高危人乳头瘤病毒 (HPV) 是一种致癌病毒，几乎与所有宫颈癌以及越来越多的肛门癌和口腔癌相关。 HPV 的两种致癌蛋白 E6 和 E7 可以使角质形成细胞永生化，对于 HPV 相关的细胞转化至关重要。目前，这些致癌蛋白对宿主蛋白质组的整体调控作用尚不完全清楚，需要进一步探索E6和E7蛋白的功能和致癌机制。我们使用实验室先前建立的平台来构建重组腺病毒质粒表达HPV16 E7基因，进一步构建表达HPV16/18 E6、E7以及E6和E7基因的重组病毒颗粒。这些重组病毒被用来感染C33A细胞以实现HPV16/18 E6/E7基因的持续表达。随后提取总RNA，利用RNA-Seq技术进行转录组测序，鉴定与宫颈癌HPV感染相关的差异表达基因。RNA-Seq分析显示，HPV16/18 E6/E7基因过表达上调GP6、CD36、 HDAC6、ESPL1 和 DNMT3B 属于与宫颈癌相关的差异表达基因 (DEG)。 Spearman 相关分析揭示了 HDAC6 和 DNMT3B 基因与关键通路（包括 DNA 复制、肿瘤增殖特征、G2M 检查点、p53 通路和 PI3K/AKT/mTOR 信号通路）之间存在统计学上显着的相关性。此外，qRT-PCR和Western blot分析表明，HPV16/18 E7均可上调与HPV感染相关宫颈癌相关基因HDAC6和DNMT3B的表达。HPV16/18 E6/E7在细胞中的成功表达表明，重组病毒保留了Ad4的复制和感染能力。此外，表达HPV16/18 E7的重组病毒可以上调参与宫颈癌通路的HDAC6和DNMT3B基因，从而影响细胞周期。此外，HDAC6 和 DNMT3B 正在成为癌症的重要治疗靶点。该研究为进一步探索HPV E6/E7的致癌机制奠定了基础，并可能为HPV相关癌症的治疗提供新的方向。版权所有©2024 Shao，Shah，Su，Li，Huang，Wang，Wang和Wu。

High-risk human papillomavirus (HPV) is a carcinogenic virus associated with nearly all cases of cervical cancer, as well as an increasing number of anal and oral cancers. The two carcinogenic proteins of HPV, E6 and E7, can immortalize keratinocytes and are essential for HPV-related cellular transformation. Currently, the global regulatory effects of these oncogenic proteins on the host proteome are not fully understood, and further exploration of the functions and carcinogenic mechanisms of E6 and E7 proteins is needed.We used a previously established platform in our laboratory for constructing recombinant adenoviral plasmids expressing the HPV16 E7 gene to further construct recombinant virus particles expressing HPV16/18 E6, E7, and both E6 and E7 genes. These recombinant viruses were used to infect C33A cells to achieve sustained expression of the HPV16/18 E6/E7 genes. Subsequently, total RNA was extracted and RNA-Seq technology was employed for transcriptome sequencing to identify differentially expressed genes associated with HPV infection in cervical cancer.RNA-Seq analysis revealed that overexpression of the HPV16/18 E6/E7 genes upregulated GP6, CD36, HDAC6, ESPL1, and DNMT3B among the differentially expressed genes (DEGs) associated with cervical cancer. Spearman correlation analysis revealed a statistically significant correlation between the HDAC6 and DNMT3B genes and key pathways, including DNA replication, tumor proliferation signature, G2M checkpoint, p53 pathways, and PI3K/AKT/mTOR signaling pathways. Further, qRT-PCR and Western blot analyses indicated that both HPV16/18 E7 can upregulate the expression of HDAC6 and DNMT3B, genes associated with HPV infection-related cervical cancer.The successful expression of HPV16/18 E6/E7 in cells indicates that the recombinant viruses retain the replication and infection capabilities of Ad4. Furthermore, the recombinant viruses expressing HPV16/18 E7 can upregulate the HDAC6 and DNMT3B genes involved in cervical cancer pathways, thereby influencing the cell cycle. Additionally, HDAC6 and DNMT3B are emerging as important therapeutic targets for cancer. This study lays the foundation for further exploration of the oncogenic mechanisms of HPV E6/E7 and may provide new directions for the treatment of HPV-related cancers.Copyright © 2024 Shao, Shah, Su, Li, Huang, Wang, Wang and Wu.