下调Tim-3对肝癌干细胞自我更新能力及Wnt信号通路的影响  

Effects of Down-Regulation of Tim-3 on Self-Renewal Ability and Wnt Signaling Pathway of Liver Cancer Stem Cells

作　　者：兰勇[1] 邢人伟[1] 陈明[1] 王旭林[1] Lan Yong;Xing Renwei;Chen Ming(Department of Hepatobiliary and Pancreatic Surgery, Taizhou Hospital, Zhejiang 318000, China)

机构地区：[1]台州市立医院肝胆胰外科

出　　处：《医学研究杂志》2019年第6期83-88,共6页Journal of Medical Research

基　　金：浙江省医药卫生科技计划项目(2018KY902)

摘　　要：目的探讨Tim-3对肝癌干细胞(liver cancer stem cells,LCSCs)自我更新能力及Wnt信号通路的影响.方法培养人肝癌细胞系HepG2、SMMC7721及正常干细胞系L02,流式细胞仪分选Nanog阳性(+)的肝癌干细胞;实时荧光定量PCR(qRT-PCR)检测Tim-3在3个细胞系中的mRNA表达水平及在肝癌干细胞中的mRNA表达水平;HepG2肝癌干细胞转染Tim-3 siRNA,荧光显微镜和光学显微镜检测转染率,Western blot法检测转染后Tim-3蛋白水平;qRT-PCR检测转染前后Tim-3、Wnt3、β-catenin的mRNA表达水平;细胞克隆形成试验和细胞成球试验检测转染前后HepG2肝癌干细胞的自我更新能力的变化.结果 qRT-PCR检测Tim-3在肝癌细胞系HepG2、SMMC7721中的mRNA表达显著高于正常肝细胞L02(P<0.05);Tim-3在HepG2肝癌干细胞的表达明显高于肝癌细胞(P<0.05);在SMMC7721肝癌干细胞与肝癌细胞比较Tim-3mRNA表达有低趋势;经荧光镜和光学显微镜观察,Tim-3 siRNA转染细胞良好;Western blot法结果表明,Tim-3蛋白水平在siRNA-451组细胞中的表达显著低于siRNA-750、siRNA-NC、未转染细胞(P<0.05);转染后HepG2干细胞Tim-3、Wnt3、β-catenin的mRNA表达水平均显著下降;克隆形成实验和细胞球形成实验显示转染Tim-3 siRNA后,细胞的克隆形成能力和细胞球生长能力均显著降低(P<0.05).结论下调Tim-3表达水平后,可通过抑制Wnt信号通路,最终减弱肝癌干细胞的自我更新能力.Objective To investigate the effects of Tim - 3 on the self - renewal ability and Wnt signaling pathway of liver cancer stem cells( LCSCs). Methods Human hepatoma cell lines HepG2, SMMC7721 and normal stem cell line L02 were cultured. Nanog positive(+) hepatoma stem cells were separated by flow cytometry. Real-time fluorescence quantitative PCR( qRT - PCR) was used to detect the mRNA expression level of Tim - 3 in three cell lines and in hepatocellular carcinoma stem cells. HepG2 liver cancer stem cells were transfected into Tim-3 siRNA. The transfection rate was detected by fluorescence microscope and optical microscope. Western blot was used to detect the level of Tim- 3 protein after transfection. qRT - PCR was used to detect the mRNA expression levels of Tim-3, Wnt3 and B - catenin before and after transfection. The self - renewal ability of HepG2 hepatocellular carcinoma stem cells before and after transfection was detected by cell clone formation test and cell sphere formation test. Results QRT - PCR test results showed that the expression of Tim - 3 mRNA in hepatocellular carcinoma cell lines HepG2 and SMMC7721 was significantly higher than that in normal hepatocytes L02( P < 0. 05 ). The expression of Tim - 3 in HepG2 liver cancer stem cells was significantly higher than that in hepatoma cells( P V 0. 05 ). There was a low trend of Tim - 3 mRNA expression in SMMC7721 hepatoma stem cells compared with hepatoma cells (0. 05 < P < 0. 1 ). Fluorescence microscopy and light microscopy showed that Tim - 3 siRNA transfected cells were good. Western blot results showed that the expression of Tim-3 protein in cells of siRNA - 451 group was significantly lower than that in siRNA - 750, siRNA - NC and non - transfected cells( P < 0. 05 ). After transfection, the mRNA expression levels of Tim - 3 , Wnt3 and p - catenin in HepG2 stem cells decreased significantly. The clone formation test and cell sphere formation test showed that, after transfection of Tim - 3 siRNA, the ability of cell clone formation ability and ce

关 键 词：Tim-3肝癌干细胞 自我更新能力 WNT信号通路 

分 类 号：R735.7[医药卫生—肿瘤]