食管癌变过程中微生物宏基因组差异及机制研究

Among the southern coastal area, Chaoshan is a rare high risk region of esophageal cancer（EC）, seriously threatening local people’s lives and health. Aimed to the EC high-risk area of Chaoshan, we systemically finished the epidemiological investigation of EC ,establishment of biological database, screened inflammation rate and microbeo infection in EC high-risk group, performed whole-genome and exome sequencing of canceration samples in different stages, preliminary established the association between infection, inflammation and carcinogenesis; and screened out a group of candidate cancer driver genes. On this foundation, this project intends to take the cross analysis of esophagus microbial genomes and canceration genomes as a breakthrough, conduct 16S r DNA and meta-genomic sequencing of endoscopic specimens (normal- atypical hyperplasia - infiltrating carcinoma) of EC cancerization, further refine and cluster the microbiota, organic combine the researches in carcinogenesis and microbial infection based on the functional analysis of these two genomes, find out the evidences to prove the associations between specific microbial inflection, esophagus inflammation and cancerization, analyze the functional relevancy and molecular mechanisms between microbial inflection and driver mutations by in-vitro cell experiment. The implementation of the project will screen out the EC high-risk microorganisms, explain the molecular mechanisms between the microorganisms and carcinogenesis, and ultimately provide new evidences for prevention and early diagnosis of esophageal cancer in Chaoshan.

广东潮汕为罕见的南方沿海食管癌高发区，严重危害潮汕人民生命与健康。课题组立足于潮汕食管癌高发区，系统地完成食管癌发病流行病学调查、生物标本与信息库构建；高危人群食管微生物感染、炎症筛查；癌变多阶段组织全基因/外显子组测序，初步确立感染、炎症与癌变的关联，并筛选出一组癌变候选驱动基因。在此基础上，本申请拟以食管微生物基因组与癌变基因组交叉分析为突破口，拟对食管癌变过程中的内镜标本（正常-非典型增生-浸润癌）进行16S rDNA与宏基因组测序对微生物进一步细化与分群，在两套基因组功能分析的基础上将癌变与微生物感染有机的结合，确定特定群组微生物感染、食管炎症与癌变共存的证据链；体外细胞实验分析微生物感染与驱动突变的具体功能关联与分子机制。本申请的实现既可筛选出食管癌变高风险微生物，又可解释其与癌变的分子机制，将为潮汕地区食管癌的预防以及早期诊断提供新的依据。

食管癌是世界上恶性肿瘤第六大常见的死亡原因，排在恶性肿瘤发病率第九位。宿主-微生物的相互作用在维持人体健康方面起着重要作用。细菌可以通过产生氧化应激、触发慢性炎症状态或操纵免疫反应等间接作用导致永久性的基因组改变，进而影响肿瘤的的发生发展。我们前期研究发现在不同癌变阶段样本中炎症等级、DNA损伤程度与食管上皮增生程度呈正相关。因此本研究从微生物感染（环境因素）以及基因组遗传变异两个方面对食管癌变不同阶段样本进行研究，以期进一步了解ESCC的癌变及其致癌机制。首先我们对癌变不同阶段样本采用全基因组与外显子测序，并用靶向测序验证，绘制了食管癌变过程的基因组动态变异图谱发现食管非典型增生与浸润癌在突变频率上无显著区别，而单纯增生中突变较少。TP53与CDKN2A为食管鳞癌癌变过程中最显著的早期驱动突变基因，其他驱动变异事件还涉及到了DNA修复、凋亡、细胞增殖、细胞黏附等功能。其次，我们对癌变不同阶段的样本采用16S rRNA扩增子测序和宏基因组测序。发现正常的食管菌群主要以厚壁菌门为主，其优势菌属以链球菌属、嗜血杆菌属、韦永氏球菌属、奈瑟菌属、放线菌属、消化链球菌属、普氏菌属为主。而在癌变过程中食管菌群组成结构发生了改变，尤其是咽峡链球菌、普氏菌属，提示这些菌属可能参与了食管鳞癌癌变的发生发展；同时在食管癌变中食管菌群也影响了宿主功能通路，主要涉及DNA复制与修复通路以及免疫相关通路（NOD样信号通路、抗原处理及呈递、Th17细胞的分化、IL-17信号通路等）。总之，咽峡链球菌、普氏菌属的增加可能是ESCC癌变的驱动因素，而ESCC癌变过程中TP53与CDKN2A最显著的早期驱动突变基因， DNA修复以及免疫相关通路在食管癌变过程中发挥重大作用。