miR-452-5p对肝癌细胞迁移和侵袭能力的影响及作用靶点预测

Effects of miR-452-5p on migration and invasion of hepatocellular carcinoma cells and its target prediction

目的观察miR-452-5p对肝癌细胞迁移、侵袭能力的影响,并预测其作用靶点。方法将肝癌SMMC7721细胞分为miR模拟组、对照1组、miR抑制组、对照2组;miR模拟组转染miR-452-5p mimic,对照1组转染miR-452-5p mimicNC,miR抑制组转染miR-452-5p inhibitor,对照2组转染miR-452-5p inhibitorNC;转染24 h后,采用细胞划痕实验和Transwell迁移实验检测肝癌细胞迁移能力,采用Transwell侵袭实验检测细胞侵袭能力。miRNA靶基因预测软件得出EPB41L33'UTR含有miR-452-5p的结合位点,利用双荧光素酶报告实验验证miR-452-5p与EPB41L33'UTR的结合;采用Westernblotting法检测miR模拟组、对照1组、miR抑制组、对照2组细胞中的EPB41L3蛋白。结果miR模拟组细胞迁移率及Transwell实验迁移细胞数、侵袭细胞数高于对照1组(P均<0.05)。miR抑制组细胞迁移率及Transwell实验迁移细胞数、侵袭细胞数低于对照2组(P均<0.05)。双荧光素酶报告实验结果提示miR-452-5p可以直接结合EPB41L33'UTR,即EPB41L3是miR-452-5p的直接靶基因。miR模拟组细胞中EPB41L3蛋白表达低于对照1组,miR抑制组细胞中EPB41L3蛋白表达高于对照2组(P均<0.01)。结论miR-452-5p高表达可提高肝癌细胞的迁移侵袭能力,作用机制可能与靶向调控EPB41L3有关。

Objective To investigate the effects of miR-452-5p on the migration and invasion of hepatocellular carci-noma(HCC)cells and to predict its target.Methods HCC SMMC7721 cells were divided into the miR mimic group,the Control group 1,the miR inhibitor group,and the Control group 2.The HCC SMMC7721 cells in the miR mimic group were transfected with miR-452-5p mimic,the Control group 1 with miR-452-5p mimic NC,the miR inhibitor group with miR-452-5p inhibitor,and the Control group 2 with miR-452-5p inhibitor NC.After transfecting for 24 h,HCC migration and invasion abilities were tested by Scratch test and Transwell assay.Bioinformatics analysis was performed to predict the target genes of miR-452-5p.The binding of miR-452-5p to EPB41L33'UTR was verified via Dual-luciferase reporter as-say.EPB41L3 protein expression level was detected by Western blotting in the miR mimic group,Control group 1,miR in-hibitor group and Control group 2.Results The migration rate in the Scratch test and the migration and invasion cells in the Transwell assay of the miR mimic group were higher than those of the Control group 1(all P<0.05),while the migra-tion rate and the migration and invasion cells of the miR inhibitor group were lower than those of the Control group 2(all P<0.05).Dual-luciferase reporter assay indicated a direct binding of miR-452-5p to EPB41L33'UTR,which certificated that EPB41L3 was the direct target gene of miR-452-5p.EPB41L3 protein expression level of the miR mimic group was lower than that of the Control group 1,while EPB41L3 protein expression level of the miR inhibitor group was higher than that of the Control group 2(both P<0.01).Conclusions High expression of miR-452-5p promotes migration and inva-sion of HCC cells by directly targeting EPB41L3.