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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 detect the expression of microRNA-186 (miRNA-186, miR-186) in hepatocellular carcinoma (HCC) tissues and cells and its effect on the biological characteristics of hepatocellular carcinoma SMMC-772 cells. Methods: The expression of miR-186 in 34 HCC tissues and its corresponding paracancerous tissues was detected by real-time fluorescence quantitative PCR. Four HCC cells (Hep G2, Hep3B, SMMC-7721 and BEL-7402) The expression of miR-186 in cell line HL-7702. The eukaryotic expression vectors carrying miR-186-mimics and miR-186-inhibitor were transfected into SMMC-7721 cells, and then transfected into SMMC-7721 cells by CCK-8 method, Invasion experiments were used to detect the changes of cell proliferation, apoptosis, migration and invasion. Real-time fluorescence quantitative PCR and Western blotting were used to further detect the effect of miR-186 on ROCK1. Results: The expression of miR-186 in HCC tissues was 0.034 ± 0.033, which was significantly lower than that in the adjacent tissues (0.077 ± 0.056, P <0.001). The results of clinicopathological analysis showed that the expression of miR-186 in HCC tissue correlated with tumor size and TNM staging (all P <0.05). The expression level of miR-186 in HCC cells was significantly lower than that in normal hepatocytes (all P <0.05), and the expression level of miR-186 in SMMC-7721 cells was the lowest. Compared with the control group, the proliferation activity of miR-186-mimics group cells at each time point was significantly decreased, while the proliferation activity of miR-186-inhibitor group cells at all time points were significantly increased (P values were <0.05) The apoptosis rate of miR-186-mimics group was significantly increased, while the apoptosis rate of miR-186-inhibitor group was significantly lower (P <0.05). The migration and invasion ability of miR-186-mimics group was obviously Decreased, while miR-186-inhibitor group cells migration and invasion ability was significantly enhanced (P values were <0.05). Overexpression of miR-186 decreased the expression of ROCK1 mRNA and protein, and inhibited the expression of ROCK1 mRNA and protein (all P <0.05). Conclusion: miR-186 is low expressed in HCC tissues and cells, and can down-regulate the expression of ROCK1 to inhibit the proliferation, migration and invasion of HCC cells and promote apoptosis.