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

doi:10.3969/j.issn.1006-5725.2024.02.002

Abstract

Abstract:

Objective To generate minichromosome maintenance protein 2 （MCM2） gene knockout cervical 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 replication 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.

Key words: MCM2, gene knockout, cervical cancer, HeLa cell, DNA replication

CLC Number:

R737.33

Ping LI,Tuo TANG,Aixue ZHENG,Luping ZHANG,Tao WANG,Xian HONG,Zhihui DENG. Generation of MCM2 gene inducible knockout cervical cancer HeLa cells and its effect on DNA replication[J]. The Journal of Practical Medicine, 2024, 40(2): 133-139.

Figures/Tables 6

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References 25

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sgRNAs	核心寡核苷酸序列 Oligonucleotide sequence（5′→3′）
Scramble-gRNA.F	CACCGGCACTACCAGAGCTAACTCA
Scramble-gRNA.R	AAACTGAGTTAGCTCTGGTAGTGCC
MCM2.KO1-gRNA.F	CACCGGATCATTGCCTCGCCGACGC
MCM2.KO1-gRNA.R	AAACGCGTCGGCGAGGCAATGATCC
MCM2.KO2-gRNA.F	CACCGGAGGATGGAGAGGAGCTCAT
MCM2.KO2-gRNA.R	AAACATGAGCTCCTCTCCATCCTCC

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Generation of MCM2 gene inducible knockout cervical cancer HeLa cells and its effect on DNA replication

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