miR-138-5p通过靶向SETD6基因调控Raf/MEK/ERK通路抑制肝癌细胞迁移、侵袭的分子机制

Molecular mechanisms of Mi-138-5p inhibiting the migration and invasion of hepatocellular carcinoma cells by targeting SETD6 gene to regulate Raf/MEK/ERK pathway

目的探究miR-138-5p通过靶向赖氨酸甲基转移酶(SETD6)基因调控Raf/MEK/细胞外信号调节激酶(ERK)通路抑制肝癌细胞迁移、侵袭的分子机制。方法qRT-PCR、Western印迹检测正常肝细胞L02、肝癌细胞HepG2、Hep3b、HuH-7细胞中miR-138-5p、SETD6的表达水平,噻唑蓝(MTT)法检测细胞增殖情况,Transwell实验检测细胞侵袭、迁移能力,Western印迹检测转染后Hep3b细胞中CyclinD1、基质金属蛋白酶(MMP)-2蛋白表达水平,双荧光素酶报告实验及Western印迹检测miR-138-5p与SETD6的联系。结果与人正常肝细胞L02比较,肝癌细胞HepG2、Hep3b、HuH-7中miR-138-5p的表达水平显著降低,SETD6水平显著升高(P<0.05),选择Hep3b细胞进行后续实验;与NC、miR-con组比较,miR-138-5p mimics组miR-138-5p的表达水平显著升高,细胞存活率、侵袭和迁移细胞数目、SETD6、CyclinD1、MMP-2水平显著降低(P<0.05);与NC、si-con组比较,si-SETD6组中SETD6蛋白水平显著降低,细胞存活率、侵袭和迁移细胞数目、CyclinD1、MMP-2水平显著减少(P<0.05);TargetScan生物信息学软件预测显示,miR-138-5p与SETD63'UTR存在结合位点,双荧光素酶报告实验显示,miR-138-5p与SETD63'UTR区域特异性结合,Western印迹进一步检测显示miR-138-5p与SETD6直接结合负向调控SETD6的表达;与miR-138-5p+pcDNA组比较,过表达SETD6后,miR-138-5p+pcDNA-SETD6组细胞存活率、侵袭和迁移细胞数目、SETD6、CyclinD1、MMP-2蛋白水平显著升高(P<0.05);与miR-con组比较,过表达miR-138-5p后,miR-138-5p组Raf、p-MEK、p-ERK蛋白水平显著降低,与miR-138-5p+pcDNA组比较,过表达SETD6后,miR-138-5p+pcDNA-SETD6组Raf、p-MEK、p-ERK蛋白水平显著升高(P<0.05)。结论肝癌细胞的增殖、侵袭和迁移受到miR-138-5p及SETD6的双重调控,miR-138-5p可能通过调节SETD6的表达调控Raf/MEK/ERK通路进而调节肝癌细胞的增殖、侵袭和迁移,可为肝癌患者分子靶向治疗研究提供思路。

Objective To explore the molecular mechanism of microRNA-138-5p inhibiting the migration and invasion of hepatocellular carcinoma cells by targeting lysine methyltransferase gene regulating Raf/MEK/ERK pathway.Methods The expressions of miR-138-5p and SETD6 in normal hepatocytes L02,HepG2,Hep3b and HuH-7 cells were detected by qRT-PCR and Western blot.Cell proliferation was detected by MTT method.Cell invasion and migration were detected by Transwell assay.CyclinD1 and MMP-2 protein expression levels in Hep3b cells after transfection were detected by Western blot.Double luciferase reporter assay and Western blot were used to detect the relationship between microRNA-138-5p and SETD6.Results Compared with human normal hepatocyte L02,the expression levels of microRNA-138-5p in hepatocellular carcinoma cells HepG2,Hep3b and HuH-7 were significantly decreased,and the level of SETD6 was significantly increased(P<0.05).Hep3b cells were selected for subsequent experiments.Compared with NC and microRNA-con groups,the expression levels of microRNA-138-5p in the microRNA-138-5p mimics group were significantly increased,and the cell survival rate,the number of invasive and migrating cells,SETD6,CyclinD1,MMP-2 were significantly increased(P<0.05).Compared with NC and si-con groups,the level of SETD6 protein in si-SETD6 group decreased significantly,cell survival rate,number of invasive and migrating cells,CyclinD1 and MMP-2 levels decreased significantly(P<0.05).TargetScan bioinformatics software predicted that there were binding sites between microRNA-138-5p and SETD63'UTR,and double luciferase report experiment showed that microRNA-138-5p was specifically bound to SETD6'UTR region,Western blot showed that microRNA-138-5p was specifically bound to SETD6'UTR region.Further detection showed that the expression of SETD6 was negatively regulated by the direct combination of microRNA-138-5p and SETD6.Compared with the microRNA-138-5p+pcDNA group,the cell survival rate,the number of invasive and migrating cells,SETD6,CyclinD1 and MMP-2 protein levels in the microRNA-138-5p+pcDNA-SETD6 group increased significantly(P<0.05).Compared with the microRNA-con group,the levels of Raf,p-MEK and p-ERK protein in the microRNA-138-5p group decreased significantly(P<0.05).Compared with the microRNA-138-5p+pcDNA group,after microRNA-138-5p-SETD6 expression,the levels of Raf,p-MEK and p-ERK protein in the microRNA-138-5p group decreased significantly,Raf,p-MEK and p-ERK protein levels were significantly increased in 138-5p+pcDNA-SETD6 group(P<0.05).Conclusions The proliferation,invasion and migration of hepatocellular carcinoma cells are regulated by both microRNA-138-5p and SETD6.MicroRNA-138-5p might regulate the Raf/MEK/ERK pathway by regulating the expression of SETD6,thus regulating the proliferation,invasion and migration of hepatocellular carcinoma cells,which might provide ideas for molecular targeted therapy of hepatocellular carcinoma patients.