OBSCN突变对于肝癌细胞增殖和迁移能力的影响  

A preliminary study on the effect of OBSCN mutations on the proliferation and migration ability of hepatocellular carcinoma

作　　者：付琳琳 王玉 赵雪梅 FU Linlin;WANG Yu;ZHAO Xuemei(School of Pharmacy,Shandong First Medical University&Shandong Academy of Medical Sciences,Taian 271016,China;School of Clinical and Basic Medicine,Shandong First Medical University&Shandong Academy of Medical Sciences,Jinan 250117,China)

机构地区：[1]山东第一医科大学(山东省医学科学院)药学院,山东泰安271016 [2]山东第一医科大学(山东省医学科学院)临床与基础医学院,山东济南250117

出　　处：《山东第一医科大学（山东省医学科学院）学报》2023年第7期485-492,共8页Journal of Shandong First Medical University ＆ Shandong Academy of Medical Sciences

摘　　要：目的构建细胞骨架蛋白OBSCN基因过表达和敲除的稳转细胞系,初步探究OBSCN突变对肝癌细胞增殖和迁移能力的影响。方法qPCR技术确定HepG2肝癌细胞有无OBSCN本底表达,慢病毒感染HepG2细胞构建过表达细胞系;Crispr cas9技术构建敲除细胞系;并利用Western blot技术检测敲除及过表达细胞Obscurin蛋白表达量,CCK-8法和Transwell小室法探究OBSCN突变细胞株的增殖和迁移能力。结果qPCR验证HepG2细胞无OBSCN本底表达,慢病毒感染得到HepG2 H21157过表达稳转细胞系和HepG2 GL119对照空载体稳转细胞系;菌落PCR、质粒PCR以及基因测序结果验证敲除重组质粒reOBSCN构建成功,转染得到OBSCN敲除的HepG2 OBSCN KO稳转细胞系和对照空载体HepG2 ecas稳转细胞系;Western blot技术验证OBSCN敲除及过表达细胞系构建成功;CCK-8法结果显示OBSCN过表达加快了HepG2细胞增殖,差异有统计学意义(P<0.0001),OBSCN基因的敲除可以减慢HepG2细胞增殖,差异有统计学意义(P<0.0001);Transwell小室法得到HepG2 H21157、HepG2 GL119的迁移细胞数分别为150.30±14.95、136.50±15.02,HepG2 OBSCN KO、HepG2 ecas的迁移细胞数分别为112.70±20.30、147.8±11.55,差异有统计学意义(P<0.01)。结论OBSCN基因的过表达会加快HepG2肝癌细胞的增殖和迁移,OBSCN基因的敲除会减慢HepG2肝癌细胞的增殖和迁移。Objective:A stable cell line with overexpression and knockout of the OBSCN gene was constructed,and the effect of OBSCN mutation on the proliferation and migration ability of hepatoma cells was preliminarily explored.Methods:qPCR technology was used to determine whether HepG2 cell had OBSCN background expression,and HepG2 cell was infected by lentivirus to construct an overexpression cell line.Crispr cas9 technology was used to construct knockout cell lines;Western blot technology was used to detect the expression of Obscurin protein in knockout and overexpressed cells,and the proliferation and migration ability of OBSCN mutant cell lines was explored by CCK-8 method and Transwell chamber method.Results:qPCR verified that HepG2 cell had no OBSCN background expression,and HepG2 H21157 overexpression and HepG2 GL119 control empty vector stable cell line were obtained by lentivirus infection.The results of colony PCR,plasmid PCR and gene sequencing verified that the knockout recombinant plasmid reOBSCN was successfully constructed,and the HepG2 OBSCN KO stable cell line and the control empty vector HepG2 ecas stable cell line were transfected to obtain OBSCN knockout.Western blot technology verified the successful construction of OBSCN knockout and overexpression cell lines.The results of CCK-8 method showed that OBSCN overexpression accelerated the proliferation of HepG2 cell(P<0.0001),and knockout of OBSCN gene could slow down the proliferation of HepG2 cell(P<0.0001);the number of migratory cells of HepG2 H21157 and HepG2 GL119 were 150.30±14.95 and 136.50±15.02,respectively,and the number of migratory cells of HepG2 OBSCN KO and HepG2 ecas were 112.70±20.30 and 147.8±11.55,respectively,with statistical differences(P<0.01).Conclusion:Overexpression of OBSCN gene accelerates the proliferation and migration of HepG2 hepatoma cells,and knockout of OBSCN gene slows down the proliferation and migration of HepG2 cell.

关 键 词：HEPG2肝癌细胞 OBSCN 过表达 基因敲除 重组质粒 

分 类 号：R735.7[医药卫生—肿瘤]