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PubMed2005

Efficient inhibition of human telomerase reverse transcriptase expression by RNA interference sensitizes cancer cells to ionizing radiation and chemotherapy.

Telomerase activation plays critical roles in tumor growth and progression in part through the maintenance of telomere structure. Indeed, the ubiquitous expression of telomerase in human cancers makes telomerase a promising target for cancer therapy. Genetic, pharmacologic, and antisense methods to inhibit telomerase have been described; however, in most cases, cancer cell death was observed only after many cell divisions. Here, using retroviral delivery of small interfering RNAs (siRNAs) specific for the human telomerase reverse transcriptase (hTERT), we successfully inhibited telomerase activity in cervical cancer cell lines. Cells lacking hTERT expression exhibited significantly decreased telomerase activity and showed shortened telomeres and telomeric 3' overhangs with passage. These cells entered replicative senescence after a considerable number of cell divisions. Notably, the proliferative rate of these cells was significantly impaired, compared with control cells with telomerase activity, even in low-passage cells (population doubling 5). Likewise, colony-forming ability and tumorigenicity in mice were attenuated in low-passage cells lacking hTERT. We further examined the effects of chemotherapy and ionizing radiation on cells in which hTERT expression was suppressed. Cells lacking hTERT showed a significantly increased sensitivity, compared with control cells, to ionizing radiation or chemotherapeutic agents that induce DNA double- strand breaks, such as topoisomerase inhibitors or bleomycin. These findings suggest that an siRNA-based strategy can be applied to the development of novel telomerase inhibitors, the antitumor effects of which may be enhanced in combination with ionizing radiation and chemotherapy.

Animals↗
PubMed2005

RNA interference in Haemonchus contortus: suppression of beta-tubulin gene expression in L3, L4 and adult worms in vitro.

We have used RNAi to target two beta-tubulin genes in the parasitic stages of Haemonchus contortus in vitro. Soaking exsheathed-L3, L4 and adult worms in medium containing dsRNA resulted in a significant decrease (greater than 1000-fold in some cases) in the expression of the specific beta-tubulin transcript, as measured by quantitative PCR. During the initial 24h exposure to the dsRNA, the gene suppression effect was quite specific to the targeted gene. Six days after initial exposure to dsRNA, treated L3 worms were less able to migrate through a filter mesh, indicating decreased motility, and showed less development to the L4 stage than control larvae. The gene suppression effect occurs in exsheathed L3 larvae despite the fact that this life stage does not have functioning mouthparts, indicating that uptake of dsRNA does not depend on its ingestion. Suppression occurred with dsRNA presented in 'naked' or liposome-encapsulated forms, indicating that a liposome formulation was not necessary for uptake to occur. Adult worms also showed significant gene suppression, however, they did not show any reduced motility compared to controls over a 3-day period. Adult worms treated with ivermectin to paralyse their pharynx, still showed significant gene suppression, again suggesting that uptake of dsRNA does not require ingestion. We have shown that soaking in dsRNA is an effective method for RNAi with the parasitic stages of H. contortus, and, hence, may offer significant potential as a tool for studying gene function in this parasite species.

Animals↗
PubMed2006

Down-regulation of Bcl-XL by RNA interference suppresses cell growth and induces apoptosis in human esophageal cancer cells.

AIM: To determine the inhibitory effect of the vector-generated small interfering RNAs (siRNAs) on the expression of the Bcl-X(L) gene in established human esophageal cancer cells, and to investigate the effect of the Bcl-X(L) siRNAs on cell growth and apoptosis in esophageal cancer cells. METHODS: Three siRNA-expressing vectors targeting different sites of the Bcl-X(L) gene were constructed from pTZ-U6+1 vector. Cultured esophageal cancer cells were transfected with the siRNA-expressing vector (or the control vector) using lipofectamine 2000. Bcl-X(L) gene expression was determined with semiquantitative RT-PCR assay and Western blotting. Among the three siRNA-expressing vectors, the most highly functional vector and its effect on cell growth and apoptosis in esophageal cancer cells was further analyzed. RESULTS: Of the three siRNA-expressing vectors, siRNA-expressing vector No.1 was the most potent one which suppressed Bcl-X(L) mRNA production to 32.5% of that in the untreated esophageal cancer cells. Western blotting analysis showed that siRNA-expressing vector No.1 markedly down-regulated the expression of Bcl-X(L) in human esophageal cancer cells. Treatment of esophageal cancer cells with siRNA-expressing vector No.1 resulted in inhibition of cell growth and induction of apoptosis. CONCLUSION: Down-regulation of Bcl-X(L) by vector-generated small interfering RNAs can suppress cell growth and induce apoptosis in human esophageal cancer cells.

Apoptosis↗
PubMed2006

[Reversion of multidrug resistance (MDR) in human glioma cells by RNA interference (RNAi)].

OBJECTIVE: To explore whether the constructed vector of short haprin in vivo can induce human glioma cell line BT325 to produce RNAi duplexes and reverse the expression of MDR1 gene. METHODS: Three 62nt oligonucleotide fragments (shRNA) were constructed according to GenBank MDR1 sequence and were cloned to the retrovirus-delivered vectors. After transfected these vectors directly into the human malignant glioma BT325 cells by lipofectamine 2000 with enhanced green fluorescence protein (EGFP) co-transfecting, the MDR1 gene silence effects were detected by the changing level of mRNA and P-glycoprotein including real time PCR (RT-PCR), Northern blot and Western blot analysis. To assess the multidrug resistance against adriamycin (ADR) and VCR, cell proliferation assays were performed by cell counting kit-8. RESULTS: The RNAi plasmid vectors were constructed successfully. RT-PCR showed MDR1 mRNA was significantly reduced (P < 0.05). Northern blot analysis showed that the gene silence became most intense at 48 hours after transfection. Western blot analysis demonstrated that P-gp expression was reduced at different time to 12.9%, 30.3% and 4.8%, respectively. The chemosensitivity assays indicated that the transfected cells showed an enhanced sensitivity to ADR and VCR. Based on the value of IC(50), BT325 cells had significantly increased sensitivity to the drugs. CONCLUSION: The sequence specific RNAi can inhibit MDR1 mRNA and P-gp expression in the glioma cell line. It may reverse multidrug resistance phenotype, therefore, may provide promising therapeutic modalities in the treatment of human glioma.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

An RNA interference model of RPS19 deficiency in Diamond-Blackfan anemia recapitulates defective hematopoiesis and rescue by dexamethasone: identification of dexamethasone-responsive genes by microarray.

Diamond-Blackfan anemia (DBA), a congenital erythroblastopenia, is a model disease for the study of erythroid differentiation but is poorly understood. RPS19 is the only gene yet to have been associated with DBA, but its relevance to erythroid differentiation is unclear. The molecular basis for the stimulation of erythropoiesis by glucocorticoids in patients with DBA has not been identified. We demonstrate that targeted degradation of the RPS19 transcript, through retroviral expression of short hairpin RNAs (shRNAs), blocks the proliferation and differentiation of erythroid progenitor cells in cultured human CD34(+) cells. Treatment of RPS19-deficient cells with dexamethasone restores erythroid differentiation to normal levels. We investigated the molecular basis of pharmacologic therapies for DBA using oligonucleotide microarrays to survey gene expression in CD34(+) cells treated with combinations of dexamethasone, erythropoietin, stem cell factor, and interleukin-3. Dexamethasone did not alter expression of RPS19 but activated a genetic program that includes a set of key hematopoietic regulatory genes. Genes specific to erythroid progenitor cells were up-regulated by dexamethasone, while genes specific to nonerythroid lineages were down-regulated. Deficiency of RPS19 therefore blocks proliferation of immature erythroid progenitor cells, and dexamethasone activates proliferation of the same cell population through mechanisms independent of RPS19.

Anemia, Diamond-Blackfan↗
PubMed2005

[Effect of silencing HIF-1alpha by RNA interference on expression of vascular endothelial growth factor in osteosarcoma cell line SaOS-2 under hypoxia].

BACKGROUND & OBJECTIVE: Hypoxia-inducible factor-1 alpha (HIF-1alpha) is a key regulator for hypoxia tolerance and angiogenesis of tumor. This study was to investigate the expression of HIF-1alpha and vascular endothelial growth factor (VEGF) in human osteosarcoma cell line SaOS-2 under hypoxia, to explore the effect of HIF-1alpha on hypoxia-activated angiogenesis regulation pathway in osteosarcoma. METHODS: CoCl2 was used as chemical hypoxia-inducing reagent to mimic tumor hypoxic microenvironment. mRNA and protein levels of HIF-1alpha and VEGF at different hypoxic culture phases were detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Small hairpin RNAs (shRNAs) eukaryotic expression vector targeting HIF-1alpha was constructed, and transfected into SaOS-2 cells. Western blot was used to detect gene silencing effect on HIF-1alpha. RT-PCR and enzyme-linked immunosorbent assay (ELISA) were used to observe the change of VEGF gene expression after HIF-1alpha gene silence. RESULTS: Under hypoxia, mRNA level of HIF-1alpha kept stable, while its protein level increased obviouslyu both mRNA and protein levels of VEGF were up-regulated. The shRNAs plasmid targeting HIF-1alpha gene was constructed successfully, and down-regulated HIF-1alpha gene in SaOS-2 cells efficiently followed by VEGF gene down-regulation. CONCLUSIONS: Hypoxia can increase protein level of HIF-1alpha in osteosarcoma. HIF-1alpha up-regulates the gene expression of VEGF via transcription activation which promotes angiogenesis in osteosarcoma under hypoxic microenvironment.

Bone Neoplasms↗
PubMed2006

Use of RNA interference to inhibit integrin subunit alphaV-mediated angiogenesis.

One of the central molecules in capillary formation during angiogenesis is the integrin alphaVbeta3. The aim of this study was to inhibit alphaV-mediated angiogenesis in vitro using RNAs (siRNA) as well as antisense oligodeoxyribonucleotides (asON). Five siRNAs, against the alphaV chain of alphaVbeta3, and three asON, which had the respective sequence of the antisense sequence of three of the siRNAs molecules, were examined. Two of the siRNAs and their respective asON were designed on the basis of computer-predicted secondary structure analysis of alphaV mRNA. The different molecules were transfected into human umbilical vein endothelials cells (HUVEC) using lipofection. Following stimulation by PMA, two siRNAs showed a dose-dependent inhibition of PMA-induced alphaV mRNA and protein upregulation, as assessed by real-time RT-PCR and flow cytometry. At a concentration of 25 nM a complete inhibition of protein upregulation was found using siRNAs while transfection of the respective asON sequences reduced the protein upregulation only by 44%. To evaluate the anti-angiogenic potential a cell culture model of human angiogenesis based on the co-cultivation of endothelial cells and dermal fibroblasts was used. Transfection of the siRNA sequence (50 nM) resulted in an inhibition of the total length of capillary-like tubules by 40.6% in comparison to 21.1% using the respective asON sequence. In conclusion, siRNA-based downregulation of alphaV expression showed a stronger inhibition of capillary tube formation in an angiogenesis in vitro assay, than asON having the same sequence as the antisense strand of the siRNAs. Therefore, siRNAs are useful tools for functional alphaV knock-down experiments and might be a therapeutic alternative for antagonists which bind directly to the integrins alphaVbeta3 or alphaVbeta5.

Apoptosis↗
PubMed2005

RNA interference is ineffective as a routine method for gene silencing in chick embryos as monitored by fgf8 silencing.

The in vivo accessibility of the chick embryo makes it a favoured model system for experimental developmental biology. Although the range of available techniques now extends to miss-expression of genes through in ovo electroporation, it remains difficult to knock out individual gene expression. Recently, the possibility of silencing gene expression by RNAi in chick embryos has been reported. However, published studies show only discrete quantitative differences in the expression of the endogenous targeted genes and unclear morphological alterations. To elucidate whether the tools currently available are adequate to silence gene expression sufficiently to produce a clear and specific null-like mutant phenotype, we have performed several experiments with different molecules that trigger RNAi: dsRNA, siRNA, and shRNA produced from a plasmid coexpressing green fluorescent protein as an internal marker. Focussing on fgf8 expression in the developing isthmus, we show that no morphological defects are observed, and that fgf8 expression is neither silenced in embryos microinjected with dsRNA nor in embryos microinjected and electroporated with a pool of siRNAs. Moreover, fgf8 expression was not significantly silenced in most isthmic cells transformed with a plasmid producing engineered shRNAs to fgf8. We also show that siRNA molecules do not spread significantly from cell to cell as reported for invertebrates, suggesting the existence of molecular differences between different model systems that may explain the different responses to RNAi. Although our results are basically in agreement with previously reported studies, we suggest, in contrast to them, that with currently available tools and techniques the number of cells in which fgf8 gene expression is decreased, if any, is not sufficient to generate a detectable mutant phenotype, thus making RNAi useless as a routine method for functional gene analysis in chick embryos.

Animals↗
PubMed2005

RNA interference of soybean isoflavone synthase genes leads to silencing in tissues distal to the transformation site and to enhanced susceptibility to Phytophthora sojae.

Isoflavones are thought to play diverse roles in plant-microbe interactions and are also potentially important to human nutrition and medicine. Isoflavone synthase (IFS) is a key enzyme for the formation of the isoflavones. Here, we examined the consequences of RNAi silencing of genes for this enzyme in soybean (Glycine max). Soybean cotyledon tissues were transformed with Agrobacterium rhizogenes carrying an RNAi silencing construct designed to silence expression of both copies of IFS genes. Approximately 50% of emerging roots were transformed with the RNAi construct, and most transformed roots exhibited >95% silencing of isoflavone accumulation. Silencing of IFS was also demonstrated throughout the entire cotyledon (in tissues distal to the transformation site) both by high-performance liquid chromatography analysis of isoflavones and by real-time reverse transcription-PCR. This distal silencing led to a nearly complete suppression of mRNA accumulation for both the IFS1 and IFS2 genes and of isoflavone accumulations induced by wounding or treatment with the cell wall glucan elicitor from Phytophthora sojae. Preformed isoflavone conjugates were not reduced in distal tissues, suggesting little turnover of these stored isoflavone pools. Distal silencing was established within just 5 d of transformation and was highly efficient for a 3- to 4-d period, after which it was no longer apparent in most experiments. Silencing of IFS was effective in at least two genotypes and led to enhanced susceptibility to P. sojae, disrupting both R gene-mediated resistance in roots and nonrace-specific resistance in cotyledon tissues. The soybean cotyledon system, already a model system for defense signal-response and cell-to-cell signaling, may provide a convenient and effective system for functional analysis of plant genes through gene silencing.

Base Sequence↗
PubMed2004

RNA interference targeting transforming growth factor-beta type II receptor suppresses ocular inflammation and fibrosis.

PURPOSE: Transforming growth factor-beta(TGF-beta) is an important mediator of wound healing responses. In the eye, TGF-beta activity has been implicated in causing corneal haze after laser surgery and subconjunctival scarring following glaucoma surgery. The purpose of the study was to determine whether small interference RNAs (siRNAs) targeting the type II receptor of TGF-beta (TbetaRII) could be used to suppress the TGF-beta action. METHODS: TbetaRII specific siRNAs designed from the human gene sequence were transfected into cultured human corneal fibroblasts. The protein and transcript levels of the receptor were determined by immunofluorescence, western blotting, and real time PCR. Immunofluorescence and immunoblotting were carried out to examine fibronectin assembly. A wound closure assay was used to assess cell migration in in vitro fibroblast cultures. In addition, the in vivo effects of TbetaRII siRNA were evaluated in a mouse model of ocular inflammation and fibrosis generated by subconjunctival injection of phosphate buffered saline and latex beads. Mouse TbetaRII siRNA was introduced into experimental eyes. Cellularity on tissue sections was evaluated after staining with hematoxylin and eosin. Collagen deposition was visualized by picrosirius red staining. RESULTS: TbetaRII siRNAs abrogated the receptor transcript and protein expression in cultured corneal fibroblasts. The gene knockdown inhibited fibronectin assembly and retarded cell migration. In the mouse model, introduction of TbetaRII specific siRNA significantly reduced the inflammatory response and matrix deposition. CONCLUSIONS: TbetaRII specific siRNAs were efficacious both in vitro and in vivo in knocking down the TGF-beta action. A direct application of siRNA into eyes to downregulate TbetaRII expression may provide a novel therapy for preventing ocular inflammation and scarring.

Adolescent↗
PubMed2004

A universal transgene silencing method based on RNA interference.

Inducible gene expression systems have contributed significantly to the understanding of molecular regulatory networks. Here we describe a simple and powerful RNA interference-based method that can silence the expression of any transgene. We first used an IRES bicistronic lentiviral vector and showed that targeting the second cistron with a specific siRNA resulted in silencing of both transgenes. We then inserted a siRNA minimal target sequence in the 3'-untranslated region (3'-UTR) of a transgene and showed that the cognate siRNA delivered by a lentiviral vector led to the partial silencing of the transgene. The multimerization of this siRNA target sequence led to the highly efficient silencing of four different transgenes. This new method to silence transgene expression is more versatile than existing methods of conditional inactivation of gene expression, such as transcriptional switches or site-specific recombination. It is applicable to a wide variety of models including primary cells, terminally differentiated cells and transgenic animals.

Animals↗
PubMed2006

Interactions between the RNA interference effector protein Ago1 and 14-3-3 proteins: consequences for cell cycle progression.

The Argonaute family member Ago1 is required for formation of pericentric heterochromatin and small interfering RNA (siRNA)-mediated post-transcriptional gene silencing in the fission yeast Schizosaccharomyces pombe. In addition, we have recently demonstrated that Ago1 function is required for enactment of cell cycle checkpoints (Carmichael, J. B., Provost, P., Ekwall, K., and Hobman, T. C. (2004) Mol. Biol. Cell 15, 1425-1435). Here, we provide evidence that the amino terminus of Ago1 binds to proteins that function in cell cycle regulation including 14-3-3 proteins. Interestingly, the amino terminus of human Ago2, the endonuclease that cleaves siRNA-targeted mRNAs, was also demonstrated to bind 14-3-3 proteins. Overexpression of the Ago1 amino terminus in yeast resulted in cell cycle delay at the G(2)/M boundary. Further investigation revealed that nuclear import of the mitosis-inducing phosphatase Cdc25 is inhibited by overexpression of the Ago1 amino terminus. Under these conditions, we found that the cyclin-dependent kinase Cdc2 is constitutively phosphorylated on tyrosine 15, thereby reducing the activity of this kinase, a situation that delays entry into mitosis. We hypothesize that 14-3-3 proteins are required for Argonaute protein functions in cell cycle and/or gene-silencing pathways.

14-3-3 Proteins↗
PubMed2002

ARGONAUTE1 is required for efficient RNA interference in Drosophila embryos.

Double-stranded RNA (dsRNA) triggers homology-dependent posttranscriptional gene interference (RNAi) in a diverse range of eukaryotic organisms, in a process mechanistically related to viral and transgene-mediated cosuppression. RNAi is characterized by the conversion of long dsRNA into approximately 21-25-nt small interfering RNAs (siRNA) that guide the degradation of homologous mRNA. Many of the genes required for siRNA production and target mRNA degradation are widely conserved. Notably, members of the Argonaute-like gene family from Arabidopsis, Caenorhabditis elegans, Drosophila, and Neurospora have been genetically and/or biochemically identified as components of the RNAi/cosuppression pathway. We show here that mutations in the Drosophila Argonaute1 (AGO1) gene suppress RNAi in embryos. This defect corresponds to a reduced ability to degrade mRNA in response to dsRNA in vitro. Furthermore, AGO1 is not required for siRNA production in vitro nor can the introduction of siRNA bypass AGO1 mutants in vivo. These data suggest that AGO1 functions downstream of siRNA production.

ATP-Binding Cassette Transporters↗
PubMed2006

Stable RNA interference in Spodoptera frugiperda cells by a DNA vector-based method.

Double-stranded RNA (dsRNA)-mediated interference (RNAi) is a powerful tool for silencing of gene expression in many organisms. To establish a DNA vector-based method for stable RNAi in Spodoptera frugiperda cells (Sf9), we created a stably transfected Sf9 cell line to express large dsRNA fragment targeting to silence the firefly luciferase gene (luc). The luc dsRNA specifically and stably suppressed the baculovirus-mediated luciferase expression. Thus, gene silencing in Sf9 cells was achieved using DNA vectors similar to the facile design described in this study.

Animals↗