研究背景及目的：宫颈癌是严重危害女性健康的常见恶性肿瘤，高危型人乳头瘤病毒（High-risk human papillomavirus, HR-HPV）持续感染是其主要致病因素。目前已有疫苗可预防HPV16、18、31、33、45、52和58型感染，但并未覆盖所有高危型别，仍有部分宫颈癌由非疫苗HR-HPV所引起。随着疫苗接种的普及，非疫苗HR-HPV在宫颈癌的相对构成比例呈现逐渐上升趋势，因此需加强对其病原学及致癌机制的研究。HPV39作为非疫苗HR-HPV之一，与HPV18同属α-7进化分支，理论上应具有相似的分子特征与致病机制，但流行病学调查数据显示，HPV18的传播能力和致癌性均显著高于HPV39，造成这些差异的潜在机制尚不清楚。此外，现有HR-HPV致癌机制研究主要集中于编码区如E6、E7等，而对上游调控区（Upstream Regulatory Region, URR）关注较少。URR虽不直接编码蛋白，但其在病毒转录调控、与宿主互作及致癌信号通路激活等方面发挥重要作用。基于以上背景，本研究拟从病毒基因组遗传变异特征、免疫表位和URR调控功能等方面对HPV39和HPV18进行系统比较，评估HPV39是否具备与HPV18相似的致癌能力，探讨在疫苗免疫选择压力下，HPV39是否有可能在未来成为新的优势型别并引发更多宫颈癌病例。

方法：本研究收集HPV39阳性临床样本，通过扩增和测序获得HPV39全长序列，并结合NCBI数据库中已报道的HPV18及39全长序列构建进化树并划分进化分支，利用MEGA软件分析和比较HPV18和HPV39基因多态性；利用生物信息学工具IEDB及Vaxijen软件预测并筛选HPV18和HPV39早期蛋白的T细胞抗原表位；进一步针对病毒基因组中突变率较高的URR片段，通过质粒稳定转染构建HPV18和HPV39 URR DNA转染的宫颈癌细胞模型，并利用转录组测序和差异表达分析，结合生物信息学工具，筛选出URR可能影响的分子信号通路并进行验证。

结果：从临床样本中成功获得50条HPV39突变株全长序列，从NCBI数据库中分别获得178条和38条HPV18和HPV39全长序列。经删除重复，最终共纳入145条HPV18和84条HPV39全长序列进行分析。进化树显示HPV18可分为三个主要进化分支（A、B、C），而HPV39分为两个（A、B两系）。基因多态性分析发现HPV18全长序列的碱基突变率（8.06%）与氨基酸突变率（11.71%）均显著高于HPV39（分别为4.92%和6.43%）；在URR区域，HPV18的突变率（10.49%）也显著高于HPV39（7.70%），并表现出更高的序列间差异。经预测和筛选发现，HPV18与HPV39的E1、E2、E6、E7蛋白的T细胞抗原表位，在HLA-I类及II类表位的总体数量和保守性方面相当。对二者表位的同源性进行比较，发现50%以上的表位同源性超70%，其中E2表位NTTPIIHLK同源性达100%。转染HPV18和HPV39 URR DNA均可显著提高HPV阴性宫颈癌细胞C33A的生长速度和细胞活力，促进细胞的迁移和侵袭；转录组测序和差异基因富集分析结果表明，URR可对C33A细胞产生多方面的影响，其中包括对Wnt信号转导通路的显著激活作用。通过实验验证发现，HPV18和HPV39 URR可促进β-catenin核转位及TCF/LEF转录活性，从而激活下游与细胞周期有关的靶基因，推动细胞向更恶性的肿瘤表型转化。但尚未明确发现HPV18与HPV39 URR对宫颈癌细胞系影响具有明显差异。

结论：本论文系统比较了HPV18和HPV39的进化和变异特征，并对两者URR在宫颈癌发生发展中的致癌机制进行了探索。研究结果表明HPV18在碱基和氨基酸层面的变异程度均高于HPV39，可能具有更强的遗传进化适应性。两者的URR本身即可调控宿主细胞的生物学行为，在促进宫颈癌细胞生长和恶性转化方面发挥了重要作用。随着疫苗的广泛应用，HPV39有可能会成为新的优势型别，因此，未来需关注HPV39等非疫苗HR-HPV的致癌性，并进一步深入探讨URR在宿主细胞内的具体作用机制，以期为宫颈癌的防治提供更全面的理论依据。

Background and objective: Cervical cancer is a common malignancy that seriously threatens women's health, with persistent infection by high-risk human papillomavirus (HR-HPV) as its primary cause. Although current vaccines can prevent infection with HPV types 16, 18, 31, 33, 45, 52, and 58, they do not cover all high-risk types, and a proportion of cervical cancer cases are still caused by non-vaccine HR-HPV types. As vaccination becomes widespread, the relative proportion of non-vaccine HR-HPV in cervical cancer cases is gradually increasing, highlighting the need for further investigation into their pathogenicity and mechanisms of carcinogenesis. HPV39, a non-vaccine HR-HPV type in the α-7 phylogenetic group alongside HPV18, is presumed to share similar molecular features. However, epidemiological studies show that HPV18 exhibits significantly higher transmissibility and oncogenicity than HPV39, and the underlying reasons for these differences remain unclear. Furthermore, most existing studies on HR-HPV carcinogenesis have focused on coding regions such as E6 and E7, while the upstream regulatory region (URR) has received comparatively less attention. Although the URR does not encode proteins directly, it plays a critical role in regulating viral transcription, interacting with host factors, and activating oncogenic signaling pathways. Given this context, this thesis aims to systematically compare HPV18 and HPV39 in terms of their genomic variation, T-cell epitopes, and URR regulatory functions, in order to evaluate whether HPV39 possesses oncogenic potential comparable to HPV18 and to explore whether HPV39 could emerge as a new predominant genotype under vaccine-induced immune selection pressure and potentially lead to more cervical cancer cases in the future.

Methods: In this study, we collected HPV39-positive clinical samples and obtained full-length HPV39 genome sequences through PCR amplification and sequencing. These sequences, along with published full-length sequences of HPV18 and HPV39 from the NCBI database, were used to construct phylogenetic trees. Genetic polymorphisms of HPV18 and HPV39 were analyzed and compared using MEGA software. To assess immunogenic potential, T cell antigenic epitopes within early viral proteins (E1, E2, E6, and E7) were predicted and screened using the IEDB and Vaxijen bioinformatics tools. Furthermore, focusing on the highly mutated URR of the viral genome, we constructed stable cervical cancer cell lines (C33A) transfected with HPV18 or HPV39 URR DNA via plasmid transfection. mRNA sequencing and differential gene enrichment analysis were performed, and bioinformatics tools were used to identify molecular signaling pathways potentially affected by the URR, which were subsequently validated through molecular biology experiments.

Results: We successfully obtained 50 full-length sequences of HPV39 mutant strains from clinical samples, and 178 and 38 full-length sequences of HPV18 and HPV39 from the NCBI database, respectively. After deletion of duplicates, a total of 145 HPV18 and 84 HPV39 full-length sequences were finally included for analysis. The evolutionary tree showed that HPV18 could be divided into three major evolutionary branches (A, B and C), while HPV39 was divided into two (A and B lineages). Gene polymorphism analysis revealed that the base mutation rate (8.06%) and amino acid mutation rate (11.71%) of the full-length sequence of HPV18 were significantly higher than that of HPV39 (4.92% and 6.43%, respectively); in the URR region, the mutation rate of HPV18 (10.49%) was also significantly higher than that of HPV39 (7.70%), and showed higher inter-sequence variation. Prediction and screening revealed that the T-cell antigenic epitopes of the E1, E2, E6, and E7 proteins of HPV18 and HPV39 were comparable in terms of the overall number and conservatism of HLA-class I and class II epitopes. Comparison of the homology of the two epitopes revealed that more than 50% of the epitopes had more than 70% homology, with 100% NTTPIIHLK homology for the E2 epitope. Transfection of both HPV18 and HPV39 URR DNA significantly increased the growth rate and cell viability of HPV-negative cervical cancer cells C33A, and promoted cell migration and invasion; the results of transcriptome sequencing and differential gene enrichment analyses indicated that the URR could have a multifaceted effect on the C33A cells, which included a significant activation of the Wnt signal transduction pathway. Experimental validation revealed that HPV18 and HPV39 URR could promote β-catenin nuclear translocation and TCF/LEF transcriptional activity, thereby activating downstream cell cycle-related target genes and driving the transformation of cells to a more malignant tumour phenotype. However, no significant difference in the effects of HPV18 and HPV39 URR on cervical cancer cell lines has been clearly identified.

Conclusions: In this thesis, we systematically compared the evolutionary and mutational characteristics of HPV18 and HPV39, and explored the oncogenic mechanisms of both URRs in cervical carcinogenesis. The results suggest that HPV18 has a higher degree of variation than HPV39 at both the base and amino acid levels, and may have stronger genetic evolutionary adaptations. Both URRs can themselves regulate the biological behaviour of host cells and play an important role in promoting cervical cancer cell growth and malignant transformation. With the wide application of vaccines, HPV39 is likely to become a new dominant type. Therefore, in the future, it is necessary to pay attention to the oncogenic potential of non-vaccine HR-HPVs, such as HPV39, and to further explore in depth the specific mechanism of action of the URRs in host cells, with a view to providing a more comprehensive theoretical basis for the prevention and treatment of cervical cancer.