TSG101-siRNA对肝癌耐药细胞株QGY/CDDP的逆转作用

目的 构建TSG101(tumor susceptibility gene101)小干扰RNA(TSG101-siRNA)的真核表达载体,观察RNA干扰沉默TSG101基因后化疗药物顺铂(CDDP)对肝癌耐药细胞株QGY/CDDP增殖的影响.方法 根据小干扰RNA的设计原则由计算机软件辅助设计TSG101的靶序列,应用DNA重组技术将目的序列克隆入小干扰RNA的表达载体;RT-PCR检测TSG101-siRNA对QGY/CDDP细胞tsg101mRNA表达的影响;Western blot检测TSG101-siRNA对TSG101基因表达蛋白的抑制效率;采用MTT法测定TSG101-siRNA联合化疗药物CDDP对QGY/CDDP的增殖抑制作用.结果 测序显示干扰载体构建成功,TSG101-siRNA能抑制tsg101 mRNA表达,使QGY/CDDP细胞TSG101蛋白的表达下降,抑制QGY/CDDP细胞的增殖,明显增强CDDP对QGY/CDDP的增殖抑制.结论 TSG101-siRNA的真核表达载体能有效地抑制QGY/CDDP细胞的增殖,提高化疗药物CDDP对QGY/CDDP的抑制率,能逆转肝癌耐药细胞株QGY/CDDP对CDDP的耐药性.

Effect of tumor susceptibitity gene 101-siRNA eukaryotic expression vector and cisplatin on proliferation of QGY/CDDP

ObjectiveTo construct the eukaryotic expression plasmids of tumor susceptibitity gene(TSG)101-siRNA and investigate the effect of RNA interference targeting tsg101 gene and cisplatin on liver cancer resistant strain QGY/CDDP.MethodsThe targeting fragments specifically against TSG101 were designed according to the principle of small interfering RNA designation using computer software.The sequences were cloned into siRNA expression plasmids through DNA recombinant technology.The expressions of tsg101mRNA and TSG101 after TSG101-siRNA transfection was detected by RT-PCR and Western blot respectively.MTT test were applied to measure the proliferation of QGY/CDDP strain.ResultsThe fragments of interest were obtained by digestion and further confirmed by DNA sequencing.TSG101-siRNA inhibited the expressions of tsg101mRNA and TSG101 and the proliferation of QGY/CDDP,especially in combination with cisplatin.ConclusionThe eukaryotic expression vector of TSG101-siRNA combined with cisplatin inhibits the proliferation of QGY/CDDP and increases the sensitivity of liver cancer resistant strain QGY/CDDP to chemotherapy.