Cathepsin B表达对肝癌细胞系HepG2转移潜能影响的观察

Influence of cathepsin B expression on metastasis potential of hepatocarcinoma HepG2 cell line

目的:探讨小干扰RNA降低组织蛋白酶B(Cat B)基因表达对HepG2细胞增殖、运动及侵袭能力的影响。方法:将肝癌HepG2细胞分为Cat B siRNA干扰组、阴性对照siRNA干扰组(阴性对照组)、空脂质体组和空白对照组。脂质体转染法将Cat B siRNA转染入HepG2细胞;半定量RT-PCR和蛋白质印迹法检测转染后48hCat B的表达变化;收集转染后48h的细胞,再分别以CCK-8法、划痕实验和Transwell侵袭实验检测各组细胞增殖、运动及侵袭能力的变化。结果:半定量RT-PCR和蛋白质印迹法检测Cat B mRNA和蛋白水平表达变化,Cat B siRNA干扰组较其余各组均明显降低,差异有统计学意义,P<0.05。CCK-8法测细胞生长曲线示,将转染后48h的细胞种入96孔板后12h,Cat B siRNA干扰组增殖能力较对照组明显降低,差异有统计学意义,P<0.05。划痕实验中,Cat B siRNA干扰组细胞迁移距离较对照组明显降低,差异有统计学意义,P<0.05。Transwell侵袭实验中,Cat B siRNA干扰组穿膜细胞数较对照组明显降低,差异有统计学意义,P<0.05。结论:特异性干扰Cat B基因表达可抑制HepG2细胞的增殖、运动及侵袭能力。

OBJECTIVE：To explore the effects of siRNA mediated downregulation of Cat B gene expression on bio- logical behavior of hepatocelluiar carcinoma HepG2 ceils. METHODS： HepG2 cells were divided into four groups：Cat B siRNA intervention group, control siRNA intervention group, empty liposome group and blank control group. The expres- sion of Cat B mRNA and protein at 48 h after transfection were detected by RT-PCR and Western Blot respectively. The transfected cell after 48 h was re-collected and the cell proliferation, motility and invasion ability were then tested by CCK-8 method, wound-healing migration assay and transwell test respectively. RESULTS： Compared with the control group,the mRNA and protein level of Cat B were significantly decreased in the Cat B siRNA intervention group（P〈0.05） ; The CCK-8 method displayed that after the transfected cell was migrated into 96 well plate,after 12 hour,the cell proliferation in Cat B siRNA intervention group was significantly inhibited compared with the control group（P〈0.05）. The wound-healing migration assay showed that the migration distance in the Cat B siRNA intervention group was much shorter than the control group（P〈0.05）. The transwell invasion assay showed that the cell that passed the transwell membrane in the Cat B siRNA intervention group was less than the control group（P〈0.05）. CONCLUSION： Knock- down of Cat B gene expression may decrease the proliferation,motility and invasion ability in HepG2 hepatocarcinoma cell line.