HPV18非随机整合在宫颈上皮细胞恶性转化中的作用及机制研究

High-risk human papillomavirus (HPV) integration into human genome is a key event in the progression of major cervical diseases. Previous studies have proved that HPV integration is observed in all HPV18-related cervical cancers, and cancers with HPV18 integration are more aggressive which threaten women’ life and health seriously. Our preliminary studies have revealed the non-random integration of HPV in human genome, and plotted the landscape of HPV18 integration loci, which confirmed that 8q24 site is the highest frequent locus of HPV18 integration. On the basis of the reliable preliminary studies, the molecular mechanism of micro-homology repair pathway mediated non-random integration will be clarified by establishing a cellular model of HPV18 non-random integration into the 8q24 locus. The three-dimensional draft culture will be applied to simulate the malignant transformation process of cervical epithelial cell induced by HPV18 non-random integration, and the effect of HPV18 non-random integration on the three-dimensional structure of host chromatin and remote interaction of chromatin will be explored by using three dimensional genomics. The animal model of non-random integration of HPV18 into the 8q24 locus will be established, which will further illustrate the molecular mechanism of malignant phenotype changes. The foundation will be laid at illuminating cervical pathogenesis deeply and establishing early diagnosis of HPV18 positive cervical cancer and the precise treatment targeted HPV18 integration sequence.

高危型人乳头状瘤病毒（HPV）整合至宿主基因组是宫颈重大疾病发生的关键事件。已证实HPV18感染的宫颈癌组织中病毒基因的整合率为100%，且此类肿瘤恶性程度更高，严重威胁妇女生命健康。我们前期研究揭示了HPV在人类基因组中的非随机整合规律，绘制了HPV18整合模式图，证实8q24是HPV18整合频率最高的位点。本项目在前期笃实的研究基础上，建立HPV18非随机整合至8q24位点的细胞模型，阐明HPV启动微同源修复介导非随机整合的分子基础；三维培养模拟HPV18非随机整合诱导宫颈上皮细胞的恶变过程,运用三维基因组学探索HPV18非随机整合导致宿主染色质三维结构改变及染色质的远程交互作用;建立HPV18非随机整合至8q24位点的动物模型，多组学分析进一步阐明恶性表型改变的分子机制，为深入明确宫颈癌的发病机制、建立HPV18阳性宫颈癌早期诊断及靶向HPV18整合序列的精准治疗奠定理论基础。

本项目基于CRISPR-Cas9介导的同源重组末端修复机制，本项目通过构建特异性靶向8q24位点的CRISPR/Cas9质粒以及HPV DNA donor质粒,在HaCaT细胞中进行共转染，通过嘌呤霉素以及EGFP荧光双重筛选得到HPV DNA在8q24位点特异性整合的单克隆细胞。RT-qPCR方法检测到了E6和E7 mRNA在单克隆细胞中的高表达，同时检测了下游相关基因的表达改变。细胞学实验证实了HPV DNA在8q24位点的敲入诱导了宿主细胞生物学行为的恶性转化。上述实验充分验证了CRISPR-Cas9技术实现细胞内定点基因敲入的可能性，我们构建了13q22位点特异性HPV整合单克隆细胞模型。体外实验证实了HPV定点整合细胞相关基因表达的改变以及恶性生物学行为进展。Hi-C，ChIP-seq以及RNA-seq多组学分析发现了HPV整合对整合位点附近染色质A/B compartment、TAD结构以及基因表达调控的影响。