【摘要】 目的 探讨微小核糖核酸-325-3p(miR-325-3p)靶向紧密连接蛋白1(CLDN1)调控人肝癌细胞增殖、侵袭与迁移的作用。方法 将对数生长期的人肝癌细胞株SMMC-7721分为四组：阴性对照组、上调组、下调组和正常组。荧光显微镜观察各组细胞转染效率；细胞计数试剂-8(CCK-8)法检测各组细胞增殖活性；侵袭小室(Transwell)实验检测各组细胞侵袭和迁移活性；实时-定量聚合酶链反应(RT-qPCR)检测各组细胞miR-325-3p、CLDN1、细胞外调节蛋白激酶1/2(ERK1/2)、E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)、基质金属蛋白酶-2(MMP-2)信使核糖核酸(mRNA)表达；免疫印迹法(WB)检测各组CLDN1、ERK1/2、E-cadherin、N-cadherin、Vimentin、MMP-2蛋白表达及p-ERK1/2水平；双荧光素酶报告基因检测验证miR-325-3p对CLDN1的作用。结果 阴性对照组、上调组和下调组细胞转染效率分别为(91.38±16.21)%、(92.17±15.93)%、(94.12±17.05)%;与正常组、阴性对照组和上调组比，下调组细胞吸光度值上升，增殖抑制率显著下降，侵袭细胞数和迁移细胞数增加，miR-325-3p表达和E-cadherin mRNA和蛋白表达显著下降，CLDN1、N-cadherin、Vimentin、MMP-2 mRNA和蛋白表达上升，ERK1/2 mRNA表达上升，p-ERK1/2水平、p-ERK1/2/ERK1/2水平上升，差异有统计学意义(P<0.05或P<0.01);双荧光素酶报告基因实验证实miR-325-3p可靶向调控CLDN1的表达。结论 miR-325-3p可抑制肝癌细胞增殖、侵袭和迁移，可能与靶向CLDN1,抑制ERK1/2表达，促进E-cadherin表达，抑制N-cadherin、Vimentin、MMP-2表达相关。更多还原

【Abstract】 Objective To investigate the effects of micro ribonucleic acid-325-3p(miR-325-3p) targeting tight junction protein 1(CLDN1) on proliferation, invasion and migration of human hepatocellular carcinoma cell line SMMC-7721.Methods Human hepatocellular carcinoma cell growing in logarithmic phase was divided into four groups: negative control group, up-regulation group and down-regulation group, normal group. Each group was set with 5 multiple wells, and the transfection efficiency of cells in each group was observed under fluorescence microscope after 48 hours of transfection. Cell counting reagent-8(CCK-8) was used to detect the proliferation activity of cells in each group. The invasion and migration activities of cells in each group were detected by the invasive chamber(Transwell) test. The expressions of miR-325-3p, CLDN1, ERK1/2, E-cadherin, N-cadherin, vimentin, matrix metalloproteinase-2(MMP-2) mRNA were detected by real-time quantitative polymerase chain reaction(RT-qPCR). Western blot(WB) was used to detect the expression of CLDN1, ERK1/2, E-cadherin, N-cadherin, Vimentin, MMP-2 protein and p-ERK1/2 level in each group. The effect of miR-325-3p on CLDN1 was verified by double luciferase reporter gene detection.Results The transfection efficiencies of negative control group, up-regulation group and down-regulation group were(91.38±16.21)%,(92.17±15.93)%,(94.12±17.05)%, respectively. Compared with normal group, negative control group and up-regulation group, the absorbance values of down-regulation group was increased, the proliferation inhibition rate was significantly decreased, the number of invading cells and migrating cells was increased, and the expressions of miR-325-3p and E-cadherin mRNA and protein was significantly decreased, the mRNA and protein expressions of CLDN1, N-cadherin, Vimentin, MMP-2 increased, ERK1/2 mRNA expression were increased, the levels of p-ERK1/2 and p-ERK1/2/ERK1/2 were increased, and the differences were statistically significant(P<0.05 or P<0.01). MiR-325-p targeting CLDN1 was confirmed by dual luciferase reporter gene assay. Conclusion MiR-325-3p inhibits the proliferation, invasion and migration of hepatocellular carcinoma cells, which may be related to targeting CLDN1, inhibition ERK1/2 expression, promotion the expression of E-cadherin, and inhibition the expression of N-cadherin, Vimentin, and MMP-2.更多还原