Livin基因和Survivin基因联合靶向siRNA抑制大肠癌细胞增殖的作用

采用1个载体编码2个shRNA技术, 构建Livin基因和Survivin基因联合靶向的siRNA重组表达载体, 研究其对人大肠癌细胞生长协同抑制的增效作用。构建Livin基因和Survivin基因联合靶向的siRNA重组表达载体并转染大肠癌细胞, 通过RT-PCR和Western blotting方法检测Livin基因与Survivin基因的表达, 利用流式细胞仪检测处理后细胞的凋亡效应。经酶切鉴定和测序分析证实Livin基因和Survivin基因联合靶向的siRNA重组表达载体构建成功, 它对大肠癌细胞Livin和Survivin mRNA的抑制率分别为27.90%和32.24%, 对Livin和Survivin蛋白表达的抑制率分别为22.28%和40.86%, 诱导肿瘤细胞凋亡率为 (11.69 ± 1.37) %, 但协同干扰作用比单独干扰Livin基因或Survivin基因弱。Livin基因和Survivin基因联合靶向的siRNA重组表达载体构建成功并能抑制Livin基因和Survivin基因的表达, 但协同抑制作用比单独干扰Livin基因或Survivin基因弱。

Inhibitory effect of multiple siRNA recombinant expression vector targeting simultaneously Livin and Survivin genes on human colon cancer cells proliferation

In order to maximize the efficiency and versatility of the vector-based siRNA approach, we have developed a novel siRNA expression vector containing multiple tandem siRNA cassettes to investigate the   synergistic inhibitory effect of it on human colon cancer cells proliferation.  Multiple siRNA recombinant    expression vector targeting simultaneously Livin and Survivin genes was constructed and transfected into human colon cancer cell.  The effect of multiple siRNA recombinant expression vector was detected by RT-PCR,  Western blotting and flow cytometry.  It was confirmed by restriction endonuclease and sequence analysis   that multiple siRNA recombinant expression vector targeting simultaneously Livin and Survivin genes was   constructed successfully.  Livin and Survivin genes inhibition ratio of Livin and Survivin siRNA at mRNA levels were 27.90% and 32.24%, at protein levels were 22.28% and 40.86%, the apoptotic ratio was (11.69 ± 1.37) %, but the synergistic effect was weaker than Livin and Survivin RNA interference, respectively.  The multiple siRNA recombinant expression vector targeting simultaneously Livin and Survivin genes has been constructed successfully.  It can inhibit the expression of Livin and Survivin genes in human colon cancer cells, but the  synergistic effect was weaker than Livin and Survivin RNA interferences, respectively.