miR-325-3p靶向CLDN1调控人肝癌细胞增殖、侵袭与迁移  

Effect of miR-325-3p targeting CLDN1 on proliferation,invasion and migration of human hepatocellular carcinoma cell

作　　者：施喆 韩艳珍[1] 周丽媛 王艳春 单铁强[3] 单铁英[4] 聂晓进[5] 刘记平 朱艳梅 袁红伟[8] SHI Zhe;HAN Yan-zhen;ZHOU Li-yuan;WANG Yan-chun;SHAN Tie-qiang;SHAN Tie-ying;NIE Xiao-jin;LIU Ji-ping;ZHU Yan-mei;YUAN Hong-wei(Affiliated Hospital of Hebei University of Engineering,Handan Hebei,056002;Quzhou County Hospital,Handan Hebei,056002;Qinhuangdao Haigang Hospital,Qinhuangdao Hebei,066000;Hebei University of Engineering,Handan Hebei,056002;Handan Central Hospital,Handan Hebei,056002;Handan Vocational College of Science and Technology,Handan Hebei,056002;Handan First Hospital,Handan Hebei,056002,China.;Handan Second Hospital,Handan Hebei,056003,China)

机构地区：[1]河北工程大学附属医院,056002 [2]邯郸市曲周县医院,056002 [3]河北省秦皇岛市海港医院,066000 [4]河北工程大学,056002 [5]邯郸市中心医院,056002 [6]邯郸科技职业学院,056002 [7]邯郸市第一医院,056002 [8]邯郸市第二医院,056003

出　　处：《现代消化及介入诊疗》2022年第7期852-858,共7页Modern Interventional Diagnosis and Treatment in Gastroenterology

基　　金：河北省卫健委立项课题(20220653)。

摘　　要：目的探讨微小核糖核酸-325-3p(miR-325-3p)靶向紧密连接蛋白1(CLDN1)调控人肝癌细胞增殖、侵袭与迁移的作用。方法将对数生长期的人肝癌细胞株SMMC-7721分为四组:阴性对照组、上调组、下调组和正常组。荧光显微镜观察各组细胞转染效率;细胞计数试剂-8(CCK-8)法检测各组细胞增殖活性;侵袭小室(Transwell)实验检测各组细胞侵袭和迁移活性;实时-定量聚合酶链反应(RT-qPCR)检测各组细胞miR-325-3p、CLDN1、细胞外调节蛋白激酶1/2(ERK1/2)、E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)、基质金属蛋白酶-2(MMP-2)信使核糖核酸(mRNA)表达;免疫印迹法(WB)检测各组CLDN1、ERK1/2、E-cadherin、N-cadherin、Vimentin、MMP-2蛋白表达及p-ERK1/2水平;双荧光素酶报告基因检测验证miR-325-3p对CLDN1的作用。结果阴性对照组、上调组和下调组细胞转染效率分别为(91.38±16.21)%、(92.17±15.93)%、(94.12±17.05)%;与正常组、阴性对照组和上调组比,下调组细胞吸光度值上升,增殖抑制率显著下降,侵袭细胞数和迁移细胞数增加,miR-325-3p表达和E-cadherin mRNA和蛋白表达显著下降,CLDN1、N-cadherin、Vimentin、MMP-2 mRNA和蛋白表达上升,ERK1/2 mRNA表达上升,p-ERK1/2水平、p-ERK1/2/ERK1/2水平上升,差异有统计学意义(P<0.05或P<0.01);双荧光素酶报告基因实验证实miR-325-3p可靶向调控CLDN1的表达。结论miR-325-3p可抑制肝癌细胞增殖、侵袭和迁移,可能与靶向CLDN1,抑制ERK1/2表达,促进E-cadherin表达,抑制N-cadherin、Vimentin、MMP-2表达相关。Objective To investigate the effects of micro ribonucleic acid-325-3p(miR-325-3p)targeting tight junction protein 1(CLDN1)on proliferation,invasion and migration of human hepatocellular carcinoma cell line SMMC-7721.Methods Human hepatocellular carcinoma cell growing in logarithmic phase was divided into four groups:negative control group,up-regulation group and down-regulation group,normal group.Each group was set with 5 multiple wells,and the transfection efficiency of cells in each group was observed under fluorescence microscope after 48 hours of transfection.Cell counting reagent-8(CCK-8)was used to detect the proliferation activity of cells in each group.The invasion and migration activities of cells in each group were detected by the invasive chamber(Transwell)test.The expressions of miR-325-3p,CLDN1,ERK1/2,E-cadherin,N-cadherin,vimentin,matrix metalloproteinase-2(MMP-2)mRNA were detected by real-time quantitative polymerase chain reaction(RT-qPCR).Western blot(WB)was used to detect the expression of CLDN1,ERK1/2,E-cadherin,N-cadherin,Vimentin,MMP-2 protein and p-ERK1/2 level in each group.The effect of miR-325-3p on CLDN1 was verified by double luciferase reporter gene detection.Results The transfection efficiencies of negative control group,up-regulation group and down-regulation group were(91.38±16.21)%,(92.17±15.93)%,(94.12±17.05)%,respectively.Compared with normal group,negative control group and up-regulation group,the absorbance values of down-regulation group was increased,the proliferation inhibition rate was significantly decreased,the number of invading cells and migrating cells was increased,and the expressions of miR-325-3p and E-cadherin mRNA and protein was significantly decreased,the mRNA and protein expressions of CLDN1,N-cadherin,Vimentin,MMP-2 increased,ERK1/2 mRNA expression were increased,the levels of p-ERK1/2 and p-ERK1/2/ERK1/2 were increased,and the differences were statistically significant(P<0.05 or P<0.01).MiR-325-p targeting CLDN1 was confirmed by dual luciferase reporter ge

关 键 词：微小核糖核酸-325-3p 紧密连接蛋白1 肝癌 增殖 侵袭 迁移 

分 类 号：R575[医药卫生—消化系统]