微RNA-186在人肝细胞肝癌中的表达及作用  

Expression of microRNA-186 and its role in human hepatocellular carcinoma

作　　者：宋丹[1] 王孜尧 黄平[1] 

机构地区：[1]重庆医科大学附属第一医院肝胆外科,重庆400016

出　　处：《肿瘤》2017年第7期750-761,共12页Tumor

摘　　要：目的:检测微RNA-186(miRNA-186,miR-186)在肝细胞肝癌(hepatocellular carcinoma,HCC)组织和细胞中的表达水平,以及其对肝癌SMMC-772细胞生物学特性的影响。方法:采用实时荧光定量PCR法检测34对HCC组织及其对应癌旁组织中miR-186的表达情况,同时检测4株HCC细胞(Hep G2、Hep3B、SMMC-7721和BEL-7402)以及正常肝细胞株HL-7702中miR-186的表达情况。将携带有miR-186-模拟物(mimics)和miR-186-抑制物(inhibitor)的真核表达载体转染至SMMC-7721细胞后,分别采用CCK-8法、FCM法以及Transwell小室迁移和侵袭实验检测细胞增殖、凋亡、迁移和侵袭能力的变化。分别采用实时荧光定量PCR和蛋白质印迹法进一步检测miR-186对靶基因ROCK1的作用。结果:miR-186在HCC组织中的表达量为0.034±0.033,明显低于其在癌旁组织中的表达量0.077±0.056(P<0.001)。临床病理学特征分析结果显示,HCC组织中miR-186的表达量与肿瘤大小和TNM分期有关(P值均<0.05)。miR-186在HCC细胞中的表达量均明显低于正常肝细胞(P值均<0.05),其中以在SMMC-7721细胞中的表达水平最低。与对照组相比,miR-186-mimics组细胞在各时间点的增殖活性均明显降低,而miR-186-inhibitor组细胞在各时间点的增殖活性均明显升高(P值均<0.05);miR-186-mimics组细胞的凋亡率明显升高,而miR-186-inhibitor组细胞凋亡率明显降低(P值均<0.05);miR-186-mimics组细胞的迁移和侵袭能力明显降低,而miR-186-inhibitor组细胞的迁移和侵袭能力明显增强(P值均<0.05)。过表达miR-186能够降低ROCK1 m RNA及蛋白的表达水平,抑制miR-186表达则会上调ROCK1 m RNA及蛋白的表达水平(P值均<0.05)。结论:miR-186在HCC组织和细胞中均低表达,并能够通过下调ROCK1的表达来抑制HCC细胞的增殖、迁移和侵袭,同时促进细胞凋亡。Objective： To investigate the expression level of microRNA-186（miR-186） in human hepatocellular carcinoma （HCC） tissues and cells,and to study its influences on the biological characteristics of hepatoma SMMC-7721 cells. Methods： The expression level of miR-186 in 34 pairs of HCC and para-cancerous tissues as well as in four kinds of HCC cell lines （including HepG2,SMMC-7721,Hep3B and Bel-7402） and the normal hepatocyte HL-7702 was detected by real-time fluorescent quantitative PCR.After the transfection with eukaryotic expression vector carrying miR-186-mimics or miR-186-inhibitor,the changes of proliferation,apoptosis,migration and invasion of SMMC-7721 cells were detected by CCK-8 assay,FCM method,Transwell chamber migration and invasion teat,respectively.Furthermore,the effect of miR-186 on target gene ROCK1 was verified by real-time fluorescent quantitative PCR and Western blotting. Results： The expression level of miR-186 in HCC tissues was significantly lower than that in the para-cancerous tissues （0.034±0.033 vs 0.077±0.056,P 〈 0.001）.The expression of miR-186 in HCC tissues was related to tumor size and TNM stage （both P 〈 0.05）.The relative expression level of miR-186 in HCC cells was significantly lower than that in normal hepatocytes （all P 〈 0.05）,especially in SMMC-7721 cells with the lowest expression level.As compared with the control,the proliferation activity of SMMC-7721 cells transfected with miR-186-mimics was decreased at each time point,while the proliferation activity of SMMC-7721 cells transfected with miR-186-inhibitor was increased （both P 〈 0.05）;the apoptosis rate of SMMC-7721 cells transfected with miR-186-mimics was increased,while the apoptosis rate of SMMC-7721 cells transfected with miR-186-inhibitor was decreased （both P 〈 0.05）;the number of SMMC-7721 migrating and invading through Transwell chamber after transfection with miR-186-mimics was decreased,while the number of migrated and invaded SMMC-7721 cells after transfection

关 键 词：癌  肝细胞 微RNAS 细胞增殖 细胞凋亡 肿瘤侵润 

分 类 号：R735.7[医药卫生—肿瘤]