上调miR-125b对肝癌Huh-7细胞系增殖及顺铂诱导凋亡的作用研究

Upregulated miR-125b changed Huh-7 proliferation and apoptosis to cisplatin

关键词:  miRNA  肝细胞癌  化疗耐药性

Key words:  miRNA  hepatocellular carcinoma  chemoresistance

摘要:

目的　研究miR-125b在肝癌发生发展中的作用机制，及其表达对于肝细胞癌化疗耐受性的影响。　方法　采用qPCR检测肝癌患者癌组织、癌旁组织、Huh-7细胞系（对照组）及转染miR-125b mimic的Huh-7细胞系（转染组）中miR-125b表达情况。采用流式细胞仪及MTT法检测对照组及转染组细胞周期及增殖情况。Western blot 检测Huh-7细胞系转染后Mcl-1蛋白表达。设3组：对照组为正常培养细胞，miRNA control组转染miRNA control，miR-125b mimic组转染miR-125b mimic。Western blot 检测Mcl-1特异性siRNA阻断Mcl-1蛋白表达后，细胞凋亡相关蛋白caspase3的表达情况。设3组：对照组细胞正常培养；顺铂组加入顺铂，终浓度50 μg/mL培养；转染+顺铂组转染Mcl-1-siRNA，48 h后加入顺铂，终浓度50 μg/mL。　结果　肝癌组织miR-125b表达较癌旁组织明显减低，为癌旁组织的（46.24±8.53）%，P＜0.05。 miR-125b mimic 转染 Huh-7细胞后，转染组细胞G1/S比例较对照组明显增高（P＜0.05)；Mcl-1蛋白表达较对照及miRNA control组下调（P＜0.05)；转染+顺铂组中的Caspase3蛋白表达较对照组及顺铂组明显增高（P＜0.05)。　结论　上调miR-125b的表达可以抑制肝癌细胞增殖，抑制靶蛋白Mcl-1表达，促进顺铂诱导凋亡，具有抑癌基因的作用。

Abstract:

Objective　Investigate miR-125b function in hepatocellular carcinoma (HCC) and its relation to chemoresistance. 　Methods 10 cases HCC specimen from clinic were chosen for detecting the miR-125b expression by qRT-PCR, same detections were performed in 3 HCC cell lines as well. Transfected Huh-7 cell line with miR-125b mimic, detected cell proliferation by TUNEL, Mcl-1 protein expression by western blot, and detected Caspase 3 expression when Mcl-1-siRNA and cisplatin were added in Huh-7 cells. 　Results HCC specimen miR-125b expression was （46.24±8.53）% of the specimen adjacent to cancer （P＜0.05). When miR-125b was up-regulated, the ratio of G1/S increased significantly compare to normal miR-125b in Huh-7 cells, Mcl-1 protein expression were decreased compare to control(P<0.05), Caspase 3 expression in Huh-7 increased significantly after Mcl-1-siRNA and cisplatin were added （P＜0.05). 　Conclusion miR-125b suppressed the proliferation of Huh-7, decreased Mcl-1 protein expression and magnified HCC apoptosis to cisplatin. miR-125b did as a cancer suppressor gene in HCC and possibly correlated with HCC chemoresistance.