Rev-erbβ基因敲除对肝癌HepG2细胞增殖和迁移侵袭的影响  

Rev-erbβ Knock-out Affectsthe Proliferation, Migration and Invasion Ability of Hepatocellular Carcinoma HepG2 Cell Line in vitro

作　　者：陈芳 赵俊丽[2] 夏海滨[2] CHEN Fang;ZHAO J un-li;XIA Hai-bin(School of Modern Agriculture and Biotechnology,Ankang University ,Ankang 725000,China;Gene Therapy Lab,College of Life Sciences ,Shaanxi Normal University,Xi'an 710062 ,China;Shaanan Eco-economy Research Center ,Ankang University ,Ankang 725000,China)

机构地区：[1]安康学院现代农业与生物科技学院,安康725000 [2]陕西师范大学生命科学学院基因治疗研究室,西安710062 [3]安康学院陕南生态经济研究中心,安康725000

出　　处：《四川大学学报（医学版）》2019年第4期520-526,共7页Journal of Sichuan University(Medical Sciences)

基　　金：国家自然科学基金(No.81471772);陕西省教育厅自然科学项目(No 16JK1014);安康市科技局科学技术研究项目(No2017AK05-07);安康学院高层次人才项目(No 2017AYQDZR05)资助

摘　　要：目的探讨核受体Rev-erbβ基因敲除对肝癌HepG2细胞增殖和迁移的影响。方法首先采用对靶向基因进行特定DNA修饰的CRISPR/Cas9基因组编辑技术构建Rev-erbβ基因敲除的HepG2细胞系。用Rev-erbβ打靶载体共转染到HepG2细胞,通过筛选、克隆化构建Rev-erbβ基因敲除HepG2细胞系,并采用PCR、测序和Western blot鉴定。以正常HepG2细胞为对照,采用实时荧光定量PCR(qRT-PCR)检测Rev-erbβ基因敲除对肿瘤迁移、侵袭相关基因表达的影响,采用MTT、细胞划痕、Transwell等实验检测Rev-erbβ基因敲除对HepG2细胞增殖、迁移及侵袭的影响。结果成功构建一株Rev-erbβ基因完全敲除单克隆细胞系(经PCR、测序和Western blot鉴定),命名为HepG2 C5(Rev-erbβ^-/-)。qRT-PCR结果显示,Rev-erbβ基因敲除可以导致基质金属蛋白酶-2,-9基因(MMP2,MMP9)、细胞外基质蛋白1基因(ECM1)表达上调(P均<0.05),细胞黏附相关基因E-钙黏蛋白基因(CDH1)下降(P=0.05);MTT、细胞划痕、Transwell实验显示,并且HepG2 C5比对照细胞有更强的增殖、迁移与侵袭能力(P<0.05)。结论 Rev-erbβ基因敲除可以改变HepG2细胞与迁移、黏附相关基因的表达,进而影响HepG2细胞增殖、迁移与侵袭能力。Objective To investigate the effect of nuclear receptor Rev-erbβ knockout on proliferation and migration ability of human hepatocellular carcinoma cell line HepG2. Methods The Rev-erbβ gene knockout HepG2 cell line was abtained by CRISPR/Cas9 genome editing technique with specific DNA modification of the target gene. The Rev-erbβ gene targeting vectors were co-transfected into HepG2 cells. Through cloning and screening, the Rev-erbβ gene knockout HepG2 cell line was constructed,PCR, sequencing and Western blot methods were carried out for the identification of the Rev-erbβ gene knockout HepG2 cell line. The expression level of tumor migration and invasion-associated gene in Rev-erbβ gene knockout cell was determined by real-time quantitative PCR(qRT-PCR) and was compared with normal cell as control.MTT, cell scratch and Transwell experiments were conducted in order to explore the effect of Rev-erbβ gene on HepG2 cell’s ability of proliferation,migration and invasion. Results A Rev-erbβ gene knockout monoclonal cell line, which was identified by PCR, sequencing and Western blot, was successfully constructed and named HepG2 C5(Rev-erbβ-/-). qRT-PCR results showed that Rev-erbβ knockout resulted in up-regulation of matrix metalloproteinase-2(MMP2), matrix metalloproteinase-9(MMP9) and extracellular matrix protein-1(ECM1) gene expression(P<0.05) and down-regulation of E-cadherin(CDH1) gene expression(P=0.05). Results of MTT, cell scratch and transwell experiments showed that HepG2 C5 had stronger proliferation, migration and invasion ability than control cells(P<0.05).Conclusion Rev-erbβ gene knockout could change the expression of migration and adhesion-associated genes in HepG2 cell, and then affect the proliferation, migration and invasion ability of HepG2 cells.

关 键 词：CRISPR/Cas9 HEPG2细胞 Rev-erbβ 基因敲除 增殖 迁移 

分 类 号：R735.7[医药卫生—肿瘤]