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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 the eukaryotic expression vector TSG101-siRNA of TSG101 and to observe the effect of L-OHP on the drug-resistant cell line of colon cancer after silencing TSG101 by RNA interference HT29 / L-OHP proliferation. Methods According to the design principle of small interfering RNA, the target sequence of TSG101 was aided by computer software. The target sequence was cloned into expression vector of small interfering RNA using DNA recombinant technology. The expression of TSG101 mRNA in HT29 / L-OHP cells was detected by RT- The inhibitory effect of TSG101-siRNA on the expression of TSG101 protein was detected by Western blot. The inhibitory effect of TSG101-siRNA and chemotherapeutic drug L-OHP on HT29 / L-OHP was determined by MTT assay. The expression of TSG101-siRNA was detected by FCM HT29 / L-OHP cells before and after the cell cycle distribution. Results TSG101-siRNA could inhibit the expression of TSG101 mRNA, decrease the expression of TSG101 protein in HT29 / L-OHP cells and significantly inhibit the proliferation of HT29 / L-OHP induced by L-OHP. TSG101-siRNA transfection HT29 / L-OHP cells after G0 / G1 phase and S phase cells increased, while G2 / M phase cells decreased. Conclusion The eukaryotic expression vector of TSG101-siRNA can effectively inhibit the expression of TSG101 mRNA and TSG101 protein in HT29 / L-OHP cells and increase the inhibitory rate of oxaliplatin on HT29 / L-OHP and reverse the effect of colon cancer Drug-resistant cell line HT29 / L-OHP resistance to L-OHP.