EB病毒再激活与鼻咽粘膜早期癌变关系及其表观遗传学机制

Epstein-Barr virus (EBV) is one of the main risk factors of nasopharyngeal carcinoma(NPC).In our previous study, we have documented that elevated EBV antibodies are strongly associated with an increased risk for NPC and the EBV reactivation levels can be divided into three groups according to the antibody levels against EBV. Combined with other studies, it is hypothesized that repeated EBV reactivation may result in accumulation of genome instability in the infected nasopharyngeal epithelial cells, and push the disrupted cells towards malignancy through epigenetic mechanism. However, there is still lack of the key epidemiological evidence to support this hypothesis. We have built up a NPC screening cohort with 28688 participants in Guangdong province, and in the preliminary experiment, we have also developed the quantitative real-time PCR (Q-PCR) of EBV DNA load and methylation-specific polymerase chain reaction (MS-PCR) for promoter hypermethylation of tumor genes. In this study, we plan to prospectively study EBV reactivation and nasopharyngeal carcinogenesis. One thousand eight hundred participants with different EBV reactivation levels will be selected from the original screening cohort and EBV DNA load, EBV invasion of nasopharyngeal epithelial cells and promoter hypermethylation of oncogenes will be consecutively detected by Q-PCR, Immunohistochemistry and MS-PCR for 3 times.The relationships between EBV reactivation, genes expression and mucosa precancerous lesion will be analyzed using longitudinal data analysis model with at least 4 years of follow-up. This study will provide prospective and population-based evidence for the relation between EBV reactivation and NPC carcinogenesis, which in turn will help development of measures for primary prevention.

EB病毒（EBV）感染是鼻咽癌（NPC）的主要危险因素之一。我们已证实EBV血清抗体与NPC存在正相关关系，并根据抗体浓度将EBV再激活分三个水平。结合其他研究，提出EBV反复再激活并感染鼻咽上皮造成基因组不稳定，通过表观遗传学机制促进癌变完成。但EBV促进鼻咽黏膜早期癌变尚缺乏流行病学证据。我们前期在广东省建立了2.8万人的NPC筛查队列及定量检测鼻咽黏膜脱落细胞中EBV DNA及基因甲基化的方法。因此，本研究拟在NPC筛查队列中分层随机选择1800不同EBV再激活水平的人群，运用定量PCR、免疫组化和MS-PCR，连续3年分别监测鼻咽粘膜脱落细胞的病毒DNA、病毒感染上皮细胞和基因甲基化状态。随访4年，运用纵向数据分析模型分析EBV再激活与基因表达和早期癌变的关系。本研究为阐明EBV再激活促进鼻咽黏膜早期癌变的机制提供以人群为基础的前瞻性研究证据，为制定NPC的预防措施提供参考。

我们首次建立高效、快速的鼻咽脱落细胞中EBV DNA的抽提和荧光定量PCR检测方法并分析了EBV再激活水平与鼻咽脱落细胞中EBV DNA水平的关系，发现EBV再激活水平与鼻咽癌脱落细胞的EBV DNA水平呈正相关关系,并可以作为鼻咽癌的诊断指标，其敏感度特异度均达到90%以上。.我们通过病例对照研究和重复的前瞻性队列研究分别分析了多种生活、环境因素与EBV再激活的关系，发现HBV感染、烟草均可以通过刺激EBV再激活，从而增加鼻咽癌的发病风险。而在分析EBV再激活与鼻咽癌的发病关系时通过多因素分析校正环境因素的影响后，证实了EBV的再激活能增加鼻咽癌的发病风险（HR=5.76），且发现在鼻咽癌患者中EBV处于持续再激活的状态而在正常人中，EBV的激活呈现一过性，阴转率高。. 而在探索EBV再激活水平与鼻咽癌脱落细胞中基因的甲基化关系时，将EBV的再激活水平分为高、中、低3组，并通过鼻咽拭子刷取脱落细胞提取DNA后通过Infinium HumanMethylation450芯片检测相关位点甲基化值然后进行线性回归分析，发现EBV的再激活可引起基因的高甲基化，涉及195个基因，包括13个肿瘤相关通路，包括RASSF1、WNT和TRAF5等，其中WNT通路的多个基因及其位点发生甲基化变化，并有趋势性。. 我们还发现HLA区域的HLA-L、HLA-DQA1、HLA-DPB1、VARS的甲基化状态与EBV的再激活状态呈相关性，提示EBV可能通过DNA甲基化影响机体对EBV感染状态的调控。相关研究正在通过大样本进行验证.. 整个项目在SCI杂志发表论文2篇，申请专利2项，培养研究生4名。