ACACB基因DNA甲基化/过表达的肝癌细胞株MHCC97H增殖侵袭迁移能力变化观察  

Proliferation,invasion and migration abilities of MHCC97H liver cancer cells with DNA methylation or over-expression of ACACB gene

作　　者：伍柳玉 胡艳玲 刘顺[1] 秦小玲 梁秋丽 刘美良 杨钰 曾小云[1] WU Liuyu;HU Yanling;LIU Shun;QIN Xiaoling;LIANG Qiuli;LIU Meiliang;YANG Yu;ZENG Xiaoyun(School of Public Health,Guangxi Medical University,Nanning 530199,China;不详)

机构地区：[1]广西医科大学公共卫生学院,南宁530199 [2]广西医科大学生命科学研究院 [3]广西中医药大学公共与管理学院

出　　处：《山东医药》2021年第33期1-5,共5页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(81760611)。

摘　　要：目的观察抑制乙酰辅酶A羧化酶(ACACB)基因DNA甲基化/感染ACACB基因过表达慢病毒dCAS9-VP64的肝癌细胞株MHCC97H增殖侵袭迁移能力变化情况。方法采用荧光定量PCR法检测肝癌细胞株SMMC7721、Hep3B、MHCC97H、MHCC97L及正常肝细胞株LO2中ACACB mRNA,采用焦磷酸测序实验测算SMMC7721、Hep3B、MHCC97H、MHCC97L、LO2细胞中ACACB基因cg06131338位点甲基化率。选取甲基化率相对较高的肝癌细胞株分为5-Aza-CdR组和对照组、ACACB过表达组和阴性表达组,5-Aza-CdR组加入DNA甲基转移酶抑制剂5-Aza-CdR,对照组加入二甲基亚砜,ACACB过表达组感染ACACB过表达慢病毒dCAS9-VP64,阴性表达组感染阴性对照慢病毒。采用焦磷酸测序实验测算5-Aza-CdR组和对照组细胞中ACACB基因cg06131338位点甲基化率,采用荧光定量PCR法检测4组细胞中ACACB mRNA,采用CCK8法观察各组细胞增殖能力(以OD值表示),采用侵袭小室观察各组细胞侵袭能力(以侵袭细胞数表示),采用迁移小室观察各组细胞迁移能力(以迁移细胞数表示)。结果肝癌细胞株SMMC7721、Hep3B、MHCC97H、MHCC97L中,ACACB mRNA的相对表达量均低于正常肝细胞株LO2(P均<0.01),ACACB基因cg06131338位点甲基化率均高于正常肝细胞株LO2(P均<0.01)。5-Aza-CdR组细胞中ACACB基因cg06131338位点甲基化率低于对照组(P<0.01),ACACB mRNA相对表达量高于对照组(P<0.01)。ACACB过表达组细胞中ACACB mRNA的相对表达量高于阴性对照组(P<0.01)。5-Aza-CdR组细胞增殖、侵袭和迁移能力均低于对照组(P均<0.01),ACACB过表达组细胞增殖、侵袭和迁移能力均低于阴性对照组(P均<0.01)。结论在肝癌细胞株SMMC7721、Hep3B、MHCC97H、MHCC97L中,ACACB mRNA表达降低、ACACB基因cg06131338位点甲基化率升高,抑制cg06131338位点的甲基化可促进ACACB mRNA表达。抑制cg06131338位点的甲基化或过表达ACACB均可降低MHCC97H细胞增殖、侵袭和迁移能力。Objective To observe the changes of the proliferation,invasion and migration abilities of liver cancer cell line MHCC97H with inhibition of the DNA methylation of ACACB gene or infection of ACACB gene over-expression lentivirus dCAS9-VP64.Methods We used the qPCR to detect ACACB mRNA in the liver cancer cell lines SMMC7721,Hep3B,MHCC97H,MHCC97L and normal liver cell line LO2.Pyrosequencing experiments were used to calculate the methylation rate of ACACB gene cg06131338 site in SMMC7721,Hep3B,MHCC97H,MHCC97L,and LO2 cells.The liver cancer cell lines with relatively high methylation rate were divided into the 5-Aza-CdR group and control group,ACACB over-expression group and negative expression group.The cells in the 5-Aza-CdR group were added with 5-Aza-CdR,and the control group with DMSO.The cells in the ACACB over-expression group were infected with the ACACB overexpression lentivirus dCAS9-VP64,and the negative expression group with the negative control lentivirus.Pyrosequencing experiments were used to calculate the methylation rate of ACACB gene cg06131338 site in the 5-Aza-CdR group and control group.The qPCR method was used to detect ACACB mRNA in the four groups,CCK8 method to observe cell proliferation ability of each group(expressed by OD value),invasion chamber to observe the invasion ability of each group(expressed by the number of invasion cells),and the migration chamber to observe the invasion ability of cells in each group(indicated by the number of migration cells).Results The relative expression of ACACB mRNA in the liver cancer cell lines SMMC7721,Hep3B,MHCC97H,and MHCC97L was lower than that of normal liver cell line LO2,and the methylation rate of ACACB gene cg06131338 site was higher than that of normal liver cell line LO2(all P<0.01).The methylation rate of ACACB gene cg06131338 site in the 5-Aza-CdR group was lower than that in the control group,and the relative expression of ACACB mRNA was higher than that in the control group(both P<0.01).The relative expression of ACACB mRNA in the ACACB

关 键 词：乙酰辅酶A羧化酶基因 肝细胞癌 DNA甲基化 细胞增殖 细胞侵袭 细胞迁移 

分 类 号：R735.7[医药卫生—肿瘤]