【摘要】：目的:檢測(cè)微RNA-186(miRNA-186,miR-186)在肝細(xì)胞肝癌(hepatocellular carcinoma,HCC)組織和細(xì)胞中的表達(dá)水平,以及其對(duì)肝癌SMMC-772細(xì)胞生物學(xué)特性的影響。方法:采用實(shí)時(shí)熒光定量PCR法檢測(cè)34對(duì)HCC組織及其對(duì)應(yīng)癌旁組織中miR-186的表達(dá)情況,同時(shí)檢測(cè)4株HCC細(xì)胞(Hep G2、Hep3B、SMMC-7721和BEL-7402)以及正常肝細(xì)胞株HL-7702中miR-186的表達(dá)情況。將攜帶有miR-186-模擬物(mimics)和miR-186-抑制物(inhibitor)的真核表達(dá)載體轉(zhuǎn)染至SMMC-7721細(xì)胞后,分別采用CCK-8法、FCM法以及Transwell小室遷移和侵襲實(shí)驗(yàn)檢測(cè)細(xì)胞增殖、凋亡、遷移和侵襲能力的變化。分別采用實(shí)時(shí)熒光定量PCR和蛋白質(zhì)印跡法進(jìn)一步檢測(cè)miR-186對(duì)靶基因ROCK1的作用。結(jié)果:miR-186在HCC組織中的表達(dá)量為0.034±0.033,明顯低于其在癌旁組織中的表達(dá)量0.077±0.056(P0.001)。臨床病理學(xué)特征分析結(jié)果顯示,HCC組織中miR-186的表達(dá)量與腫瘤大小和TNM分期有關(guān)(P值均0.05)。miR-186在HCC細(xì)胞中的表達(dá)量均明顯低于正常肝細(xì)胞(P值均0.05),其中以在SMMC-7721細(xì)胞中的表達(dá)水平最低。與對(duì)照組相比,miR-186-mimics組細(xì)胞在各時(shí)間點(diǎn)的增殖活性均明顯降低,而miR-186-inhibitor組細(xì)胞在各時(shí)間點(diǎn)的增殖活性均明顯升高(P值均0.05);miR-186-mimics組細(xì)胞的凋亡率明顯升高,而miR-186-inhibitor組細(xì)胞凋亡率明顯降低(P值均0.05);miR-186-mimics組細(xì)胞的遷移和侵襲能力明顯降低,而miR-186-inhibitor組細(xì)胞的遷移和侵襲能力明顯增強(qiáng)(P值均0.05)。過(guò)表達(dá)miR-186能夠降低ROCK1 m RNA及蛋白的表達(dá)水平,抑制miR-186表達(dá)則會(huì)上調(diào)ROCK1 m RNA及蛋白的表達(dá)水平(P值均0.05)。結(jié)論:miR-186在HCC組織和細(xì)胞中均低表達(dá),并能夠通過(guò)下調(diào)ROCK1的表達(dá)來(lái)抑制HCC細(xì)胞的增殖、遷移和侵襲,同時(shí)促進(jìn)細(xì)胞凋亡。
[Abstract]:Aim: to investigate the expression of microRNA-186 (miRNA-186,miR-186) in hepatocellular carcinoma (hepatocellular carcinoma,HCC) and its effect on the biological characteristics of SMMC-772 cells. Methods: the expression of miR-186 in 34 pairs of HCC and its adjacent tissues was detected by real-time fluorescence quantitative PCR. The expression of miR-186 in four HCC cells (Hep G2Hep3BnSMMC-7721 and BEL-7402) and the normal hepatocyte line HL-7702 was also detected. After transfection of eukaryotic expression vector carrying (mimics) and miR-186- inhibitor (inhibitor) into SMMC-7721 cells, the changes of cell proliferation, apoptosis, migration and invasion were detected by CCK-8 assay, FCM assay and Transwell chamber migration and invasion assay, respectively. The effect of miR-186 on target gene ROCK1 was further detected by real-time fluorescence quantitative PCR and Western blotting. Results: the expression of miR-186 in HCC was 0.034 鹵0.033, which was significantly lower than that in paracancerous tissues (0.077 鹵0.056). The results of clinicopathological analysis showed that the expression of miR-186 in HCC was related to tumor size and TNM stage (P < 0. 05). The expression of miR-186 in HCC cells was significantly lower than that in normal hepatocytes (P = 0. 05), especially in SMMC-7721 cells. Compared with the control group, the proliferative activity of miR-186-mimics group was significantly lower than that of control group, while the proliferative activity of miR-186-inhibitor group was significantly increased at each time point (P < 0. 05), and the apoptosis rate of miR-186-mimics group was significantly higher than that of control group. The apoptosis rate of miR-186-inhibitor group was significantly lower than that of miR-186-mimics group (P < 0. 05), but the migration and invasion ability of miR-186-mimics group was significantly lower than that of miR-186-inhibitor group (P < 0. 05). Overexpression of miR-186 could decrease the expression of ROCK1 m RNA and protein, but inhibit the expression of miR-186 could up-regulate the expression of ROCK1 m RNA and protein (P < 0 05). Conclusion: the expression of miR-186 is low in HCC tissues and cells, and it can inhibit the proliferation, migration and invasion of HCC cells by down-regulating the expression of ROCK1, and promote the apoptosis of HCC cells.
【作者單位】： 重慶醫(yī)科大學(xué)附屬第一醫(yī)院肝膽外科;
【分類(lèi)號(hào)】：R735.7

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