靶向cyclinE小干扰RNA对肝癌HepG2细胞增殖和凋亡的影响

目的:以cyclinE基因编码区为靶位,构建表达小干扰RNA(siRNA)的质粒载体,观察转染后对HepG2细胞的影响.方法:针对cyclinE基因序列构建表达siRNA的真核表达载体pSilencer3.1-H1hygro,利用脂质体Metafectene转染CyclinE基因高表达的肝癌细胞株HepG2.流式细胞仪检测细胞周期及凋亡率,MTT法检测细胞增殖活性,RT-PCR和Western blot法观察转染后细胞cyclinE基因表达.结果:成功构建了表达siRNA的真核质粒载体,转染后cyclinE基因mRNA及蛋白表达水平分别下降了79%和65%.结论:靶向cyclinE基因的siRNA可有效沉默HepG2细胞高表达的cyclinE基因,从而抑制肝癌细胞的增殖并促进凋亡.

Effects of small RNA interference to target cyclinE gene on apoptosis and proliferation of HepG2 hepatocellular carcinoma cells

AIM:To design pSilencer3.1-H1hygro plasmid expressing small interfering RNA(siRNA)that targets the coding region of cyclinE,and to evaluate the inhibitory effect of this siRNA on HepG2 cells after transfection. METHODS:pSilencer3.1-Hlhygro vector of the siRNA based on the coding region of cyclinE was constructed.The plasmid and liposome metafectene were cotransfected into HepG2 cells,which overexpressed cyclinE.Cell prolif- eration and apoptosis were assessed by MTT assay and flow cytometry,respectively,and ex- pression of cyclinE was detected by RT-PCR and Western blotting. RESULTS:The plasmid expressing siRNA targeted on the coding region of cyclinE was successfully constructed.Expression of cyclinE mRNA and protein was suppressed by up to 79% and 65%,respectively. CONCLUSION:siRNA targeted on cyclinE can silence cyclinE overexpression,and subsequent- ly suppress proliferation and promote apoptosis of HepG2 cells.