中国女性高危HPV58病毒基因的致癌特点及其机制研究

It is well known that infection with high-risk HPVs is very necessary for cervical carcinogenesis. HPV16 and HPV18 are the most common genotypes, while HPV58 is much more prevalent in east Asia especially China. HPV58 has been paid wide attention recently because of its special global geographical distribution and regional carcinogenicity. However, there are few reports about gene expression and oncogenic characteristics of HPV58. The applicant has finished high throughput analysis of HPV58 including whole genome and transcriptome analysis, and cloning of the full length of viral genome. From pre-experiment results, it seemed that HPV58 was different from HPV16 with respect to oncogenic potentials. It indicated that miRNA (miR-99a) could contribute to the progress of carcinogenesis induced by viral proteins of HPV58, E6 or E7. Thus, we will study on regulation of cellular behaviors by HPV58 E6/E7 which would be different from HPV16/18, and the roles of related molecules including viral genes, miRNAs and cellular genes during HPV58 infection in raft cultures in vitro and cervical lesions in vivo. This study aims to determine characteristics of HPV58 and roles of miRNAs which would facilitate cervical carcinogenesis. From this study it will help us to find effective targets to prevent HPV58 infection and provide scientific evidents to develep new vaccine suitable for east Asian.

高危HPV 感染是宫颈癌发生的必需因素，其中HPV16,18是全球最常见的型别，但在中国等东亚地区HPV58呈较高流行频率。HPV58特殊的地理分布及区域致癌高危性正在受到密切关注。申请人已通过高通量技术及全基因组和转录组分析，完成对HPV58全长基因克隆测序，并通过预实验发现HPV58可能存在与HPV16不同的致癌机制，miR-99a可能参与HPV58 E6/E7的致癌过程。本项目拟在前期研究的基础上，进一步通过功能实验明确HPV58 E6/E7在调控细胞生物学行为中的作用及其与HPV16 E6/E7间的差异；再借助角化上皮细胞的三维器官筏式培养在体外真实再现HPV58的感染过程，并结合宫颈病变组织中病毒癌基因与各种相关分子表达的相关性分析，揭示HPV58致癌作用及特点和miRNA在其中的作用及调控机制，旨在探寻阻遏HPV58致癌的有效靶点和研发适合东亚地区新一代HPV疫苗提供科学依据。

本项目通过设计HPV58 E6和E7特异性多肽片断，将多肽交联至三种不同载体蛋白，免疫兔子，获得免疫血清，取血清做ELISA 确定滴度，分离脾细胞、融合，取细胞上清检测，筛选出阳性克隆，将阳性克隆制备重组抗体并进行测序分析，从而获得特异性HPV58 E6和E7兔单克隆抗体。此外，通过构建pFLAG-CMV5.1-HPV58E6/E7和pFLAG-CMV5.1-HPV16E6/E7真核生物表达质粒，转染293T和U2OS细胞，观察HPV58E6/E7和HPV16E6/E7基因对细胞的周期、凋亡、侵袭能力的差异及下游分子的表达差异，比较两者致癌能力的差异。从我们的结果可以看出，相比于HPV58E6/E7，HPV16E6/E7具有更强的促细胞周期进程、抑制细胞凋亡、促进细胞侵袭的能力，为进一步揭示HPV致癌过程中的重要分子所起的关键作用，且初步阐述了HPV在自然界的分布规律和不同病变中分布特征。为更好的了解HPV的致癌过程和致癌的关键环节，有针对性的提供治疗方案和预防措施打下了基础，也为探索阻断宫颈癌发生的新靶点提供了科学证据，丰富了宫颈癌病因学理论。