Tim-3在肝癌细胞上皮间质转化及转移中的作用机制  

Mechanism of Tim-3 in epithelial-mesenchymal transition of hepatocellular carcinoma

作　　者：林华鹏[1] 段艳飞[1] 马长林[1] 乔森[1] LIN Hua-peng;DUAN Yan-fei;MA Chang-lin;QIAO Sen(Department of Hepatobiliary Surgery,Jining No.1 People’s Hospital,Jining 272000,China)

机构地区：[1]济宁市第一人民医院肝胆外科

出　　处：《中国现代普通外科进展》2019年第9期673-677,682,共6页Chinese Journal of Current Advances in General Surgery

基　　金：山东省医药卫生科技发展计划（2016WS0147)

摘　　要：目的:探讨Tim-3与肝癌细胞发生上皮间质转化(EMT)的关系及对肝癌细胞侵袭和转移能力的影响。方法:培养正常肝细胞L02和肝癌细胞SMMC-7721,RT-PCR检测Tim-3表达差异,将SMMC-7721分为3组,对照组:未接受转染的SMMC-7721;实验组转染Tim-3 siRNA细胞低表达Tim-3;阳性对照组转染PEX-3-hTim-3过表达质粒细胞高表达Tim-3。应用RT-PCR、Western blot检测3组肝癌细胞Tim-3及EMT相关基因:E-cadherin、N-cadherin、MMP-9、Twistl、Slug、Snail、Smad mRNA和蛋白的表达。通过Transwell小室侵袭实验检测3组肝癌细胞迁移和侵袭能力改变;分析Tim-3表达与EMT相关基因表达的相关性。结果:(1)与正常肝细胞L02比较;Tim-3在肝癌细胞中高表达(P<0.05);(2)RTPCR和Western bolt结果提示,实验组上皮标志物E-cadherin表达较对照组明显上升(P<0.05),而间质标志物N-cadherin、MMP-9、Twistl、Snail、Slug、Smad表达较对照组下降(P<0.05),提示EMT受到抑制;阳性对照组中E-cadherin表达较对照组下降(P<0.05),N-cadherin、MMP-9、Twist1、Snai1、Slug、Smad表达较对照组上升(P<0.05),促进了肝癌EMT;(3)Transwell迁移实验中,实验组与对照组和阳性对照组相比较,肝癌细胞迁移和侵袭细胞数明显减少,3组之间比较差异均有统计学意义(P<0.05),实验说明Tim-3表达水平的改变,影响肝癌的迁移和侵袭性;(4)Tim-3的表达与EMT标志物的表达做相关性分析显示,Tim-3的表达与E-cadherin的表达呈负相关(P<0.05),与N-cadherin、MMP-9、Twist1、Snai1、Slug、Smad的表达呈正相关(P<0.05)。结论:Tim-3是肝癌细胞EMT的潜在诱导者,抑制Tim-3的表达,同时抑制肝癌细胞EMT的发生,肝癌细胞迁移和侵袭能力下降。因此,Tim-3有望成为今后的临床抗肿瘤药物新的治疗靶点用于改善和评估患者预后。Objective:To investigate the relationship between Tim-3 and epithelial-mesenchymal transition(EMT) in hepatocarcinoma cells and its effect on invasion and metastasis of hepatoma cells.Method:Culture normal liver cell L02 and hepatocellular carcinoma cell SMMC-7721,detection of Tim-3 expression by RT-PCR.The hepatocellular carcinoma cells were divided into three groups,experimental group control group HCC cells without transfection;experimental group:HCC transfected with Tim-3 siRNA,low expression of Tim-3;positive control group:HCC transfected with PEX-3-hTim-3 high expression plasmid,high expression of Tim-3.RT-PCR and Western blot were used to detect the expression of mRNA and protein of Tim-3 and EMT related genes in different groups of hepatoma cells:E-cadherin,N-cadherin,MMP-9,Twistl,Slug,Snail,Smad.The MTT assay,Transwell chamber invasion assay were used to detect the proliferation of liver cancer cells and the migration and invasion.Analysis of the correlation between Tim-3 expression and EMT-related gene expression.Results:Tim-3 was highly expressed in hepatocellular carcinoma compared to normal liver cell L02(P<0.05).The results of RT-PCR and Western blot indicated that the expression of Tim-3 was inhibited in the experimental group,and the expression of E-cadherin was significantly increased,while the expression of interstitial markers:N-cadherin,MMP-9,Twistl,Snail,Slug and Smad decreased.The epithelial-mesenchymal transition of hepatoma cells was inhibited;the expression of epithelial marker E-cadherin was decreased in the positive control group,while the expression of the interstitial markers:N-cadherin,MMP-9,Twist1,Snail,Slug,Smad increased.Promotes epithelial-mesenchymal transition in liver cancer cells.Transwell small migration experiment:Compared with the control group and the positive control group,the number of liver cancer cell migration and invasion cells was significantly reduced in the experimental group,and there were significant differences between the three groups(P<0.05).The experiment i

关 键 词：肝癌细胞 上皮间质转化 T细胞免疫球蛋白及黏蛋白分子3 上皮型钙黏附蛋白 神经型钙黏附蛋白 

分 类 号：R735.7[医药卫生—肿瘤]