TSG101-siRNA表达载体对结肠癌HT29／L-OHP细胞株耐药的逆转作用

TSG101-siRNA expression vector reverses anti-drug resistanse of human colon cancer cell line HT29 against oxaliplatin

目的构建TSG101(tumour susceptibility gene101,TSG 101)小干扰RNA(TSG101-siRNA)的真核表达载体,观察RNA干扰沉默TSG101基因后化疗药物奥沙利铂(L-OHP)对结肠癌耐药细胞株HT29/L-OHP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HT29/L-OHP细胞TSG101 mRNA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L-OHP对HT29/L-OHP的增殖抑制作用;FCM分析TSG101-siRNA转染HT29/L-OHP细胞前后的细胞周期分布情况。结果测序显示干扰载体构建成功,TSG101-siRNA能抑制TSG101mRNA表达,使HT29/L-OHP细胞TSG101蛋白的表达下降,明显增强L-OHP对HT29//L-OHP的增殖抑制,TSG101-siRNA转染HT29/L-OHP细胞后G0/G1期和S期细胞数有增加,而G2/M期细胞数有减少。结论TSG101-siRNA的真核表达载体能有效地抑制HT29/L-OHP细胞的TSG101 mRNA、TSG101蛋白的表达,提高化疗药物奥沙利铂对HT29/L-OHP的抑制率,能逆转结肠癌耐药细胞株HT29/L-OHP对L-OHP的耐药性。

Objective To construct an siRNA expression plasmid against tumor susceptibility gene 101 （tsglOl） and investigate its effect of RNA interference on oxaliplatin （L-OHP）-resistant human colon cancer cell line HT29/L-OHP. Methods The targeting fragments specifically against tsgl01 were designed according to the principle of small interfering RNA designation using an online software. The sequences were cloned into siRNA expression plasmids, and the plasmids were transfected into HT29/L-OHP ceils. The expression of tsgl01 mRNA after the transfection of TSG101-siRNA plasmid was detected by RT-PCR. The expression of TSG101 after TSG101-siRNA transfection was detected by Western blotting. MTT test was applied to measure the inhibition combined with L-OHP on the proliferation of HT29/L-OHP cells. Distribution of cell cycle was analyzed using flow cytometry after RNA interference to HT29/L-OHP cells. Results The expected fragments were designed, and the TSG101-siRNA plasmid was confirmed by DNA sequencing. The TSG101-siRNA plasmid inhibited the expression of tsg101 mRNA, and the expression of TSG101 protein, and suppressed the proliferation of HT29/L-OHP cells obviously when combined with oxaliplatin. The analysis of cell cycle indicated that the TSG101-siRNA plasmid reduced the cells in G2/M phases and increased the cells in G0/G1 and S phases. Conclusion The expression vector of TSG101-siRNA combined with oxaliplatin inhibits the proliferation of HT29/L-OHP ceils and increases the sensitivity to chemotherapy.