miR-590-5P通过下调靶基因S100A 10表达、抑制Wnt信号通道调控肝癌细胞增殖

MiR-590-5P Inhibits Growth of HepG2 Cells via Decreasing S100A 10 Expression and Inhibiting Wnt Pathway

目的:研究miR-590-5P对肝癌细胞增殖能力的影响以及参与HCC发生发展的机制。方法:利用QPCR对肝癌细胞株mi R-590-5P的表达谱系进行分析和验证,生物信息技术预测S100A10可作为miR-590-5P的潜在靶基因,并通过实时定量PCR以及Western blot进行验证。通过慢病毒载体在肝癌细胞株HepG2中过表达miR-590-5P,CCK-8法检测转染后细胞增殖,流式细胞技术检测转染后细胞周期变化,Western blot检测转染后细胞Wnt信号相关蛋白表达水平。结果:荧光定量检测结果显示,miR-590-5P在人肝癌细胞中低表达,而S100A10蛋白在肝癌细胞中高表达。S100A10 3’UTR luciferase报告系统验证表明,mi R-590-5P可以通过结合S100A10 3’UTR而抑制报告基因的表达。通过慢病毒系统在HepG2细胞中高表达miR-590-5P,可有效抑制S100A10基因的表达。在肝癌细胞HepG2中过表达mi R-590-5P,可通过调控细胞周期,明显抑制Wnt信号相关蛋白:Wnt5a、cMyc、Cyclin D1的表达水平,促进E-cadherin、Caspase3的表达,加速β-catenin蛋白的磷酸化,而调控细胞增殖。结论:mi R-590-5P在人肝癌细胞中低表达,而S100A10基因在人肝癌细胞中过表达,S100A10为mi R-590-5P的靶基因。mi R-590-5P在肝癌细胞中过表达,可有效抑制S100A10基因的表达,抑制Wnt通路的激活,使肿瘤细胞停滞在G1期,从而参与肝癌细胞增殖进程。

Objective：To study the effect of miR-590-5P on the proliferation of hepatocellular carcinoma cells and the mechanism involved in the occurrence and development of HCC.Method：The QPCR was used on hepatocellular carcinoma cell line mi R-590-5P expression spectrum analysis and verification,the bioinformaticsprediction S100A10 as mi R-590-5P potential target genes and verified by quantitative real-time PCR and Western blot.By lentiviral vector in hepatocellular carcinoma cell line Hep G2 cells over expressing mi R-590-5P,detected by CCK-8 assay after transfection cell proliferation,flow cytometry was transfected into the cell cycle.After transfection,the cells Wnt signaling associated protein expression levels were detected by Western blot.Result：The results of fluorescence quantitative analysis showed that miR-590-5P was low expressed in human hepatocellular carcinoma cells,while S100A10 protein was highly expressed in hepatocellular carcinoma cells.3＇UTR luciferase S100A10 reporting system validation showed that miR-590-5P can inhibit the expression of the reporter gene by binding to 3＇UTR S100A10.The expression of S100A10 gene could be effectively inhibited by high expression of mi R-590-5P in HepG2 cells through the slow virus system.In human hepatoma HepG2 cells overexpressing miR-590-5P,through the regulation of cell cycle,inhibit Wnt signaling related proteins：Wnt5 a,cmyc and Cyclin D1 expression level,promoted the expression of E-cadherin and Caspase3,acceleration of beta catenin protein phosphorylation,and regulation cell proliferation.Conclusion：Mi R-590-5P in human hepatocellular carcinoma cells in low expression,and S100A10 gene in human hepatocellular carcinoma cells over expression,S100A10 for the mi R-590-5P target gene.Overexpression of mi R-590-5P in hepatocellular carcinoma cells can effectively inhibit the expression of S100A10 gene,inhibit the activation of Wnt pathway,so that the tumor cells in the G1 phase of stagnation,so as to participate in the process of proliferation of hepatocellular carcinoma cells.