miRNA-124-3p靶向EZH2调控肝癌细胞转移和侵袭能力的机制研究

microRNA-124-3p regulates migration and invasive ability of liver cancer cells through targeting EZH2 gene

目的探讨miRNA-124-3p靶向果蝇Zeste基因增强子人类同源物2(EZH2)调控肝癌细胞转移和侵袭能力的机制。方法H22细胞分为Con组、mimic组、inhibitor组,mimic组和inhibitor组中分别转染miRNA-124-3p类似物mimic和抑制物inhibitor。实时荧光定量PCR法检测EZH2 mRNA表达水平。PI染色-流式细胞术检测细胞周期的变化,划痕实验检测细胞迁移能力,Transwell小室检测细胞转移和侵袭能力,平板克隆形成实验检测细胞克隆形成能力,免疫荧光法检测细胞EZH2的表达变化,Western blot法检测细胞中EZH2、细胞外因子1(wnt-1)和β-连环蛋白(β-catenin)蛋白表达水平。采用荧光素酶报告基因法验证miRNA-124-3p和EZH2的靶向关系。结果与Con组比较,mimic组细胞EZH2蛋白和mRNA表达水平均下调,细胞24 h迁移率下降,转移细胞数、侵袭细胞数和克隆形成数均减少,EZH2的荧光强度下调,wnt-1和β-catenin蛋白表达水平均上调,差异均有统计学意义(均P<0.05)。与Con组和mimic组比较,inhibitor组EZH2蛋白和mRNA表达水平均上调,细胞24 h迁移率上升,转移细胞数、侵袭细胞数和克隆形成数均增加,EZH2的荧光强度上调,wnt-1和β-catenin蛋白表达水平均下调,差异均有统计学意义(均P<0.05)。荧光素酶报告基因结果显示miRNA-124-3p是EZH2的调控非编码RNA,miRNA-124-3p可以靶向结合EZH2。结论miRNA-124-3p可以靶向抑制EZH2表达,下调wnt-1、β-catenin蛋白水平,改善肝癌细胞的转移和侵袭。

Objective To investigate the mechanism of microRNA-124-3p in regulating the migration and invasion ability of liver cancer cells.Methods Mouse liver cancer H22 cells were were transfected with miRNA-124-3p analog mimic and inhibitor,respectively.Real-time fluorescence quantitative PCR was used to detect the expression of EZH2 mRNA,PI staining-flow cytometry was used to detect the cell cycle,scratch test Transwell assay was used to detect cell migration and invasion ability,plate clone formation assay was used to detect cell clone formation ability,immunofluorescence assay was used to detect EZH2 expression,Western blot was used to detect the activation of wnt-1/β-catenin signal pathway in cells.The luciferase reporter gene method was used to verify the targeting relationship between miRNA-124-3p and EZH2 gene.Results Compared with the control group,the expression levels of EZH2 protein and mRNA in the mimic group were down-regulated,the 24 h migration rate of the mimic group decreased,the number of metastatic cells,invasion cells and colony forming cells decreased,the expression levels of wnt-1 andβ-catenin protein in the mimic group were significantly up-regulated(all P<0.05).Compared with the control group and the mimic group,the expression levels of EZH2 protein and mRNA in the inhibitor group were up-regulated,the 24 h migration rate of the inhibitor group increased,the number of metastatic cells,invasive cells and colony forming cells increased,the fluorescence intensity of EZH2 in the inhibitor group was up-regulated,and the expression levels of wnt-1 andβ-catenin protein in the inhibitor group were significantly down-regulated(all P<0.05).The results of the luciferase reporter gene showed that miRNA-124-3p was the regulatory non-coding RNA of EZH2 gene,and the two had a targeted binding relationship.Conclusion miRNA-124-3p can target and inhibit EZH2 expression,down-regulate wnt-1,β-catenin signal pathway,and inhibit the metastasis and invasion of liver cancer cells.