摘要
目的采用CRISPR/Cas9系统敲除肝癌细胞系HepG2的高迁移率蛋白A2(HMGA2)基因,探讨HMGA2敲除对于肝癌细胞生长、增殖、迁移以及侵袭的影响。方法根据GenBank数据库搜索人HMGA2序列,利用在线sgRNA设计软件针对HMGA2的第一外显子以及第二外显子各设计一条sgRNA,构建靶向HMGA2的sgRNA重组敲除载体sgE1和sgE2,将sgRNA载体转染细胞,通过有限稀释法筛选得到HMGA2^-/-细胞株。通过CCK8实验以及平板克隆形成实验检测HMGA2敲除对于HepG2细胞生长以及克隆形成能力的影响;采用Transwell实验检测HMGA2敲除对于HepG2细胞迁移与侵袭的影响。结果相比于野生型,HMGA2^-/-细胞株的生长速率减慢,克隆形成能力(121.83±21.68 vs 59.50±20.68,P<0.01)、迁移(359.67±32.53 vs 245.61±24.23,P<0.05)与侵袭(251.33±43.43 vs 47.00±10.00,P<0.01)能力显著下降。结论HMGA2敲除降低了肝癌细胞HepG2的体外生存能力以及转移能力,HMGA2基因可以作为一个肝癌治疗的潜在靶点。
Objective To knock out the high-mobility group AT-hook 2(HMGA2)gene using the CRISPR/Cas9 system in HepG2 cells and investigate the effect of HMGA2 knockout on the growth,proliferation,migration,and invasion of hepatoma carcinoma cells.Methods The human HMGA2 gene sequence was obtained from GenBank.Two sgRNAs were designed for each of the first and second exons of the HMGA2 gene using online sgRNA design software.The recombinant sgRNA vectors sgE1 and sgE2 were constructed and transfected into HepG2 cells to obtain the HMGA2^-/-cell line.The effects of HMGA2 knockout on the growth and proliferation of HepG2 cells were investigated via CCK8 and clone formation assays,while the migration and invasion abilities were measured using Transwell assays.Results Compared with wild-type HepG2 cells,the knockout cell line showed reduced proliferation,clone formation(121.83±21.68 vs 59.50±20.68,P<0.01),invasion(359.67±32.53 vs 245.61±24.23,P<0.05),and migration(251.33±43.43 vs 47.00±10.00,P<0.01).Conclusions HMGA2 knockout in hepatoma carcinoma cells inhibited both cellular proliferation and tumor metastasis in vitro.Therefore,HMGA2 may constitute a target for hepatoma gene therapy.
作者
徐涛
顾鹏
陈傍柱
叶行
梁春锦
张映辉
顾为望
XU Tao;GU Peng;CHEN Bangzhu;YE Xing;LIANG Chunjin;ZHANG Yinghui;GU Weiwang(Laboratory Animal Center&Institute of Comparative Medicine,Southern Medical University,Guangzhou 510515,China;Songshan Lake Pearl Laboratory Animal Science and Technology Ltd.,Dongguan 523808;School of Biotechnology and Health Sciences,Wuyi University,Jiangmen 529020)
出处
《中国比较医学杂志》
CAS
北大核心
2020年第12期23-29,共7页
Chinese Journal of Comparative Medicine
基金
广东省科技计划项目(2016A020208004)
国家自然科学基金项目(31271042)
国家科技部国际合作项目(2011DFA33290)
江门市创新团队项目(2017TD02)
广州市科技计划项目(201704020012)。
