shRNA沉默HuR抑制肝细胞癌细胞系SMMC-7721的增殖、迁移及侵袭能力  

shRNA Silencing HuR Inhibits Proliferation, Migration and Invasion of SMMC-7721 Hepatocellular Carcinoma Cells

作　　者：湛钊 周莉莉 高义沙 余畅 陈相屹 胡桐 孙达权 ZHAN Zhao;ZHOU Li-Li;GAO Yi-Sha;YU Chang;CHEN Xiang-Yi;HU Tong;SUN Da-Quan(Department of Biochemistry and Molecular Biology,School of Basic Medical Sciences,Guizhou Medical University,Guiyang 550025,China;Research Center for Basic Sciences of Medicine,Guizhou Medical University,Guiyang 550025,China)

机构地区：[1]贵州医科大学基础医学院生物化学与分子生物学教研室,贵阳550025 [2]贵州医科大学基础医学科学研究中心,贵阳550025

出　　处：《中国生物化学与分子生物学报》2020年第8期952-960,共9页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金(No.81560390);贵州省科学技术基金(黔科合基础No.[2017]1147)资助。

摘　　要：人抗原R (human antigen R,HuR)基因在肺癌、乳腺癌等多种肿瘤组织中高表达。推测HuR基因也参与肝癌的发展过程。为探索HuR对肝细胞癌细胞系SMMC-7721的增殖、迁移和侵袭的作用,本研究通过蛋白质印迹实验,检测HuR在肝细胞癌细胞系和正常肝细胞中蛋白质的表达水平。结果显示,肝细胞癌细胞系SMMC-7721 HuR的表达量显著高于正常肝细胞HL-7702。合成特异性靶向HuR基因的shRNA,转染肝细胞癌细胞系SMMC-7721,检测结果发现,HuR基因表达量下调90%。沉默HuR,实时细胞分析技术(real time cell analysis,RTCA)结果显示,细胞增殖能力降低55%,侵袭能力下降75%;细胞划痕实验结果显示,迁移能力下降80%;克隆形成实验中细胞克隆数减少85%;此外,在HuR敲低稳转肝细胞癌细胞系SMMC-7721细胞中过表达Bcl-2,其细胞学现象部分获得逆转。激光共聚焦扫描系统检测结果显示,Bcl-2主要定位于肝细胞癌细胞系SMMC-7721的核膜及胞质。蛋白质印迹法检测结果显示,沉默HuR,Bcl-2下调92%,Survivin下调55%,Twinst1下调69%,N-钙黏着蛋白(N-cadherin)下调48%,E-钙黏着蛋白(E-cadherin)上调1.5倍。以上结果表明,HuR可能通过调控定位于核膜及胞质上的Bcl-2来参与肝细胞癌细胞系SMMC-7721的增殖、迁移、侵袭及克隆形成过程,HuR基因有望成为临床上治疗肝癌的一个新的潜在靶点。Human antigen R(HuR) gene is highly expressed in lung cancer, breast cancer and other tumor tissues. This research speculates that HuR gene is also involved in the development of liver cancer. In order to explore the effects of HuR on the proliferation, migration and invasion of hepatocellular carcinoma cell line SMMC-7721, this study examined the protein expression level of HuR in hepatocellular carcinoma cells and human live cells by Western blotting. The results showed that the expression level of HuR in SMMC-7721 hepatocellular carcinoma cells was significantly higher than that in HL-7702 cells. Consequently, the shRNA specifically targeting the HuR gene was synthesized and transfected into SMMC-7721 hepatocellular carcinoma cells, and the result showed that HuR gene expression was down-regulated by 90%. Cytological results of real-time cell analysis(RTCA) indicated that after HuR was silenced, the cell proliferation capacity decreased by 55% and the invasion capacity decreased by 75%, the migration capacity decreased by 80% in wound healing assays, and the number of clones decreased by 85% in colony formation assays. In addition, the overexpression of Bcl-2 in HuR knockdown and stably transfected SMMC-7721 hepatoma cells partially reversed its cytology. The results of the confocal laser scanning microscope indicated that Bcl-2 was mainly located in the nuclear membrane and cytoplasm of SMMC-7721 cells. Western blotting showed that silencing HuR reduced Bcl-2 by 92%, Survivin by 55%, Twinst1 by 69%, N-cadherin by 48%, and E-cadherin by 1.5 times. The above results indicate that HuR may participate in the proliferation, migration, invasion and colony formation of SMMC-7721 hepatocellular carcinoma cells by regulating Bcl-2 localization on the nuclear membrane and cytoplasm. Therefore, HuR gene is expected to become a new potential target of a clinical treatment for liver cancer.

关 键 词：人抗原R SMMC-7721 实时细胞分析技术 增殖 激光共聚焦系统 

分 类 号：R735.7[医药卫生—肿瘤]