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目的探讨小干扰RNA(small interfering RNA,siRNA)抑制p15基因对肝癌耐药的影响。方法采用浓度递增法用顺铂(cisplatin,CDDP)诱导肝癌细胞HepG2建立动态耐药模型HepG2/CDDP/2.0。脂质体法将针对p15 mRNA的siRNA(Y1、Y2、Y3)和阴性对照siRNA-F转染至HepG2/CDDP/2.0细胞,筛选最佳转染浓度和最有效的干扰序列。MTT法检测HepG2/CDDP/2.0对CDDP的半数抑制浓度(IC50)、流式细胞仪分析HepG2/CDDP/2.0细胞周期分布、RT-PCR和Western blot分别检测p15 mRNA和p15蛋白、P-糖蛋白(P-glycoprotein,P-gp)的表达。结果成功建立肝癌耐药模型HepG2/CDDP/1.6,系中度耐药。RT-PCR显示siRNA Y3干扰效果最佳。转染后G1期细胞比例分别为53.8%(24 h),51.6%(48 h),48.8%(72 h)(P<0.05);RT-PCR和Western blot显示p15表达降低,p-gp表达增加。结论在肝癌耐药的动态过程中,随着耐药时间的延长,G1期的比例增加。抑制p15的表达可降低G1期的比例,增加肿瘤耐药性。
Objective To investigate the effect of small interfering RNA (siRNA) on the resistance of hepatocarcinoma to p15 gene. Methods HepG2 cells were induced by cisplatin (CDDP) in a concentration - increasing manner. HepG2 / CDDP / 2.0 was established. Liposome method siRNA (Y1, Y2, Y3) against p15 mRNA and negative control siRNA-F were transfected into HepG2 / CDDP / 2.0 cells to screen the optimal transfection concentration and the most effective interference sequences. The half inhibitory concentration (IC50) of HepG2 / CDDP / 2.0 on CDDP was detected by MTT assay. The cell cycle distribution of HepG2 / CDDP / 2.0 was analyzed by flow cytometry. The expressions of p15 mRNA and p15 protein, P-glycoprotein (P-glycoprotein, P-gp) expression. Results The HepG2 / CDDP / 1.6 model was established successfully, which was moderately drug-resistant. RT-PCR showed that siRNA Y3 had the best interference. The percentage of cells in G1 phase was 53.8% (24 h), 51.6% (48 h) and 48.8% (72 h) respectively after transfection (P <0.05). The expression of p15 was decreased and the expression of p-gp increase. Conclusions In the dynamic process of drug resistance of liver cancer, the proportion of G1 phase increases with the extension of drug resistance time. Inhibition of p15 expression can reduce the proportion of G1 phase, increase tumor drug resistance.