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Results for “RNA interference”

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PubMed2006

[RNA interference inhibits VEGF expression and growth of PC-3 in prostate carcinoma].

OBJECTIVE: To construct a eukaryotic expression vector carrying human VEGF RNAi and to study the effect of RNA interference on VEGF expression in prostate carcinoma. METHODS: VEGF RNAi was synthesized, inserted into the RNA interference eukaryotic expression vector, and confirmed by the result sequencing. The vector was transfected into prostate cancer PC-3, the VEGF expression detected by Western blot and the cell inhibiting rate determined by MTT. RESULTS: The VEGF RNAi eukaryotic expression vector was successfully constructed. Compared with the empty vector group and the control group, the amount of VEGF protein expression was obviously decreased in the VEGF RNAi group. The inhibiting rates were 23.5% , 33. 5% and 40. 8% at 24, 48 and 72 h respectively. CONCLUSION: VEGF RNAi can inhibit the protein expression and growth of PC-3, which provides an experimental base for the biological therapy of prostate cancer.

Cell Line, Tumor↗

PubMed2006

Impact of RNA interference on gene networks.

Small endogenous RNAs such as microRNAs (miRNAs) and small interfering RNAs (siRNAs) have been found to post-transcriptionally control cellular gene networks by targeting complementary mRNAs for translation impairment (miRNA) or destruction (siRNA). We have developed a computational model, coordinated to molecular and biochemical parameters of RNA interference pathways, to provide (semi-) quantitative insight into the molecular events managing siRNA-mediated gene expression silencing in native and synthetic gene networks. Based on mass-conservation principles and kinetic rate laws, we converted biochemical RNA interference pathways into a set of ordinary differential equations that describe the dynamics of siRNA-mediated translation-regulation in mammalian cells. Capitalizing on mechanistic details of synthetic transactivator operation, we wired this model into a transcription control circuitry in which the siRNA and its target mRNA are independently regulated at the transcriptional level. In this context, we studied the impact of siRNA transcription timing on the onset of target gene transcription and production kinetics of target mRNA-encoded proteins. We also simulated the rate of siRNA-induced mRNA depletion and demonstrated that the relative concentrations of interacting siRNAs/mRNAs and the number of siRNA-specific target sites on a transcript modulate (i) the rate of target mRNA disappearance, (ii) the steady-state mRNA levels and (iii) induction dynamics of mRNA-encoded protein production. As our model predictions are consistent with available biochemical parameters, extrapolations may improve our understanding of how complex regulatory gene networks are impacted by small endogenous RNAs.

Gene Expression Regulation↗

PubMed2006

Suppression of EGFR expression by antisense or small interference RNA inhibits U251 glioma cell growth in vitro and in vivo.

Epidermal growth factor receptor (EGFR) had been reported as one of the major responsible genes for malignant progression and phenotype reversion of gliomas, and has been used as one of the most important therapeutic targets. In the present study, small interference RNA (siRNA) and antisense EGFR expression constructs, which target sequences of human EGFR catalytic domain (2400-2420) and the 3'-coding region, respectively, were used to examine the growth inhibition effects on U251 glioma cells. Cell growth was significantly inhibited and G2/M arrest was observed in antisense- and siRNA-treated groups. Matrigel matrix demonstrated spotted cell clustering pattern in antisense- and siRNA-transfected U251 cells, indicating poor cell growth activities. In addition, the tumor volumes in U251 subcutaneous mice model treated with antisense and siRNA were significantly smaller than those treated with control siRNA and phosphate-buffered saline. Also, glial fibrillary acidic protein expression was upregulated in antisense- and siRNA-treated groups than the control groups. Our results demonstrated that antisense- or siRNA-targeting intracellular region of EGFR can inhibit EGFR expression, exerted growth inhibition effect on U251 glioma cells in vitro and in vivo. Consequently, siRNA expression plasmid-mediated gene therapy would be a new strategy in treatment of gliomas.

Animals↗

PubMed2006

RNA interference in the mouse vascular endothelium by systemic administration of siRNA-lipoplexes for cancer therapy.

RNA interference (RNAi) entails the potential for novel therapeutic strategies through the silencing of disease-causing genes in vivo. However, recent studies have raised an issue regarding applicable routes of administration for small interfering RNA (siRNA) molecules as therapeutics. In this study, we demonstrate that liposomally formulated siRNA molecules, the so-called siRNA-lipoplexes, but not naked siRNAs, are delivered to the tumor endothelial cells in vivo by microscopy. In addition, functional intracellular delivery of formulated siRNA targeting the tumor suppressor PTEN is shown in endothelial cells of the liver and tumor. Finally, the therapeutic potential of systemically administered siRNA(CD31)-lipoplexes is established by inhibition of tumor growth in two different xenograft mouse models. Our findings corroborate the applicability of this liposomal siRNA delivery technology for inducing RNAi to modulate gene expression levels in angiogenesis-dependent processes. In addition, our results advocate CD31 as a promising therapeutic target for antiangiogenic intervention. Therefore, our study provides a basis for the development of antiangiogenic cancer therapies based on RNAi.

3T3 Cells↗

PubMed2003

RNA interference: new mechanisms for targeted treatment?

Nucleic acid-based sequence-specific therapeutic intervention offers the potential for treatment of particular cancers without side effects. RNA interference (RNAi) induced by small interfering RNA (siRNA) (19-21 bp) is a normal cellular mechanism leading to highly specific and extraordinarily efficient degradation of the corresponding mRNA. The mechanism of RNAi as well as strategies for the design and delivery of siRNA are described. The growing role of RNAi in target validation for cancer-specific genetic aberrations is discussed. We attempt an early assessment of the potential for using RNAi technologies to treat cancer directly, especially hematologic malignancies. Promising targets for specific gene silencing in hematologic oncology include oncogenic fusion proteins and oncogenes activated by point mutations. Potency and specificity of gene silencing are the major advantages of the new RNAi technology over other nucleic acid-based gene targeting approaches. Crucial questions for pharmaceutical interventions remain. Advances in the areas of delivery, systemic spreading and duration of the silencing effect are necessary before the methodology can enter clinical oncology.

Animals↗

PubMed2006

Engineering mucosal RNA interference in vivo.

Mucosal surfaces serve as a gateway to disease. Here, we demonstrate that RNA interference can be used to manipulate mucosal gene expression in vivo. Using a murine model, we show that direct application of liposome-complexed siRNA mediates gene-specific silencing in cervicovaginal and rectal mucosa. A single vaginal or rectal administration of siRNA targeting hematopoietic or somatic cell gene products reduced corresponding mRNA levels by up to 90%. Using a murine model of inflammatory bowel disease, we found that the rectal application of siRNA targeting TNF-alpha led to relative mucosal resistance to experimental colitis. Liposomal siRNA formulations proved nontoxic, did not elicit a nonspecific interferon response, and provide a means for genetic engineering of mucosal surfaces in vivo.

Animals↗

PubMed2007

Knockdown of aminopeptidase-N from Helicoverpa armigera larvae and in transfected Sf21 cells by RNA interference reveals its functional interaction with Bacillus thuringiensis insecticidal protein Cry1Ac.

Aminopeptidase-N (APN) and cadherin proteins located at the midgut epithelium of Helicoverpa armigera have been implicated as receptors for the Cry1A subfamily of insecticidal proteins of Bacillus thuringiensis. Ligand blot analysis with heterologously expressed and purified H. armigera Bt receptor with three closely related Cry1A proteins tentatively identified HaAPN1 as an interacting ligand. However, to date there is no direct evidence of APN being a functional receptor to Cry1Ac in H. armigera. Sf21 insect cells expressing HaAPN1 displayed aberrant cell morphology upon overlaying with Cry1Ac protein. Down-regulating expression of HaAPN1 by RNA interference using double-stranded RNA correlated with a corresponding reduction in the sensitivity of HaAPN1-expressing cells to Cry1Ac protein. This clearly establishes that insect cells expressing the receptor recruit sensitivity to the insecticidal protein Cry1Ac, and their susceptibility is directly dependent on the amount of HaAPN1 protein expressed. Most importantly, silencing of HaAPN1 in H. armigera in vivo by RNA interference resulted in reduced transcript levels and a corresponding decrease in the susceptibility of larvae to Cry1Ac. BIAcore analysis of HaAPN1/Cry1Ac interaction further established HaAPN1 as a ligand for Cry1Ac. This is the first functional demonstration of insect aminopeptidase-N of H. armigera being a receptor of Cry1Ac protein of B. thuringiensis.

Animals↗

PubMed2005

A loxP-containing pol II promoter for RNA interference is reversibly regulated by Cre recombinase.

Several DNA vectors for RNA interference in mammalian cells have been described. These express a short hairpin RNA (shRNA) that is subsequently processed into mature small interfering RNAs (siRNAs). We previously developed the siRNA-expressing vector psiUx based on the polII promoter of the U1 small nuclear RNA gene. Here we describe the conversion of such construct into an inducible system. The starting construct psiUStuff contains a loxP-Stuffer-loxP cassette just upstream the transcription initiation site and does not express the shRNA until the two canonical loxP sites undergo Cre-mediated recombination. If sustained expression of the recombinase is maintained, transcription is repressed and shRNA synthesis is abolished. Therefore, in our system the Cre recombinase exhibits the dual function of activator and repressor allowing the on/off regulation of siRNAs production. Using a Cre recombinase whose transcription is under the control of a tetOn system, we show the temporally controlled expression of an shRNA directed towards the lamin A/C mRNA, as well as the regulated knockdown of its target.

DNA Polymerase II↗

PubMed2006

[Effects of RNA interference targeting angiotensin 1a receptor on the blood pressure and cardiac hypertrophy of rats with 2K1C hypertension].

OBJECTIVE: To investigate the effects of RNA interference (RNAi) targeting angiotensin 1a (AT1a) receptor on the blood pressure and cardiac hypertrophy of rats with 2K1C (2-kidney, 1-clip) hypertension. METHODS: Two kinds of RNAi plasmids, pAT1a-shRNA1 carrying an U6 promoter and an AT1a-specific shRNA-coding template sequence corresponding the sites 928 - 946 and pAT1a-shRNA2 carrying an U6 promoter and an AT1a-specific shRNA-coding template sequence corresponding the sites 978 - 996, and a blank plasmid pCon carrying a nonspecific shRNA-coding sequence were constructed. Thirty Sprague-Dawley rats underwent clipping of the left renal artery so as to establish two-kidney, one-clip (2K1C) hypertension models and then were randomly divided into 5 equal groups: pAT1a-shRNA1 group (injected with pAT1a-shRNA1 4 mg/kg only one time), pAT1a-shRNA2 group (injected with pAT1a-shRNA2 4 mg/kg only one time), pCon group (injected with pCon 4 mg/kg only one time), valsartan group (perfused into the stomach with valsartan, a AT1 receptor inhibitor 30 mg.kg(-1).d(-1), for 3 weeks), and control blank group (without any treatment). Three weeks later, the systolic pressure of the caudal artery was measured, catheterization through carotid artery was conducted to measure the systolic blood pressure (SBP) and diastolic blood pressure (DBP), and the left ventricular pressure curve was drawn. Then the rats were killed; the weight of the heart was measured, the ratio of left ventricle weight to body weight (LV/BW) was calculated, and pathological examination of the heart and thoracic aorta was performed. Western blotting was used to detect the protein expression of AT21 in the ventricle and aorta. Six age-matched healthy rats were used as normal controls. RESULTS: There was no significant difference in the caudal artery pressure among the 5 groups (all P > 0.05) before intervention. Three weeks later the caudal artery pressures of the blank control group and pCon group continued to significantly increase by about 25 mm Hg compared to the values before the intervention (both P < 0.001) and without significant difference between these 2 groups; however, the caudal artery pressures of the pAT1a-shRNA1, pAT1a-shRNA2, and valsartan groups were 15.1 mm Hg +/- 5.4 mm Hg, 16.4 mm Hg +/- 8.4 mm Hg, and 30.6 mm Hg +/- 18.2 mm Hg lower than those before the intervention respectively (all P < 0.01); and were also significantly lower than those of the blank groups (P < 0.01 or P < 0.05). There was no significant differences in the +/- dp/dt value and indicators of renal function among these groups. The carotid artery pressure of the pAT1a-shRNA1, pAT1a-shRNA2, and valsartan groups were 194 mm Hg +/- 5 mm Hg, 200 mm Hg +/- 5 mm Hg, and 164 mm Hg +/- 5 mm Hg, all significantly lower than those of the blank and pCon groups (234 mm Hg +/- 10 mm Hg and 232 mm Hg +/- 7 mm Hg respectively, all P < 0.01). The LV/BW of the pAT1a-shRNA1, pAT1a-shRNA2, and valsartan groups were 2.27 +/- 0.37, 2.31 +/- 0.26, and 2.26 +/- 0.39, all significantly lower than that of the blank and pCon groups (3.24 +/- 0.38 and 2.94 +/- 0.06, respectively, all P < 0.01), similar to that of the normal control group (P > 0.05). The myocardiocytes were significantly hypertrophic and the arterial tunica media was significantly thickened in the blank group and such changes were all improved to different degrees in the pAT1a-shRNA1, pAT1a-shRNA2, and valsartan groups. The protein expression levels of AT1 receptor in the myocardium of the pAT1a-shRNA and pAT1a-shRNA2 groups were lower by 53.3% and 47.8% respectively than that of the blank group, and the protein expression levels of AT1 receptor in the thoracic aorta of the pAT1a-shRNA and pAT1a-shRNA2 groups were lower by 58.7% and 49.3% respectively than that of the blank group (all P < 0.01); however, there were no significant difference in the protein expression levels of AT1 receptor in the myocardium and thoracic aorta between the valsartan and blank groups (both P > 0.05). CONCLUSION: RNA interference targeting AT1a receptor inhibits the development of renovascular hypertension and the accompanying cardiac hypertrophy. The RNAi technology may become a new strategy of gene therapy for hypertension.

Animals↗

PubMed2005

A doubly inducible system for RNA interference and rapid RNAi plasmid construction in Trypanosoma brucei.

The most rapid method for the generation of conditional mutants in Trypanosoma brucei is the use of RNA interference. A single copy of the target sequence is cloned between two opposing T7 promoters bearing tet operators, and the resulting plasmid is integrated into the genome of cells expressing both the tet repressor and T7 RNA polymerase. Upon addition of tetracycline, double-stranded RNA is synthesised from the two T7 promoters. Unfortunately, repression of T7 promoter activity may sometimes be insufficient to prevent expression of toxic amounts of double-stranded RNA. We describe here cell lines in which the expression of T7 polymerase is under tetracycline control, and show that regulation of polymerase expression can modulate transcription from a constitutive T7 promoter. In addition we describe a construct containing two copies of the tn10 Tet repressor for easy creation of repressor-expressing trypanosomes, and an RNA interference vector which allows "TA" cloning of unmodified PCR products and blue/white selection.

Animals↗

PubMed2005

Use of RNA interference to dissect the roles of trans-acting factors in alternative pre-mRNA splicing.

RNA interference (RNAi) is becoming a popular method for analyzing gene function in a variety of biological processes. We have used RNAi in cultured Drosophila cells to identify trans-acting factors that regulate the alternative splicing of endogenously transcribed pre-mRNAs. We have generated a dsRNA library comprising approximately 70% of the Drosophila genes encoding RNA binding proteins and assessed the function of each protein in the regulation of alternative splicing. This approach not only identifies trans-acting factors regulating specific alternative splicing events, but also can provide insight into the alternative splicing regulatory networks of Drosophila. Here, we describe this RNAi approach to identify alternative splicing regulatory proteins in detail.

Alternative Splicing↗

PubMed2001

Analysis of Drosophila 26 S proteasome using RNA interference.

We have utilized double-stranded RNA interference (RNAi) to examine the effects of reduced expression of individual subunits of the 26 S proteasome in Drosophila S2 cells. RNAi significantly decreased mRNA and protein levels of targeted subunits of both the core 20 S proteasome and the PA700 regulatory complex. Cells deficient in any of several 26 S proteasome subunits (e.g. d beta 5, dRpt1, dRpt2, dRpt5, dRpn2, and dRpn12) displayed decreased proteasome activity (as judged by hydrolysis of succinyl-Leu-Leu-Val-Tyr-aminomethylcoumarin), increased apoptosis, decreased cell proliferation without a specific block of the cell cycle, and accumulation of ubiquitinated cellular proteins. RNAi of many individual 26 S proteasome subunits promoted increased expression of many non-targeted subunits. This effect was not mimicked by chemical proteasome inhibitors such as lactacystin. Reduced expression of most targeted subunits disrupted the assembly of the 26 S proteasome. RNAi of six of eight targeted PA700 subunits disrupted that structure and caused accumulation of increased levels of uncapped 20 S proteasome. Notable exceptions included RNAi of dRpn10, a polyubiquitin binding subunit, and dUCH37, a ubiquitin isopeptidase. dRpn10-deficient cells showed a significant increase in succinyl-Leu-Leu-Val-Tyr-aminomethylcoumarin hydrolyzing activity of the 26 S proteasomes but accumulated polyubiquitinated proteins. d beta 5-Deficient cells had a phenotype similar to that of most PA700-deficient cells but also accumulated low molecular mass complexes containing subunits of the 20 S proteasome, probably representing unassembled precursors of the 20 S proteasomes. Cells deficient in several of the 26 S proteasome subunits were more resistant to otherwise toxic concentrations of various proteasome inhibitors. Our data suggest that those cells adapted to grow in conditions of impaired ubiquitin and proteasome-dependent protein degradation.

Amino Acid Sequence↗

PubMed2006

Global down-regulation of gene expression in the brain using RNA interference, with emphasis on monoamine transporters and GPCRs: implications for target characterization in psychiatric and neurological disorders.

RNA interference (RNAi) is a natural mechanism for regulating gene expression, which exists in plants, invertebrates, and mammals. We investigated whether non-viral infusion of short interfering RNA (siRNA) by the intracerebroventricular route would enable a sequence-specific gene knockdown in the mouse brain and whether the knockdown translates into disease-relevant behavioral changes. Initially, we targeted enhanced green fluorescent protein (EGFP) in mice overexpressing EGFP. A selective knockdown of both EGFP protein and mRNA was observed throughout the brain, with lesser down-regulation in regions distal to the infusion site. We then targeted endogenous genes, encoding the dopamine (DAT) and serotonin transporters (SERT). DAT-siRNA infusion in adult mice produced a significant down-regulation of DAT mRNA and protein and elicited hyperlocomotion similar, but delayed, to that produced on infusion of GBR-12909, a potent and selective DAT inhibitor. Similarly, SERT-siRNA infusion resulted in significant knockdown of SERT mRNA and protein and elicited reduced immobility in the forced swim test similar to that obtained on infusion of citalopram, a very selective and potent SSRI. Application of this non-viral RNAi approach may accelerate target validation for neuropsychiatric disorders that involve a complex interplay of gene(s) from various brain regions.

Animals↗

PubMed2005

[RNA interference silencing expression of survivin gene and reversing drug resistance of ovarian cancer cell line SKOV3/ADM].

OBJECTIVE: To examine expression of survivin gene in ovarian epithelial carcinoma drug resistant cell line SKOV3/ADM and its parental cell line SKOV3, and induction of cells apoptosis and reversal of drug resistance in SKOV3/ADM after RNA interference (RNAi) silencing survivin gene. METHODS: The transcription of survivin gene in cells was detected by semi-quantitative RT-PCR, the protein expression level of survivin gene was analyzed by immunofluorescence staining. SKOV3/ADM cells were treated with pshRNA-survivin and paclitaxel (Taxol), and acridine orange (AO)/ethidium bromide (EB) staining was performed to evaluate the apoptosis of cells. RESULTS: Survivin gene mRNA expressed by 99.1% and 75.3% respectively in cell lines SKOV3/ADM and SKOV3, while fluorescent cells were 59 +/- 5 and 42 +/- 3 (P < 0.05). After the introduction of pshRNA-survivin into SKOV3/ADM, mRNA transcription level of survivin gene decreased distinctly from 99.1% to 7.9%. The apoptotic cells of control group detected by AO/EB staining was 3.6 +/- 0.6, of Taxol group 10.2 +/- 1.0, of RNAi group 48.5 +/- 4.9, of RNAi + Taxol group 71.5 +/- 6.8. Apoptosis ratio between RNAi + Taxol group and RNAi group had significant difference (P < 0.05), and that between RNAi + Taxol group and Taxol group also had significant difference (P < 0.05). CONCLUSIONS: Both survivin gene mRNA and its protein are over-expressed in ovarian epithelial carcinoma cell lines SKOV3 and SKOV3/ADM, the level of survivin gene expressed in SKOV3/ADM is obviously different compared with that in its parental cell line SKOV3. RNA interference targeted against specific sequences of survivin in SKOV3/ADM cell could significantly reduce the level of survivin mRNA transcripts and protein, effectively induce the cells apoptosis and restore the sensitivity of cell to conventional chemotherapeutic agents Taxol.

Animals↗

PubMed2005

Inhibition of replication and infection of severe acute respiratory syndrome-associated coronavirus with plasmid-mediated interference RNA.

Severe acute respiratory syndrome (SARS) is a newly emerged infectious disease caused by a novel coronavirus (SARS-CoV), which spread to over 30 countries in early 2003. Until recently, no specific vaccines and effective drugs have been available to protect patients from infection by this virus. To exploit a new strategy to fight this disease, we investigated the effect of interference RNA (RNAi) on the virus infection and replication with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT), plaque-forming, Western-blot and real-time PCR assays. Results showed that the plasmid-derived siRNAs targeting the non-structural protein 1 (NSP1) sequence of the SARS-CoV genome could specifically inhibit the expression of the NSP1 sequence and effectively suppress the replication and propagation of SARS-CoV in cultured Vero E6 cell lines. The expression of the Spike and Nucleoprotein genes of SARS-CoV at mRNA and protein levels in small interfering (si)RNA-expressing cells was significantly less than that in controls when analysed with PCR and Western-blot assays, 3 days post SARS-CoV infection. Our study provides strong evidence that the NSP1 sequence in the SARS-CoV genome is a valid target for RNAi and the effect of the siRNAs probably mainly resulted from effects on global reduction of subgenome synthesis and subsequent protein expression of SARS-CoV.

Animals↗

PubMed2005

Analysis of short interfering RNA function in RNA interference by using Drosophila embryo extracts and schneider cells.

The realization that short double-stranded RNA (dsRNAs) 21-25 bp in length represent the basis for posttranscriptional gene silencing (PTGS) in plants, quelling in N. crassa, and RNA interference (RNAi) in C. elegans and Drosophila has given insight into one of the most evolutionarily conserved pathways in eukaryotes. dsRNA that arises due to viral infection, transposon mobilization, random insertion of transgenes near active promoters, transcripts from repetitive elements in the genome, or introduction of exogenous dsRNA directly is processed by one of the RNase III-related enzymes, known as the Dicers, to produce 21- to 25-bp short dsRNAs or short interfering RNAs (siRNAs) that target the degradation of the cognate RNA sequence (Denli and Hannon, 2003; Hannon, 2002; Plasterk, 2002). Proteins in the RNAi pathway and siRNA-like RNAs have also been recently demonstrated to play a role in the formation and maintenance of heterochromatin in S. pombe as well as in transgene-induced PTGS in Drosophila (Hall et al., 2002; Pal-Bhadra et al., 2004; Volpe et al., 2002). An understanding of siRNA function in these crucial regulatory pathways requires biochemical approaches to study siRNAs and their role in gene silencing as well as the formation and maintenance of heterochromatin. This chapter describes simple methods for using Drosophila embryo extracts and cultured insect cells to study siRNA function in the RNAi pathway in vivo and in vitro. We describe the most recent protocols for the preparation and use of Drosophila embryo extracts used in gene targeting studies. We present methods we have used to assay siRNA function in Drosophila embryo extracts and in cultured SL2 cells that demonstrate a combined role for siRNAs and RNA-dependent RNA polymerase (RdRp) activity in Drosophila RNAi.

Animals↗

PubMed2007

Target validation using RNA interference in solid tumors.

Reverse genetics is one strategy that is currently used to establish a link between a target gene and a disease phenotype. In this process, the function of a gene is inhibited and the consequence of its loss on a desired biological function, such as tumor growth and metastasis, is monitored. RNA interference (RNAi) has been found to be the most effective method to specifically inhibit gene expression. Notably, interactions between cancer cells, stromal cells, and the extracellular matrix (ECM) are crucial to angiogenesis and tumorigenesis. Tumor cells and the surrounding stroma are the principle source of growth factors and cytokines, which induce remodeling of the ECM mediated by metalloproteases (MMPs) secreted by macrophages. The production of macrophages is regulated by colony-stimulating factor (CSF)-1, which is overexpressed in several tumors. When short-interfering RNAs (siRNAs) targeting either the CSF-1 or its receptors were delivered into colon and breast cancer xenografts in mice, tumor growth was inhibited. Associated with this suppression, we observed decreased tumor vascularity, reduced expression of angiogenic factors and MMPs, and decreased macrophage recruitment to the tumors. The suppression of CSF-1 by RNA interference is therefore a powerful tool to block gene function and influence tumor-stroma interactions in solid tumor development.

Animals↗

PubMed2005

Use of the giant multinucleate plasmodium of Physarum polycephalum to study RNA interference in the myxomycete.

The plasmodium of Physarum polycephalum harbors billions of synchronized nuclei in a single cell of complex structure. Due to its synchrony and extreme size, it is used as a model to study events on a single cell level, such as cell cycle and differentiation. We show here for the first time that this model, despite its enormous size and structural complexity, is accessible to RNA interference by simple injection of dsRNA or siRNA. The targeted gene is that of polymalatase, an intracellular adapter of poly(beta-l-malate) involved in the maintenance of the synchrony and functioning as an extracellular hydrolase of this polymer. Real-time reverse transcriptase polymerase chain reaction analysis revealed that the specific mRNA was knocked down to about 10% of the original level. The suppression of a single injection lasted for approximately 14 cell cycles (144 h) and could be prolonged for any time by repeated dsRNA injections. Western blots indicated that the knockdown of RNA was paralleled by a strong reduction in polymalatase synthesis. However, a change in the phenotype of the plasmodium could not be clearly observed. In principle, the plasmodium offers an easy system for studying gene knockdown by RNA interference.

Animals↗