miR-193b-3p、PAK4过表达对人结肠癌细胞增殖凋亡和迁移侵袭的影响及靶向关系

Effects of overexpression of miR-193b-3p and PAK4 on proliferation,apoptosis,migration,and invasion of human colon cancer cells and their targeting relationships

目的观察微小核糖核酸193b-3p(miR-193b-3p)、P21活化蛋白激酶4(PAK4)过表达对人结肠癌细胞系HCT116细胞增殖凋亡和迁移侵袭的影响,并验证两者的靶向关系。方法体外培养人正常结肠黏膜上皮细胞FHC及人结肠癌细胞HCT116、SW480、LoVo,采用RT-PCR检测各细胞miR-193b-3p、P21活化蛋白激酶4(PAK4)mRNA。取生长状态良好的对数生长期HCT116细胞,分为4组,miR-NC组转染miRNA阴性对照、miR-193b-3p组转染miR-193b-3p模拟物、miR-193b-3p+PAK4-NC组转染miR-193b-3p模拟物+P21活化蛋白激酶4(PAK4)空载体、miR-193b-3p+PAK4组转染miR-193b-3p模拟物+PAK4过表达载体,分别采用RT-PCR、CCK-8试验、流式细胞术、Transwell实验检测各组细胞miR-193b-3p、PAK4 mRNA表达及增殖活性、凋亡率、迁移细胞数、侵袭细胞数。采用生物信息学软件Target Scan7.2预测miR-193b-3p与PAK4的靶向关系,并采用双荧光素酶报告基因实验进行验证。结果过表达miR-193b-3p后,HCT116细胞增殖活性、迁移及侵袭能力均明显下降,凋亡率明显升高,PAK4 mRNA表达水平降低(P均<0.05)。上调PAK4表达能够部分逆转过表达miR-193b-3p对HCT116细胞增殖活性、迁移及侵袭能力的抑制及对凋亡的促进作用(P均<0.05)。双荧光素酶报告基因显示miR-193b-3p能靶向结合PAK4的3'UTR区,抑制其荧光素酶活性(P<0.05)。结论miR-193b-3p过表达可抑制HCT116细胞增殖、迁移及侵袭,并促进其凋亡,miR-193b-3p与PAK4存在靶向关系。

Objective To investigate the effects of microRNA 193b-3p(miR-193b-3p)and P21-activated protein kinase 4(PAK4)overexpression on the proliferation,apoptosis,migration and invasion of human colon cancer cell line HCT116,and to verify the targeting relationship between them.Methods Human normal colon mucosal epithelial cells FHC and human colon cancer cells HCT116,SW480,LoVo were cultured in vitro,and the mRNA expression of miR-193b-3p and PAK4 was detected by RT-PCR.HCT116 cells in the logarithmic growth phase with good growth status were divided into four groups:miR-NC group(transfected with miRNA negative control),miR-193b-3p group(transfected with miR-193b-3p mimics),miR-193b-3p+PAK4-NC group(transfected with miR-193b-3p mimics+PAK4 empty vector),and miR-193b-3p+PAK4 group(transfected with miR-193b-3p mimics+PAK4 overexpression vector),respectively.The mRNA expression of miR-193b-3p and PAK4,the proliferation activity,apoptosis rate,the number of migrating cells and the number of invasive cells in each group were detected by RT-PCR,CCK-8 assay,flow cytometry,and Transwell experiment,respectively.The targeting relationship between miR-193b-3p and PAK4 was predicted by bioinformatics software Target Scan7.2,and verified by double luciferase reporter gene experiment.Results After overexpressing miR-193b-3p,the proliferation activity,migration and invasion abilities of HCT116 cells significantly decreased,the apoptosis rate significantly increased,and the expression level of PAK4 mRNA decreased(all P<0.05).Up-regulation of PAK4 expres-sion could partially reverse the inhibitory effect of overexpressing miR-193b-3p on proliferation,migration and invasion and the promotion of apoptosis of HCT116 cells(all P<0.05).The dual luciferase reporter gene assay showed that miR-193b-3p could target the 3'UTR region of PAK4 and inhibit its luciferase activity(both P<0.05).Conclusion Overex-pression of miR-193b-3p can inhibit the proliferation,migration and invasion of HCT116 cells,and promote their apopto-sis,and there is a targeting relationship between miR-193b-3p and PAK4.