人乳头瘤病毒16整合感染宫颈上皮细胞系恶性转化后的转录组学分析

Transcriptomic analysis of human papillomavirus type 16 immortalized cervical epithelial cell line H8 after malignant transformation

目的探讨人乳头瘤病毒16(human papillomavirus type 16,HPV16)整合感染宫颈上皮细胞(H8细胞)恶性转化的相关基因、信号通路和可能机制。方法构建H8细胞恶性转化的细胞模型,采用Transwell试验检测恶性转化后H8细胞的细胞侵袭能力、细胞迁移能力的变化,平板克隆形成实验法检测恶性转化后H8细胞克隆形成能力的变化。提取恶性转化的H8细胞、H8细胞的总RNA,采用Illumina Novaseq 6000测序平台对两组细胞进行转录组学测序,鉴定和分析差异表达基因(DEGs),并进行基因本体(gene ontology,GO)功能富集分析和京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)通路富集分析以及蛋白互作网络分析。结果恶性转化的H8细胞侵袭能力[(6503±62.43)个比(298±32.19)个,P<0.001]、迁移能力[(15241±93.83)个比[(7753±76.32)个,P<0.001],克隆形成能力[(428.3±5.03)个比[(281.3±12.10)个,P<0.001]较H8细胞显著升高。转录组学测序结果显示,与H8细胞组相比,恶性转化的H8细胞组共有203个基因存在表达差异,其中98个上调,105个下调。GO富集分析表明DEGs主要参与了细胞内进程、生物调节和代谢过程等生物过程,KEGG分析发现主要与丙氨酸、天冬氨酸和谷氨酸代谢通路、甘氨酸、丝氨酸和苏氨酸代谢通路、P53信号通路、TGF-β信号通路、PI3K-Akt信号通路相关。PPI分析筛选得到DDIT3、TRIB3、ASNS等10个关键基因。结论与H8细胞相比,恶性转化的H8细胞在转录水平出现大量差异表达基因和通路,可进一步为恶性转化致癌的机制提供新思路,以及为预防恶性转化寻找新靶点。

Objective To investigate the related genes,signaling pathways and possible mechanisms of malignant transformation of human papillomavirus type 16(HPV-16)immortalized cervical epithelial cell line H8.Methods The malignant transformed H8 cell model was constructed,and the changes of cell invasion ability and cell migration ability of H8 cells after malignant transformation were detected by Transwell assay,and the changes of clone formation ability of H8 cells after malignant transformation were detected by plate clone formation assay.Total RNA was extracted from malignant transformed H8 cells and H8 cells,and the two groups of cells were sequenced by transcriptome using Illumina novaseq 6000 sequencing platform,differentially expressed genes(DEGs)were identified and analyzed,and Gene Ontology(GO)function enrichment analysis,Kyoto Encyclopedia of genes and genomes(KEGG)pathway enrichment analysis and protein-protein interaction were performed.Results The invasion ability,migration ability and clone formation ability of malignant transformed H8 cells significantly increased as compared to H8 cells.A total of 203 differentially expressed genes were identified in H8 cells before and after malignant transformation,of which 98 were up-regulated and 105 down-regulated.GO enrichment analysis showed that DEGs were mainly involved in biological processes such as cellular processes,biological regulation,and metabolic processes.KEGG pathway enrichment analysis showed that DEGs were mainly enriched in alanine,aspartate and glutamate metabolic pathway,glycine,serine and threonine metabolism pathway,p53 signaling pathway and TGF-βsignaling pathway,PI3K-Akt signaling pathway.PPI analysis screened 10 hub genes including DDIT3,TRIB3 and ASNS.Conclusions Compared with H8 cells,malignant transformed H8 cells have a large number of differentially expressed genes and pathways at the transcriptional level,which could further provide new ideas for the mechanism of malignant transformation and carcinogenesis as well as finding new targets for the prevention of malignant transformation.