RNA干扰抑制结肠癌细胞内Bcl-2/C-Raf的表达

目的]构建Bcl-2/C-Raf的双靶点的RNAi质粒表达载体与单靶点表达载体比较,研究RNAi对各组癌细胞增殖的抑制作用。方法]分别构建了针对Bcl-2、C-Raf和Bcl-2/C-Raf靶基因的质粒表达载体,通过Lipofec-tamineTM2000介导转染人结肠癌细胞系HCT-8后,检测相应转染组靶基因的mRNA和蛋白质表达量,测定各组细胞活性。结果]分别转染3种质粒表达载体后,3组结肠癌细胞中相应靶基因的mRNA和蛋白质表达量均降低;转染双靶点干扰质粒的试验组;其细胞活性低于单靶点组;对于针对Bcl-2,C-Raf和Bcl-2/C-Raf基因的3组干扰实验,RNAi对结肠癌细胞增殖的抑制率分别为43.87%、40.64%和63.85%。结论]Bcl-2/C-Raf双靶点的表达载体对结肠癌细胞增殖的抑制作用要明显优于单靶点表达载体。双靶点质粒表达载体在结肠癌的基因治疗中具有很大优势。

INHIBITION OF BCL-2/C-RAF GENE EXPRESSION WITH RNA INTERFERENCE IN HUMAN COLONIC CANCER CELL

Objective] To explore the effects of RNAi with plasmid expression vector that has Bcl-2 /C-Raf two targets with single targets in inhibiting the proliferation of human colonic cancer cells. Methods] Three kinds of plasmid expression vectors corresponding to the endogenous Bcl-2, C-Raf and Bcl-2 / C-Raf genes respectively were constructed, and then were separately transfected into three groups of colonic cancer cell line HCT-8 by LipofectamineTM2000. For the three groups, the ex-pressions of mRNA and protein related to the target genes were studied, the cell activity was assessed, and the effect of RNAi on proliferation of the cells was determined. Results] The results indicated that the expression of mRNA and protein related to the target genes decreased in all three groups for the transfection of the specific interference plasmid. The cell activity in the group transfected with the two targets interference plasmid was lower than that in other two groups transfected with only single target plasmid. For the groups transfected with interference plasmids targeting to Bcl-2, C-Raf and Bcl-2 / C-Raf genes, the in-hibition rates of RNAi on the proliferation of the HCT-8 cells were 43. 87%, 40. 64% and 63. 85%, respectively. Conclu-sion] RNAi is a functional pathway in human colonic cancer cells and compared to the plasmid that expresses shRNA targeting to Bcl-2 or C-Raf single, plasmid targeting to Bcl-2 / C-Raf simultaneously can more significantly inhibit the proliferation of the colonic cancer cells, providing a potential approach to colonic cancer gene therapy.