小干扰RNA沉默尿激酶型纤溶酶原激活物受体和组织蛋白酶B基因对肝癌细胞迁移的影响及其机制

摘要

目的 探讨小干扰RNA(siRNA)沉默尿激酶型纤溶酶原激活物受体(uPAR)和组织蛋白酶B(CB)基因对肝癌细胞迁移的影响及其机制.方法 培养肝癌SMMC-7721细胞后,用荧光标记的低、中、高浓度的siRNA转染肝癌细胞,在荧光显微镜下计算siRNA转染效率,筛选出最佳siRNA转染浓度.将肝癌细胞分成空白对照组、阴性对照组(LC组)、uPAR-siRNA转染组(si-uPAR组)、CB-siRNA转染组(si-CB组)和uPAR-siRNA/CB-siRNA联合转染组(si-UC组).si-uPAR组、si-CB组、si-UC组细胞分别加入uPAR-siRNA、CB-siRNA、uPAR-siRNA/CB-siRNA转染试剂复合物进行转染,LC组仅加入转染试剂,空白对照组不进行干预.转染72 h后通过细胞划痕实验检测癌细胞迁移能力;转染48 h后,用流式细胞术分析细胞周期,实时荧光定量PCR法检测各组肝癌细胞整合素α6和基质金属蛋白酶9(MMP9)基因表达水平.结果 24 h后,用荧光标记的低浓度的siRNA转染肝癌细胞其转染效率在85％以上,细胞状态良好.与空白对照组及LC组相比,si-uPAR组、si-CB组、si-UC组细胞的迁移能力均下降、G0/G1期细胞比例增加、整合素α6及MMP9 mRNA表达水平均降低(均P<0.05);与si-uPAR组、si-CB组相比,si-UC组细胞的迁移能力均下降、G0/G1期细胞比例增加、整合素α6及MMP9 mRNA表达水平均降低(均P<0.05).结论 siRNA沉默uPAR或CB基因均可抑制肝癌细胞的迁移,且联合沉默抑制效果更明显,其可能通过抑制整合素α6和MMP9 mRNA的表达、诱导细胞G0/G1期阻滞而发挥作用.%Objective To explore the effect of small interfering RNA(siRNA) silencing urokinase-type plasminogen activator receptor(uPAR) and cathepsin B(CB) genes on the migration of hepatocellular carcinoma(HCC) cells and its mechanism. Methods HCC SMMC-7721 cells were cultured,then were transfected with fluorescent labeled siRNA of low,moderate and high concentrations. The transfection efficiency of siRNA was calculated under fluorescence microscope and the optimal transfection concentration of siRNA was screened out. The HCC cells were divided into blank control group,negative control group(LC group),uPAR-siRNA transfection group ( si-uPAR group) ,CB-siRNA transfection group( si-CB group) and uPAR-siRNA/CB-siRNA combined transfection group( Si-UC group) . The cells in the si-uPAR group,si-CB group and si-UC group were transfected by the compounds of uPAR-siRNA/transfection reagent, CB-siRNA/transfection reagent and uPAR-siRNA/CB-siRNA/transfection reagent respectively. The LC group was treated only by transfection reagent,and no treatment was performed in the blank control group. After 72h of transfection, the migration ability of HCC cells was detected by cell scratch assay. After 48h of transfection,the cell cycle was analyzed by flow cytometry. And the expression levels of integrinα6 mRNA and matrix metalloprotein 9 ( MMP9 ) mRNA were detected by real-time quantitative PCR assay. Results After 24 h of transfection with fluorescent labeled siRNA of low concentration,the transfection efficiency of HCC cells was more than 85％,and the cells were in good condition. Compared to the blank control group and LC group,the migration ability of the cells significantly decreased,the proportion of the cells at G0/G1 phase increased and the expression levels of integrin α 6 mRNA and MMP9 mRNA were lower in the si-uPAR group,si-CB group and si-UC group(all P<0. 05). Compared to the si-uPAR group and si-CB group,the migration ability of the cells significantly decreased,the proportion of the cells at G0/G1 phase increased and the expression levels of integrin α 6 mRNA and MMP9 mRNA were lower in the si-UC group(all P<0. 05). Conclusion Either uPAR or CB gene silencing by siRNA can inhibit the migration of HCC cells,and the combined silencing is more effective. The main mechanism might be related to inhibiting integrinα6 mRNA and MMP9 mRNA expressions and inducing cell arrest at G0/G1 phase.