B族链球菌基因cpsG抑制人类宫颈癌细胞增殖的研究

Inhibition of human cervical cancer cells proliferation by group B Streptococcus gene CpsG

目的 探讨B族链球菌基因cpsG影响人类宫颈癌细胞增殖的作用及其机制。方法 通过人工合成方法将cpsG的读码框序列(ORF)融合HA标签连接到质粒pLVX-mCMV-ZsGreen的限制性内切酶EcoRⅠ和BamHⅠ之间构建稳定表达cpsG人类宫颈癌细胞系。通过CCK8和集落形成实验进行生长曲线和集落形成能力分析,检测cpsG对人类宫颈癌细胞体外生长能力的影响。通过高通量RNA测序进行基因表达水平的定量分析,分析cpsG对人类宫颈癌细胞转录组学的影响,包括基因本体论(GO)分析、京都基因与基因组百科全书(KEGG)分析和融合基因分析。结果 成功构建稳定表达cpsG人类宫颈癌细胞系之后,CCK8和集落形成实验显示cpsG基因抑制人类宫颈癌细胞体外生长。高通量RNA测序技术发现cpsG能够显著调控人类宫颈癌细胞部分基因的转录能力;GO分析和KEGG分析发现cpsG调节了人类宫颈癌细胞中部分基因的功能和信号通路。主要通过上调DSC3、IGFBP4、PLCL1和下调KRT6A、COL8A1、KRT17等基因的转录,从而抑制G蛋白偶联受体活性、PI3K-Akt信号通路、TNF信号通路、NF-κB信号通路和激活脂肪酸降解、p53信号通路。结论 cpsG可能影响了人类宫颈癌细胞中部分基因的融合,主要影响了B3GAT3-ATP8、LEPROT-LEPR、MRPS6-SON融合基因的形成。

Objective To investigate the effect of group B Streptococcus(GBS) gene cpsG on human cervical cancer cell proliferation and its mechanism. Methods Human cervical cancer cell line with stable expression of cpsG was constructed by synthesizing the cpsG ORF fusing HA tag between EcoRI and BamHI of plasmid plVX-MCMV-ZsGreen. The proliferation and colony formation ability of human cervical cancer cells were analyzed by CCK8 and colony formation test, respectively. Gene expression levels were detected by high-throughput RNA sequencing, the effect of cpsG on human cervical cancer cell transcriptomics was examined with gene ontology(GO) analysis, Kyoto Encyclopedia of Genes and Genomes(KEGG) analysis and fusion gene analysis. Results After successfully constructing a human cervical cancer cell line with stable expression of cpsG, CCK8 and colony formation assays showed that the growth of human cervical cancer cells was inhibited by cpsG in vitro. High-throughput RNA sequencing showed that cpsG could significantly regulate the transcription capacity of some genes in human cervical cancer cells. GO analysis and KEGG analysis found that cpsG regulated the function and signaling pathways of some genes in human cervical cancer cells. Through upregulation of DSC3, IGFBP4, PLCL1 and down-regulation of KRT6 A, COL8 A1, KRT17 gene transcription, the G protein-coupled receptor activity, PI3 K-Akt signaling pathway, TNF signaling pathway, NF-κB signaling pathway were inhibited;and the fatty acid degradation and p53 signaling pathway were activated. Conclusion CpsG may affect the fusion of some genes in human cervical cancer cells, mainly affecting the formation of b3 GAT3-ATP8, leprot-LEPR and MRPS6-son fusion genes, resulting in the inhibition of cell proliferation.