环指蛋白187诱导上皮间变促进肝癌细胞株转移的研究  

Ring finger protein 187 mediated epithelial-mesenchymal transition pathway to promote metastasis in hepatocellular carcinoma cell lines

作　　者：唐荣[1] 张震生[1] 郑林媚[2] 武金才[1] 郑进方[1] 刘哲豪 陈平平[1] 曾勇超 章家超 陈良[1] Tang Rong;Zhang Zhensheng;Zheng Linmei;Wu Jincai;Zheng Jinfang;Liu Zhehao;Chen Pingping;Zeng Yongchao;Zhang Jiachao;Chen Liang(Department of Hepatopancreatobiliary Surgery,Hainan Provinicial People’s Hospital,the Hainan Hospital of Hainan Medical University,Haikou 570311,China;Department of Obstetrics,Hainan Provinicial People’s Hospital,the Hainan Hospital of Hainan Medical University,Haikou 570311,China)

机构地区：[1]海南省人民医院海南医学院附属海南医院肝胆胰外科,海口570311 [2]海南省人民医院海南医学院附属海南医院产科,海口570311

出　　处：《中华实验外科杂志》2020年第10期1797-1800,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金项目(81660489、81260367、81160310);海南省重点研发计划(ZDYF2019120);海南省自然科学基金项目(819QN356)。

摘　　要：目的:探讨环指蛋白187(RNF187)表达与肝癌侵袭转移和上皮间变的关系。方法:2019年4月至2019年10月,利用实时荧光定量聚合酶链反应(RT-qPCR)和蛋白质印迹法(Western blot)分别检测高转移潜能肝癌细胞株(MHCC97-H)和低转移潜能的肝癌细胞株(PLC/PRF/5、HepG2,中国科学院上海生物所)中RNF187的表达。通过慢病毒转染技术沉默RNF187后,RT-qPCR和Western blot法分别检测RNF187 mRNA及蛋白的表达水平及上皮-间充质转化(EMT)分子标志物E-钙黏蛋白(E-cadherin)、黏结合蛋白多糖-1(Syndecan-1)、N-钙黏蛋白(N-cadherin)和波形纤维蛋白(Vimentin)的表达情况,噻唑蓝(MTT)法和Transwell法分别检测MHCC97-H细胞增殖和侵袭能力的影响。计量资料以均值±标准差(Mean±SD)表示,组间比较采用t检验。结果:高转移潜能组MHCC97-H的RNF187的mRNA表达(0.082±0.006)明显高于低转移潜能组PLC/PRF/5(0.014±0.003)、HepG2(0.017±0.002,t=41.755、36.248,P均<0.05),MHCC97-H的RNF187蛋白表达(2.8±0.3)明显高于PLC/PRF/5(1.8±0.2)、HepG2(1.7±0.2,t=11.030、11.667,P均<0.05)。MHCC97-H-Mock和MHCC97-H-短发卡RNA(shRNA)-RNF187的EMT分子标志物的表达分别为,上皮标志物E-cadherin(0.81±0.08,0.31±0.06)、Syndecan-1(0.70±0.08,0.22±0.07);间皮标志物N-cadherin(0.28±0.04,0.73±0.09)和Vimentin(0.26±0.03,0.68±0.08)。两组细胞株EMT各分子标志物的表达比较差异有统计学意义(t=22.312、24.634、19.424、17.854,P均<0.05),提示MHCC97-H-shRNA-RNF187细胞株EMT过程显著下调。MTT法示,72 h后,MHCC97-H-shRNA-RNF187的增殖能力(2.2±0.4)明显低于MHCC97-H-Mock(4.5±0.6,t=2.428,P<0.05)。Transwell小室法示,MHCC97-H-shRNA-RNF187组细胞穿过微孔滤膜的细胞数量[(178±67)个]明显少于MHCC97-H-Mock组[(482±73)个,t=14.563,P<0.05]。结论:RNF187表达可诱导EMT并参与肝癌细胞的侵袭与转移过程。Objective To investigate the association of ring finger protein 187(RNF187)expression with hepatocellular carcinoma(HCC)invasion,metastasis,and epithelial-mesenchymal transition.Methods Between April 2019 and October 2019,the mRNA and protein expression levels of RNF187 in HCC cell lines with high metastatic potential(MHCC97-H)and low metastatic potential(PLC/PRF/5,HepG2)(Purchased from Shanghai Institute of Biology Chinese Academy of Sciences)were detected by quantitative real-time PCR(RT-qPCR)and Western blotting,as well as epithelial-mesenchymal transition(EMT)molecular markers e-cadherin,syndecan-1,Vimentin and Fibronactin.Cell proliferation was assessed by methyl thiazolyl tetrazolium(MTT)assay and the invasion ability of MHCC97-H cells was evaluated by Transwell assay.The data were presented as mean±SD and analyzed using two-tailed Student’s t-test.Results The expression of RNF187 mRNA in high-metastasis potential cell line MHCC97-H(0.082±0.006)was significantly higher than that in low-metastasis potential cell lines PLC/PRF/5(0.014±0.003)and HepG2(0.017±0.002,t=41.755,36.248,P<0.05).The expression of RNF187 protein(2.8±0.3)in MHCC97-H was significantly higher than that in PLC/PRF/5(1.8±0.2)and HepG2(1.7±0.2,t=11.030,11.667,P<0.05).The expression of RNF187 was positively correlated with the metastasis of HCC cells.The expression levels of EMT markers in MHCC97-H-Mock and MHCC97-H-short hairpin RNA(shRNA)-RNF187 were as follows:E-cadherin(0.81±0.08,0.31±0.06),syndecan-1(0.70±0.08,0.22±0.07),N-cadherin(0.28±0.04,0.73±0.09)and Vimentin(0.26±0.03,0.68±0.08).There were statistically significant differences between two groups in the expression of various molecular markers of EMT of cell lines(t=22.312,24.634,19.424,17.854,P<0.05),suggesting that RNF187 knockdown inhibited EMT process of MHCC97-H cells.The MTT assay showed that after 72 h,the proliferation ability of MHCC97-H-shRNA-RNF187(2.2±0.4)was significantly lower than MHCC97-H-Mock(4.5±0.6,t=2.428,P<0.05).Transwell assay showed that the n

关 键 词：肝细胞癌 环指蛋白187 上皮间质转变 转移 

分 类 号：R735.7[医药卫生—肿瘤]