miR-365靶向调控USP22促进大肠癌细胞组蛋白修饰和EMT

MiR-365 promotes histone modification and EMT of colorectal cancer by targetedly reguluating USP22

目的探讨miR-365对大肠癌细胞组蛋白修饰和上皮间质转化(epithelial-mesenchymal transition,EMT)的影响及其机制。方法采用反转录-聚合酶链反应(reverse transcription polymerase chain reaction,RT-PCR)检测大肠癌细胞株(高转移细胞系HCT-116、LoVo和中低转移细胞系SW480)中miR-365 mRNA表达量。通过转染miRNA模拟物、抑制物构建miR-365过表达和敲低模型,SW480细胞分为miR-365模拟物组、miR-365抑制物组和NC组;采用RT-PCR检测miR-365和特异性泛素肽酶22(ubiquitin-specific peptidase 22,USP22)mRNA表达水平;采用双荧光素酶报告基因检测miR-365与USP22的相互作用;Western blot法检测Vimentin、N-cadherin、E-cadherin蛋白表达水平;采用斑点杂交法检测H2A和H2B蛋白泛素化修饰;采用Transwell法检测各组细胞迁移情况,细胞划痕实验检测细胞转移能力。结果HCT-116和LoVo细胞系的miR-365 mRNA表达量显著低于SW480细胞系(P<0.05)。与NC组比较,miR-365抑制物组miR-365 mRNA的表达显著降低,而miR-365模拟物组则升高(P<0.05)。双荧光素酶报告基因检测结果显示,转染后miR-NC+USP22-wt组荧光素酶活性明显高于miR-365+USP22-wt组(P<0.05);而miR-365+USP22-mut组和miR-NC+USP22-mut组比较差异无统计学意义(P>0.05)。RT-PCR法检测结果显示,与NC组比较,miR-365抑制物组SW480细胞中USP22 mRNA的表达量显著升高,而miR-365模拟物组则降低(P<0.05)。斑点杂交法结果显示,与NC组比较,miR-365抑制物组H2AK119ub表达量显著升高,而miR-365模拟物组则降低(P<0.05);与NC组比较,miR-365抑制物组H2BK120ub表达量显著升高,而miR-365模拟物组则降低(P<0.05)。Western blot结果显示,与NC组比较,miR-365抑制物组SW480细胞中N-cadherin蛋白表达量显著降低,而miR-365模拟物组则升高(P<0.05);与NC组比较,miR-365抑制物组SW480细胞中Vimentin、E-cadherin蛋白表达量显著升高,而miR-365模拟物组则降低(P<0.05)。细胞划痕实验结果显示,与NC组和miR-365模拟物组比较,miR-365抑制物组愈合率明显升高(P<0.05);视野下miR-365抑制物组SW480细胞穿模数量明显高于NC组和miR-365模拟物组(P<0.05)。结论miR-365可通过靶向调控USP22的表达,继而调节组蛋白泛素化修饰,诱导上皮-间质转化的发生,最终引起大肠癌细胞转移。

Objective To explore the effect of miR-365 on histone modification and EMT of colorectal cancer cells and its mechanism.Methods Reverse transcription polymerase chain reaction(RT-PCR)was used to detect the miR-365 mRNA level in colorectal cancer cell lines HCT-116,LoVo and SW480.SW480 cells were divided into miR-365 mimics group,miR-365 inhibitor group and NC group.The expression levels of miR-365 and USP22 mRNA were detected by RT-PCR.Double luciferase reporter gene experiment was used to detect the interaction between miR-365 and USP22.The expression levels of Vimentin,N-cadherin and E-cadherin were detected by Western blot.Dot blot method was used to detect H2A and H2B protein modification.Transwell method was used to detect cell migration and cell scratch test was used to detect the ability of cell metastasis.Results The expression of miR-365 mRNA in HCT-116 and LoVo cell lines was significantly lower than that in SW480 cell line(P<0.05).Compared with NC group,the expression of miR-365 mRNA was decreased significantly in miR-365 inhibitor group,and increased in miR-365 mimics group(P<0.05).The results of double luciferase reporter gene detection showed that after transfection,the luciferase activity of miR-NC+USP22-wt was significantly higher than that of miR-365+USP22-wt(P<0.05),but there was no significant difference between miR-365+USP22-mut group and miR-NC+USP22-mut group(P>0.05).Compared with NC group,the expression of USP22 mRNA in SW480 cells was significantly increased in miR-365 inhibitor group,and decreased in miR-365 mimics group(P<0.05).The results of dot blot hybridization showed that the expression of H2AK119ub in miR-365 inhibitor group was significantly higher than that in NC group,but it was lower in miR-365 mimics group than that in NC group(P<0.05).Compared with NC group,the expression of H2BK120ub was significantly increased in miR-365 inhibitor group,but decreased in miR-365 mimics group(P<0.05).Compared with NC group,the expression of N-cadherin protein in SW480 cells was decreased significantly in miR-365 inhibitor group,but increased in miR-365 mimics group(P<0.05).Compared with NC group,the expression of Vimentin and E-cadherin in SW480 cells was significantly increased in miR-365 inhibitor group,but decreased in miR-365 mimics group(P<0.05).The results of cell scratch test showed that the healing rate in miR-365 inhibitor group was significantly higher than that in NC group and miR-365 mimics group(P<0.05).In the visual field,the number of SW480 cells in miR-365 inhibitor group was significantly higher than that in NC group and the miR-365 mimics group(P<0.05).Conclusion MiR-365 can induce the colorectal cancer cell metastasis by regulating histone ubiquitination and inducing epithelial-mesenchymal transition via targeted regulation of USP22 expression.