects of RNA interference on expression of matrix metalloproteinase-9 gene and invasiveness and adhesion in ovarian cancer cells].

OBJECTIVE: To investigate the inhibitory effects of RNA interference (RNAi) on the expression of matrix metalloproteinase-9 (MMP-9) gene and invasiveness and adhesion of ovarian cancer cells. METHODS: Four groups of different specific target sequence in coding region of MMP-9 and one non-specific sequence were chosen, which were Site1, Site2, Site3, Site4 and Site5. Small interference RNA (siRNA) expression cassettes (SEC) were constructed by PCR and transfected into ovarian cancer HO-8910PM cells. RT-PCR and western blot were used to detect mRNA and protein expression of MMP-9 gene; the abilities of invasion and adhesion were detected by Matrigel invasion assay and cell adhesion assay. RESULTS: The expression of MMP-9 was inhibited and the inhibitory effects of different sequence were varied. The mRNA expression was 0.64 +/- 0.06, 0.47 +/- 0.07, 0.55 +/- 0.10 in Site1, Site2, Site3 group, and protein expression was 0.30 +/- 0.09, 0.27 +/- 0.08, 0.37 +/- 0.12, respectively. Site2 group had the most efficient inhibitory effect, followed by Site1 and Site3 groups. Cell growth curve revealed that cell growth was significantly inhibited in Site2 group. Invasiveness and adhesion were significantly reduced, the inhibitory rate on invasion in Site1, Site2, Site3 groups were 50.0%, 50.0% and 37.5%, respectively; the inhibitory rate on adhesion in Site1, Site2, Site3, Site4 groups were 43.8%, 48.8%, 33.9%, 24.2% at 60 min and 41.6%, 40.2%, 35.1%, 16.0% at 90 min, respectively. CONCLUSIONS: RNAi exists in ovarian HO-8910PM cells. MMP-9 siRNA can specifically down-regulate MMP-9 expression and lead to the inhibition of invasiveness and adhesion in ovarian cancer cells.

Blotting, Western↗
PubMed2003

RNA interference and mRNA silencing, 2004: how far will they reach?

The discoveries of RNA interference and RNA-mediated posttranscriptional gene silencing have opened an unanticipated new window on the regulation of gene expression as well as a facile and highly effective tool for knocking down gene expression in many organisms and cells. In addition, RNA interference and RNA silencing may conceivably be exploited for human therapeutics sometime in the future, possibly bringing greater clinical impact than have the so far disappointing antisense endeavors. This essay summarizes recent developments and offers some personalized perspectives, with emphasis on what we do not yet know.

Animals↗
PubMed2006

shRNA transcribed by RNA Pol II promoter induce RNA interference in mammalian cell.

RNA interference is a powerful tool for gene functional analysis in mammals. Permanent gene suppression can be achieved by siRNAs as stem-loop precursors transcribed from RNA Pol III promoter such as H1 and U6 based on vector. This approach, however, has a major limitation: inhibition can not be controlled in a time or tissue specific manner because the RNA Pol III promoter is not time or tissue specific. To overcome these limitations, we designed a strategy that allows synthesis of small hairpin RNAs in a GFP-fused form mediated by RNA Pol II promoter CMV to efficiently and specifically knock down expression of both exogenous and endogenous genes in mammalian cells. As assayed by both fluorescence observing and quantitative RT-PCR, the protein and mRNA products of exogenous gene RFP were efficiently and specifically inhibited; quantitative RT-PCR and western blotting results respectively demonstrated that endogenous lamin B2 mRNA and protein was suppressed without global down-regulation of protein synthesis. Furthermore, GFP-fused shRNA efficacy for RNAi is dependent on target position based on this vector system. This method may provide a novel approach for the application of RNAi technology in suppressing gene expression in mammalian system.

Animals↗
PubMed2001

Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans.

Double-stranded RNAs can suppress expression of homologous genes through an evolutionarily conserved process named RNA interference (RNAi) or post-transcriptional gene silencing (PTGS). One mechanism underlying silencing is degradation of target mRNAs by an RNP complex, which contains approximately 22 nt of siRNAs as guides to substrate selection. A bidentate nuclease called Dicer has been implicated as the protein responsible for siRNA production. Here we characterize the Caenorhabditis elegans ortholog of Dicer (K12H4.8; dcr-1) in vivo and in vitro. dcr-1 mutants show a defect in RNAi. Furthermore, a combination of phenotypic abnormalities and RNA analysis suggests a role for dcr-1 in a regulatory pathway comprised of small temporal RNA (let-7) and its target (e.g., lin-41).

Alleles↗
PubMed2005

RNA interference in biology and disease.

RNA interference (RNAi) is a conserved biologic response to double-stranded RNA that results in the sequence-specific silencing of target gene expression. Over the past 5 years, an intensive research effort has facilitated the rapid movement of RNAi from a relatively obscure biologic phenomenon to a valuable tool used to silence target gene expression and perform large-scale functional genomic screens. In fact, recent studies reported in this journal and others have demonstrated success using RNAi to address the role of oncogene expression in leukemia cell lines and to validate the therapeutic potential of RNAi for treating these blood disorders. In order to advance these applications and gain an appreciation for the future of RNAi both in basic research and in the treatment of diseases caused by aberrant gene expression, it is important to have an understanding of the process of RNAi and its limitations.

Gene Expression Regulation, Neoplastic↗
PubMed2005

A genetic strategy to treat sickle cell anemia by coregulating globin transgene expression and RNA interference.

The application of RNA interference (RNAi) to stem cell-based therapies will require highly specific and lineage-restricted gene silencing. Here we show the feasibility and therapeutic potential of coregulating transgene expression and RNAi in hematopoietic stem cells. We encoded promoterless small-hairpin RNA (shRNA) within the intron of a recombinant gamma-globin gene. Expression of both gamma-globin and the lariat-embedded small interfering RNA (siRNA) was induced upon erythroid differentiation, specifically downregulating the targeted gene in tissue- and differentiation stage-specific fashion. The position of the shRNA within the intron was critical to concurrently achieve high-level transgene expression, effective siRNA generation and minimal interferon induction. Lentiviral transduction of CD34(+) cells from patients with sickle cell anemia led to erythroid-specific expression of the gamma-globin transgene and concomitant reduction of endogenous beta(S) transcripts, thus providing proof of principle for therapeutic strategies that require synergistic gene addition and gene silencing in stem cell progeny.

Anemia, Sickle Cell↗
PubMed2005

The prospect of silencing disease using RNA interference.

The discovery of RNA interference (RNAi), an endogenous cellular gene-silencing mechanism, has already provided a powerful tool for basic science researchers to study gene function. The subsequent finding that RNAi also operates in mammalian cells has generated excitement regarding potential therapeutic applications. In this article we discuss the basic mechanism of RNAi and the therapeutic opportunities and obstacles for harnessing RNAi for therapy of human disease.

Animals↗
PubMed2005

RNA interference: past, present and future.

RNA interference (RNAi) is the sequence-specific gene silencing induced by double-stranded RNA. RNAi is mediated by 21-23 nucleotide small interfering RNAs (siRNAs) which are produced from long double-stranded RNAs by RNAse II-like enzyme Dicer. The resulting siRNAs are incorporated into a RNA-induced silencing complex (RISC) that targets and cleaves mRNA complementary to the siRNAs. Since its inception in 1998, RNAi has been demonstrated in organisms ranging from trypanosomes to nematodes to vertebrates. Potential uses already in progress include the examination of specific gene function in living systems, the development of anti-viral and anti-cancer therapies, and genome-wide screens. In this review, we discuss the landmark discoveries that established the contextual framework leading up to our current understanding of RNAi. We also provide an overview of current developments and future applications.

Animals↗