HPV-16 E6 mutation and viral integration related host DNA methylation implicate the development and progression of cervical cancer

HPV-16 infection and viral-host integration are the most important risk factors for cervical cancer (CC). The aim of this study is to develop a new molecular strategy integrated both the viral and host genome variations identifying and monitoring CC. A total of 312 methylation and 538 RNA-seq datasets were collected from public databases to identify differentially methylated and expressed genes. HPV associated virus integration sites (VISs) were analysed using the ViMIC database. From September 2020 to August 2021, the 70 HPV-16 positive cases retrospectively collected from multi-centre cohorts were subjected to HPV-16 E6 deep sequencing and PCR-based host gene (ASTN1, DLX1, ITGA4, RXFP3, SOX17, ZNF671) methylation detection. RNAseq and expression validation (NNF671) were performed in C-33A cell line harbouring HPV D32E. Lasso and logistic regression algorithm were used to construct the CC diagnostic model. A positive correlation was observed between the average methylation level of CC patients and their pathological features including tumour stage (<i>p</i> = 0.0077) and HPV subtype (<i>p</i> &lt; 0.001). ZNF671 was identified as a CC-specific methylation marker, with an impressive 93% sensitivity. Both HPV-16 D32E mutation and integration of HPV-16 down-regulated the ZNF671 expression. Finally, a CC diagnostic nomogram was developed by integrating ZNF671 methylation level and HPV E6 mutation feature, yielding an exceptional AUC of 0.997 (95% CI: 0.934–1.000). Our study demonstrated HPV viral mutations are closely related to host gene epigenetic alterations in CC. Integration of the viral and host genetic information might be a new promising strategy for CC screening.

HPV-16感染（HPV-16 infection）与病毒-宿主整合是宫颈癌（cervical cancer, CC）最为关键的危险因素。本研究旨在开发一种整合病毒与宿主基因组变异的新型分子策略，以实现宫颈癌的识别与监测。
本研究从公共数据库中共收集312份甲基化（methylation）数据与538份RNA测序（RNA-seq）数据集，用于筛选差异甲基化基因与差异表达基因。采用ViMIC数据库分析HPV相关病毒整合位点（virus integration sites, VISs）。
2020年9月至2021年8月，研究团队从多中心队列中回顾性收集70例HPV-16阳性病例，对其开展HPV-16 E6深度测序，并采用基于聚合酶链式反应（PCR）的方法检测宿主基因（ASTN1、DLX1、ITGA4、RXFP3、SOX17、ZNF671）的甲基化水平。在携带HPV D32E的C-33A细胞系中完成RNA测序与NNF671的表达验证。
采用Lasso回归与逻辑回归算法构建宫颈癌诊断模型。
研究发现，宫颈癌患者的平均甲基化水平与肿瘤分期（*p*=0.0077）、HPV亚型（*p*<0.001）等病理特征呈显著正相关。ZNF671被鉴定为宫颈癌特异性甲基化标志物，其检测灵敏度高达93%。
HPV-16 D32E突变与HPV-16整合均可下调ZNF671的基因表达。
最终，本研究整合ZNF671甲基化水平与HPV E6突变特征，构建了宫颈癌诊断列线图（nomogram），其曲线下面积（AUC）可达0.997（95%置信区间：0.934~1.000）。
本研究证实，HPV病毒突变与宫颈癌宿主基因的表观遗传改变密切相关。整合病毒与宿主遗传信息有望成为宫颈癌筛查的新型极具应用前景的策略。