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

作　　者：李萍 汤拓 郑皑雪 张鲁平 王涛 洪鲜 邓志会 LI Ping;TANG Tuo;ZHENG Aixue;ZHANG Luping;WANG Tao;HONG Xian;DENG Zhihui(Institute of Medicine and Pharmacy,Qiqihar Medical University,Qiqihar 161006,China)

机构地区：[1]齐齐哈尔医学院医药科学研究院,黑龙江齐齐哈尔161006

出　　处：《实用医学杂志》2024年第2期133-139,共7页The Journal of Practical Medicine

基　　金：国家自然科学基金项目(编号:32071409);齐齐哈尔市科技局联合引导项目(编号:LSFGG-2022045);齐齐哈尔医学院研究生创新基金项目(编号:QYYCX2022-07)。

摘　　要：目的利用诱导性CRISPR/Cas9技术建立微小染色体维持蛋白2(MCM2)基因敲除的宫颈癌HeLa细胞系,并探究MCM2对DNA复制及复制压力的影响。方法使用诱导性CRISPR/Cas9系统TLCV2,构建MCM2敲除HeLa细胞系;并分为对照组(Control)、敲除1组(KO1)和敲除2组(KO2)。通过Western blot、EdU掺入实验、实时定量PCR、免疫荧光、CCK-8等实验,分析MCM2敲除对DNA复制和羟基脲诱导复制压力的影响。结果经过诱导后CRISPR/Cas9系统有效地敲除MCM2基因,并影响MCM2-7复合物的稳定。与对照细胞相比,MCM2敲除细胞DNA复制能力显著下降,同时Cyclin A1、Cyclin E1和CDK4的mRNA表达水平降低。在DNA复制压力下,MCM2敲除降低了细胞的存活能力、DNA损伤修复能力,并增加基因组的不稳定性。结论MCM2基因敲除降低正常条件下HeLa细胞的DNA复制能力,并降低复制压力后细胞的存活能力。本研究成功地构建了诱导性MCM2基因敲除HeLa细胞系,为进一步研究MCM2基因在宫颈癌中的作用及其生物学功能奠定了基础。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 

分 类 号：R737.33[医药卫生—肿瘤]