基于比较基因组学探寻潮汕食管鳞癌高危H. pylori基因型及其意义

Esophageal cancer is one of the most common malignancies with poor prognosis in the world, among which, esophageal squamous cell carcinoma (ESCC) remains the predominant esophageal cancer in China. Epidemiological study had shown that Chaoshan littoral region in Guangdong province presented an extremely high incidence and mortality of ESCC. Based on our previous studies, the positive correlation between chronic inflammation-associated genomic instability and esophageal carcinogenesis had been illustrated. In addition, the existence of Helicobacter pylori（Hp）in esophagus had been found in esophageal tissue and the infection rate is high by histopathological study and 16S rDNA sequencing. Meanwhile, esophageal immortallized epithelial cells (NE3) could undergo DNA damage when treated with Hp culture filtrates. Heretofore, the relationship between Hp and ESCC is still under debate internationally. Since Hp strains that carry virulent factors, such as CagA and VacA, had been shown with strong correlation with gastric cancer cell development in vitro and in vivo, we hypothesize that perhaps Hp with specific virulence genes, possibly different from those which cause gastric cancer, could induce the development of ESCC..In this study, we will isolate and culture 50 Hp strains from esophageal or gastric tissue from patients who suffer from ESCC in Chaoshan esophageal cancer high risk littoral region. Meanwhile we will isolate other 50 Hp strains from gastric tissue from patients without esophageal diseases in areas with low esophageal cancer incidence as controls. The genetic material of the isolated Hp strains will be purified and subsequently sequenced for comparative analysis. This study is aimed to determine specific genomic features and virulence genotypes linking to ESCC. The identified virulence genotypes or virulent factors will be verified on esophageal epithelial cell models. By understanding the mechanism of Hp in the development of ESCC and preventing the roles of Hp, we believe the prevalence of ESCC can be controlled and further prevented by precision medicine.

潮汕地区是我国食管鳞状细胞癌（ESCC）高发区之一。我们前期研究发现ESCC的发生发展和食管慢性炎症的程度呈正相关，16S rDNA和组织病理学显示食管组织存在幽门螺杆菌（H. pylori，Hp）感染且感染率较高，Hp培养滤液可造成食管永生化上皮细胞DNA损伤。有关ESCC与Hp的关系一直存在争议，根据胃癌与Hp的前沿研究，我们推测特定毒力型Hp（而非所有Hp）是食管慢性炎症及ESCC发生发展的重要原因。为此，我们将从潮汕地区ESCC患者食管和（或）胃窦组织中分离培养50株Hp菌株，并从ESCC非高发区无食管疾病的上消化道患者胃窦组织中分离培养50株Hp作为对照。以患者病理诊断及病变程度为背景，通过对上述100株Hp进行全基因组测序和比较基因组学分析，探索ESCC高危Hp毒性基因型，并将假说在细胞模型上进行验证，探索其内在分子机制。为精准医学背景下根治Hp及防治ESCC提供依据和新思路。

我国食管鳞癌（ESCC）高发，潮汕是食管癌高发区之一。我们前期研究发现，潮汕地区 ESCC 和慢性炎症有着较明显的相关性，潮汕食管组织存在幽门螺杆菌（Helicobacter pylori，Hp）感染，Hp培养滤液可造成食管永生化上皮细胞DNA损伤。本研究基于分离培养ESCC高发区Hp菌株，进行全基因组测序和公共数据库Hp比较基因组学分析，并从人体病理标本和细胞实验进行验证，探索SCC高危Hp毒性基因型及其分子机制。首先我们成功分离培养了55株潮汕食管鳞癌高发地区Hp菌株并进行全基因组测序，结果发现55株潮汕Hp菌株cagA阳性率100%，毒性最强的ABD型占98.2%；vacA阳性率100%，毒性最强的s1m1占36.4%，毒性次之的s1m2占63.6%，无s2m2；oipA基因阳性且处于开放状态的菌株占89.1%；合作方西澳大利亚大学研究员Alfred Chin Yen Tay提供了低发区115个Hp基因组；我们从NCBI获取全球范围内可查到Hp宿主来源地和人群的Hp基因组数据472个；根据菌株来源的不同地区、不同种族、不同疾病及基因组的质量（contigs<80）进一步筛选，最后筛选出369个（其中95个来自中国，包含本课题组的55株）有代表性的Hp基因组进行比较基因组学分析，系统发育树分析发现分离自潮汕的菌株大部分ABD型与西方ABC型的菌株在进化树上可以明显的区分开来，和其他东亚型的菌株进化距离较近。最后人体病理样本与体外细胞实验以及电镜研究揭示Hp可以诱发食管与食管胃交界上皮慢性炎症，Hp可以黏附甚至插入食管上皮细胞，引起DNA损伤、细胞器变性、细胞坏死。总之，本课题组首次利用全基因组测序与比较基因组学较系统研究了潮汕食管鳞癌高发区Hp毒性基因型，与国际菌株以及国内其他地区菌株进行分析比较，人体病理标本与体外细胞培养结果均提示高毒性Hp感染是食管鳞癌发病的风险因素。