基于CRISPR-Cas9系统敲除YTHDF2对子宫颈癌HeLa细胞生物学行为影响的研究

Effect of YTHDF2 Knockout Based on CRISPR-Cas9 System on Biological Be-havior of Cervical Cancer HeLa Cells

目的:探讨使用CRISPR-Cas9系统敲除YTH结构域m6A RNA结合蛋白2(YTHDF2)后对子宫颈癌HeLa细胞生物学行为的影响。方法:培养子宫颈癌HeLa细胞,使用CRISPR-Cas9系统构建YTHDF2敲除细胞系;分为对照组、敲除1组和敲除2组,行克隆形成实验、CCK8实验、裸鼠移植瘤模型分析YTHDF2在体内、体外增殖中的作用,RNA测序探讨作用机制。结果:双sgRNA片段敲除策略成功敲除YTHDF2,敲除1组、敲除2组增殖曲线明显低于对照组,表现为时间依赖性,与对照组相比,敲除1组、敲除2组的HeLa细胞增殖能力显著下降,细胞克隆形成数明显降低,皮下移植瘤质量明显减小,差异均有统计学意义(P<0.001);RNA水平负调控细胞周期G2/M期转换的基因集上调,细胞黏附分子基因集下调,Ras和Wnt信号通路下调,差异均有统计学意义(FDR<0.05)。结论:敲除YTHDF2通过上调负调控细胞周期G2/M期转换的基因,抑制子宫颈癌细胞增殖、克隆形成及皮下成瘤能力。

Objective:To investigate the effect of knocking out YTH N6-methyladenosine RNA binding protein 2(YTHDF2)using the CRISPR-Cas9 system on the biological behavior of HeLa cells in cervical cancer.Meth-ods:HeLa cervical cancer cells were cultured,and YTHDF2 knockout cell line was constructed using the CRISPR-Cas9 system.Divided into control group,knockout group 1 and knockout group 2,the role of YTHDF2 in proliferation in vivo and in vitro was analyzed by cloning formation experiments,CCK8 experiments,and xenograft nude mouse models,and the mechanism of action was explored by RNA sequencing.Results:The double-sgRNA fragment knockout strategy successfully knocked out YTHDF2.The proliferation curves of knockout group 1 and knockout group 2 were significantly lower than that of the control group,showing a time dependence.Com-pared with the control group,the proliferation capacity of HeLa cells in knockout group 1 and knockout group 2 was significantly reduced,the number of cell clones was considerably reduced and the mass of subcutaneous xenograft tumors was remarkably reduced,with a statistically significant difference(P<0.001).RNA levels nega-tively regulate the up-regulation of genes involved in the cell cycle G2/M phase transition,the gene set of cell ad-hesion molecules was down-regulated,Ras and Wnt signaling pathways were down-regulated,with a statistically significant difference(FDR<0.05).Conclusions:Knockout of YTHDF2 inhibits the proliferation,clonal formation and subcutaneous tumorigenesis of cervical cancer cells by upregulating genes that negatively regulate cell cycle G2/M phase transition.