实用医学杂志2024，Vol.40Issue(2)：133-139,7.DOI:10.3969/j.issn.1006-5725.2024.02.002
微小染色体维持蛋白2基因诱导性敲除宫颈癌HeLa细胞系的建立及对DNA复制的影响
Generation of MCM2 gene inducible knockout cervical cancer HeLa cells and its effect on DNA replication
摘要
Abstract
Objective To generate minichromosome maintenance protein 2(MCM2)gene knockout cervi-cal cancer HeLa cell lines using inducible CRISPR/Cas9 technology,and to explore the effect of MCM2 on DNA replication and replication stress.Methods The inducible CRISPR/Cas9 system,TLCV2,was used to construct MCM2 knockout HeLa cell lines.And the cell lines were divided into control group(Control),knockout group 1(KO1),and knockout group 2(KO2).Western blot,Edu incorporation experiment,real-time quantitative PCR(qPCR),immunofluorescence and MTT assay were used to analyze the effects of MCM2 knockout on DNA replica-tion and replication stress induced by hydroxyurea.Results The CRISPR/Cas9 system successfully knocked out the MCM2 gene after induction,and MCM2 knockout affected the stability of MCM2-7 complex.Compared with the control cells,MCM2 knockout cells had a dramatic decrease in the capacity of DNA replication,and the mRNA levels of Cyclin A1,Cyclin E1 and CDK4.Under DNA replication stress,MCM2 knockout cells decreased cell viability,DNA damage repair capacity,and increased genomic instability compared with control cells.Conclusion Knockout of MCM2 gene reduces the DNA replication capacity of HeLa cells under normal conditions and cell viability under replication stress.This study successfully generates MCM2 gene inducible knockout HeLa cell lines,laying the foundation for further research on the role and biological function of MCM2 gene in the occurrence and progression of cervical cancer.关键词
微小染色体维持蛋白2/基因敲除/宫颈癌/HeLa细胞/DNA复制Key words
MCM2/gene knockout/cervical cancer/HeLa cell/DNA replication分类
医药卫生引用本文复制引用
李萍,汤拓,郑皑雪,张鲁平,王涛,洪鲜,邓志会..微小染色体维持蛋白2基因诱导性敲除宫颈癌HeLa细胞系的建立及对DNA复制的影响[J].实用医学杂志,2024,40(2):133-139,7.基金项目
国家自然科学基金项目(编号:32071409) （编号:32071409）
齐齐哈尔市科技局联合引导项目(编号:LSFGG-2022045) （编号:LSFGG-2022045）
齐齐哈尔医学院研究生创新基金项目(编号:QYYCX2022-07) （编号:QYYCX2022-07）
