宫颈上皮内瘤变中HPV于12q15区域整合导致基因组结构改变及其基因调控的机制研究

The integration of high-risk human papillomavirus (HPV) into the host genome is a key event in the progression of cervical intraepithelial neoplasia (CIN), and cause changes in the expression of adjacent genes. Our previous study showed that 12q15 is a genomic region of high frequency of HPV integration and the expression of HMGA2 in this region is increased. Analysis of Hi-C sequencing and ChIP-seq of CIN cell line S12 showed that, there were three-dimensional structure and epigenetics changes in the loci of HPV integration. Therefore, we hypothesized that HPV integration in the 12q15 region can change the three-dimensional structure of the genome, resulting in changes in HMGA2 gene expression. Based on the previous study, we choose HPV integrated into 12q15 region as a cut-point. 3C technology is employed to validate the key interaction of chromatin. We also study the epigenetic changes of the region caused by changes of the three-dimensional structure. We will investigate the changes of HMGA2 and related signal pathways caused by HPV integration into 12q15 and their mechanism of tumorigenesis. Furthermore, we would employ the CRISPR technology to knock out HPV sequence and study the reversal phenotype of tumor cell, the key interaction of chromatin and gene expression of HMGA2 and its related signal pathways. This study would provide some new methods for the precise screening of CIN.

HR-HPV整合到宿主基因组是宫颈上皮内瘤变（cervical intraepithelial neoplasia，CIN）进展的重要事件，整合导致临近基因表达异常。前期研究发现，基因组12q15区域是HPV高频整合位点，该区域HMGA2基因表达升高。通过对S12细胞Hi-C测序及ChIP-seq结果分析得出，整合位点附近存在着基因组三维结构及表观遗传学的改变。因此我们推测：HPV整合在12q15区域后可以改变该区域基因组三维结构，从而上调该区域的HMGA2基因表达。基于前期研究，本项目选择HPV整合至12q15这一关键事件作为切入点，拟采3C技术对关键染色质相互作用进行验证，并结合该区域表观遗传学改变，探索HPV整合至12q15对HMGA2基因表达的影响。最后，通过CRISPR技术敲除HPV，对上述结果进行反向验证。本项目为CIN的精准防治提供新的筛选靶点。

前期研究发现，基因组12q15区域是HR-HPV高频整合位点，该区域HMGA2基因表达升高。本课题通过收集CIN标本证明，HMGA2是HR-HPV的高频整合位点，并且随着疾病进展，HPV整合等频率逐步增加。选择HR-HP于HMGA2位点整合的S12 细胞进行研究，联合Hi-C、ChIP-seq及 RNA-seq多组学方法，对S12细胞及对照细胞进行研究。证明了HPV整合能够通过改变染色质TAD，从而导致基因表达的异常。通过对临床样本中HPV整合以及HMGA2拷贝数以及HMGA2蛋白表达情况进行深入分析，发现从CIN进展到宫颈癌后，其HPV整合的频率明显升高。但是HMGA2的拷贝数在这个过程中并没有明显变化。从正常宫颈上皮，进展到CIN再到宫颈癌，HMGA2蛋白的表达升高明显，提示其在宫颈病变进展中可能起到重要作用。HMGA2表达量可以作为CIN进展成为宫颈癌预测的重要生物学指标。通过对HMGA2的生物学作用进行深入研究，发现抑制HMGA2表达能够抑制宫颈癌细胞系的凋亡。本研究揭示了HMGA2在宫颈病变进展中起到重要的作用，并且提示其作为宫颈病变治疗的靶点的潜力。