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

Jiang WANG, Anmin CHEN, Fengjin GUO

Abstract


OBJECTIVE    To explore the inhibition of ACHN cells via shRNA expression vector mediated cyclinE1 gene silencing. 
METHODS    The shRNA targeting at cyclinE1 gene was designed and synthesized. By ligation, the fragment was inserted into pGenesil-1-U6 to construct the recombinant plasmid pGenesil-1-U6-cyclinE1. The identifi ed recombinant plasmid was introduced into ACHN cells with lipofectamine 2000. The inhibition of cyclinE1 mRNA and protein expression were 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 and speed of cell migration were detected by transwell chamber invasive models and cell scratch method. 
RESULTS    The inhibition of expression of cyclinE1 in ACHN cells mediated by recombinant vector (0.0933 ± 0.05) was significantly lower 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 in the G1 phase and the apoptotic ratio was increased significantly (11.15 ± 4.00)% (P  < 0.05). The curves of cell growth indicated that the proliferation of cell transfected with recombinant plasmid was inhibited significantly compared with that in control group ( P  < 0.05). The results of transwell and cell scratch suggested that the abilities of invasion and migration of the cells transfected with recombinant plasmid were decreased conspicuously (P<0.05).

CONCLUSION    The expression of cyclinE1 could be inhibited successfully by RNA interference induced by shRNA expression vector. This consequently inhibits the cell growth and induces apoptosis. Our study provided a preliminary result in searching of RNA interference (RNAi) therapy for renal cell carcinoma.


Keywords


renal ACHN cells, RNA interference, cyclinE1, cell proliferation, apoptosis.

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