OBJECTIVE To explore the inhibition of ACHN cells via shRNAexpression vector mediated cyclinE1 gene silencing.METHODS The shRNA targeting at cyclinE1 gene was designedand synthesized. By ligation, the fragment was ins...OBJECTIVE To explore the inhibition of ACHN cells via shRNAexpression vector mediated cyclinE1 gene silencing.METHODS The shRNA targeting at cyclinE1 gene was designedand synthesized. By ligation, the fragment was inserted intopGenesil-1-U6 to construct the recombinant plasmid pGenesil-1-U6-cyclinE1. The identified recombinant plasmid was introducedinto ACHN cells with lipofectamine 2000. The inhibition ofcyclinE1 mRNA and protein expression were analyzed by RT-PCRand western-blotting. MTT method was used for observing cellproliferation and drawing growth curve. The cell cycle and ratiosof apoptotic cell were assessed by flow cytometric detection. Theability of invasion and speed of cell migration were detected bytranswell chamber invasive models and cell scratch method.RESULTS The inhibition of expression of cyclinE1 in ACHN cellsmediated by recombinant vector (0.0933 ± 0.05) was significantlylower than that in the group of transfected with empty vector(0.8827 ± 0.04) and the control group (0.9021 ± 0.03) (P < 0.05).Flow cytometry showed that recombinant cells were blocked inthe G_1 phase and the apoptotic ratio was increased significantly(11.15 ± 4.00)% (P < 0.05). The curves of cell growth indicated thatthe proliferation of cell transfected with recombinant plasmid wasinhibited significantly compared with that in control group (P <0.05). The results of transwell and cell scratch suggested that theabilities of invasion and migration of the cells transfected withrecombinant plasmid were decreased conspicuously (P < 0.05).CONCLUSION The expression of cyclinE1 could be inhibitedsuccessfully by RNA interference induced by shRNA expressionvector. This consequently inhibits the cell growth and inducesapoptosis. Our study provided a preliminary result in searching ofRNA interference (RNAi) therapy for renal cell carcinoma.展开更多
基金supported by a grant from Major State Basic Research Development Program,China(No.2002CB513107).
文摘OBJECTIVE To explore the inhibition of ACHN cells via shRNAexpression vector mediated cyclinE1 gene silencing.METHODS The shRNA targeting at cyclinE1 gene was designedand synthesized. By ligation, the fragment was inserted intopGenesil-1-U6 to construct the recombinant plasmid pGenesil-1-U6-cyclinE1. The identified recombinant plasmid was introducedinto ACHN cells with lipofectamine 2000. The inhibition ofcyclinE1 mRNA and protein expression were analyzed by RT-PCRand western-blotting. MTT method was used for observing cellproliferation and drawing growth curve. The cell cycle and ratiosof apoptotic cell were assessed by flow cytometric detection. Theability of invasion and speed of cell migration were detected bytranswell chamber invasive models and cell scratch method.RESULTS The inhibition of expression of cyclinE1 in ACHN cellsmediated by recombinant vector (0.0933 ± 0.05) was significantlylower than that in the group of transfected with empty vector(0.8827 ± 0.04) and the control group (0.9021 ± 0.03) (P < 0.05).Flow cytometry showed that recombinant cells were blocked inthe G_1 phase and the apoptotic ratio was increased significantly(11.15 ± 4.00)% (P < 0.05). The curves of cell growth indicated thatthe proliferation of cell transfected with recombinant plasmid wasinhibited significantly compared with that in control group (P <0.05). The results of transwell and cell scratch suggested that theabilities of invasion and migration of the cells transfected withrecombinant plasmid were decreased conspicuously (P < 0.05).CONCLUSION The expression of cyclinE1 could be inhibitedsuccessfully by RNA interference induced by shRNA expressionvector. This consequently inhibits the cell growth and inducesapoptosis. Our study provided a preliminary result in searching ofRNA interference (RNAi) therapy for renal cell carcinoma.
