宫颈癌中HPV16整合至3q28位点的鉴定及对该位点基因p63的调控机理研究

Our basic study had found that HPV16 integrated into the human genomic 3q28 region with high frequency in the cervical cancer tissue samples, and the copy number of p63 gene which located at 3q28 region was significantly amplified according to the CGH array in the matched pair study, we speculated that HPV integration may affect the expression of p63 on 3q28. In this project, we will use DIPS-PCR to screen the integration site 3q28 in a large number of HPV16 positive cervical cancer tissue samples, and then the human genomic 3q28 region will be determined as the hotspot site of HPV16 integration combining with the ordinary PCR sequencing, target exons capture sequencing and fluorescence in situ hybridization (FISH). After constructing the zinc finger nuclease (ZFNs) plasmids targeting human genomic 3q28 region specifically and corresponding donors plasmids containing the HPV16 E6E7 sequences, the human normal cervical epithelial cells will be cotransfected with ZFNs and donors plasmids. The effective transfected cells will be selected to further culture using the three-dimensional organotypic raft culture system for imitating the integration of HPV16 in vitro. The interaction of HPV16 E6, E7 oncoproteins and p63 protein which located at 3q28 region will be dected by laser Confocol, CO-IP and GST-pull down, and the motif between HPV16 oncoproteins and p63 will be identified. After that, constructing the specific ZFNs targeting the HPV16 E6, E7 or the motif between them to knockdown the expression of HPV16 E6, E7 or the motif in vitro and in vivo, the purpose is to investigate the role of HPV16 integration into 3q28 region in regulating the expression of p63, and the molecular mechanisms of the occurrence and development of cervical cancer led by HPV16 integrated into the host genome. Finally, we will study the value of HPV16 integration site 3q28 in screening of cervical cancer and assessing of the prognosis of patients.

本项目前期工作发现宫颈癌组织中HPV16整合至3q28位点频率较高，CGH芯片检测配对标本中3q28位点基因p63拷贝数明显扩增。本项目拟采用大样本DIPS-PCR筛选结合普通PCR测序和目标外显子捕获测序以及双色荧光原位杂交FISH检测以鉴定3q28是HPV16的热点整合位点；构建针对3q28位点的锌指核酶及含有HPV序列的供体质粒同时转入正常人宫颈角化上皮细胞，筛选插入成功的细胞进行三维培养以模拟HPV整合；运用激光共聚焦、免疫共沉淀和GST-pull down等技术阐明HPV16 E6、E7蛋白与3q28位点p63蛋白之间的相互作用，并鉴定它们之间作用的功能域；合成针对HPV16 E6、E7及与p63作用的功能域的锌指核酶，在体内外阐明HPV16特异整合至3q28位点对p63的调控作用及在宫颈癌发生发展中的分子机制；初步探索HPV16整合位点3q28在宫颈癌筛查及在预后评估中的价值。

本项目在大样本宫颈癌标本中，筛选HPV16阳性者制备出宫颈癌组织芯片并检测TP63的表达，证实随着CIN的进展，TP63的表达逐渐升高，HPV16整合位点3q28位点处表达的蛋白质TP63的表达与宫颈癌的发生发展具有一定的相关性。采用外显子捕获测序和RNA测序，分析得到HPV16基因组在不同分子水平的整合特征，以及HPV在人类基因组的高频位点。通过引入双色荧光原位杂交（FISH）技术，直观的证明人类基因组3q28位点处有HPV16的序列片段，即HPV16在3q28位点上与人类基因组发生整合。运用锌指核酶技术在宫颈正常原代上皮细胞中成功敲入HPV16基因，检测到HPV16E7和TP63蛋白的上调，证实HPV的整合与TP63蛋白表达的升高有密切关系。在此基础上，利用锌指核酶技术靶向敲除E7，在SiHa细胞中观察到生长增殖以及克隆形成能力受到了明显的抑制。最后，在大样本宫颈癌前病变的TCT标本中进一步验证HPV16的整合特征,从而为宫颈癌及癌前病变的初筛及早期诊断提供新的方法。