Inhibition of Proliferation Induced by Cyclin D1 Gene Silence in Human Renal Carcinoma ACHN Cells

Objective： To confirm feasibility of Cyclin D1 gene as a new target for cancer gene therapy and to verify the effectiveness of shRNA expression vector-mediated gene silencing. Methods： A RNA interference DNA template targeting Cyclin D1 gene was designed and synthesized. By ligation, the fragment was inserted into Pgenesil-1-U6 to constract the recombinant plasmid Pgenesil-shRNA-Cyclin D1. The identified recombinant plasmid was transfected into ACHN cells with lipofactamine. Cyclin D1 mRNA and protein expression was analyzed by RT-PCR and western-blotting. MTT method was used for observing cell proliferation and drawing growth curve. The cell cycle and ratios of apoptotic cell were assessed by flow cytometric detection. The ability of invasion of cell migration was detected by Transwell chamber invasive models. Results： The plasmid was constructed successfully. After interference, The expression rate of Cyclin D1 mRNA decreased to 0.10±0.04 in Cyclin D1-shRNA（experimental） group and were significantly lower than Pgenesil-NC （negative） group （0.92±0.03） and ACHN （blank control） group（0.94±0.04）（P0.05）. As well, the expression rate of Cyclin D1 protein was decreased evidently in experimental group. The results of flow cytometric detection showed that, including early and late apoptotic cells, the apoptotic ratio of experimental group increased to （37.26±0.60）% significantly, while, the negative group and blank control group were only （4.62±0.40）% and （5.95±1.20）%, respectively. The cell growth curves indicated that the proliferation of experimental group cells was inhibited significantly（P0.05） and Transwell results suggested that the abilities of invasion cells transfected with Pgenesil–CyclinD1-shRNA decreased conspicuously（P0.05）. Conclusion： The shRNA can inhibit Cyclin D1 expression, specifically and persistently. The down-regulation of Cyclin D1 expression can inhibit the proliferation and induce the apoptosis of renal cell adenocarcinoma cell line ACHN.

Study on Inhibitionof Growth of ACHN Cells via shRNA Expression Vector-Mediated Cycline1 Gene Silencing

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.

Detection of the expression of heat shock protein 70 using quantum dots as fluorescence probe in human renal carcinoma

Objective： To detect the expression of heat shock protein 70 （HSP70） in human renal carcinoma tissues and cultured ACHN cells by using quantum dots-tagged fluorescence technology and its significance. Methods： Using the fluorescence property of quantum dots, indirect immunofluorescence method and immunocytochemical method were used to detect the expression of HSP70 tagged by quantum dots in renal carcinoma tissues and ACHN cells cultured in vitro. Results： Confocal fluorescence microscopy showed that HSP70 were significantly expressed in renal carcinoma tissues and ACHN cells cultured in vitro characterized by homogeneous distribution of intensive salmon pink fluorescence. Compared with FITC tagging, quantum dots tagged fluorescence had good specificity and signal to background. There was no notable quenching after excitation by quantum dots for 30 rain. Conclusion： Quantum dots can be used to label subcellular proteins and have obvious advantages compared with the traditional fluorescence methods. The quantum dots-tagged fluorescence could be applied as a new method for clinical labeling detection.