基于多重置换扩增技术的胃癌患者胃内微生物的宏基因组学研究

Metagenomic survey of gastric microbiota in gastric adenocarcinoma patients based on multiple displacement amplification

目的 通过多重置换扩增技术(multiple displacement amplification,MDA)与宏基因组鸟枪法测序相结合的方法研究胃癌患者和健康受试者胃内微生物的组成特征。方法 纳入2017年9月—2017年12月在医院就诊的胃腺癌患者4例,健康受试者4例,收集受试者胃液标本,经过标本预处理、DNA提取、MDA扩增提取的DNA、构建测序文库、高通量测序和生物信息学分析,研究2组受试者胃内微生物的组成特征,寻找与胃癌显著相关的菌种,并评估该方法用于胃内微生物宏基因组学研究的可行性。结果 MDA能显著扩增从胃液样本中提取的微量微生物DNA(207.27±33.17)倍,扩增后的DNA量能满足构建高质量测序文库的要求;总体上看,所有样本共鉴定出131个菌种,胃癌患者胃内菌群多样性显著低于健康受试者( P <0.01, t =4.189);在细菌门水平,受试者胃液菌群主要由变形菌门、拟杆菌门、厚壁菌门、梭杆菌门组成;优势物种的组间差异分析表明,与健康受试者相比,牙髓卟啉单胞菌、口腔链球菌、干燥奈瑟菌在胃癌患者胃液中富集( P <0.05)。结论 MDA能有效用于扩增胃内微量微生物DNA;胃癌患者的胃液菌群构成与健康人存在显著差异;部分口腔及上呼吸道条件致病菌与胃癌显著相关,是潜在的胃癌生物学标志物;通过全基因组扩增和高通量测序相结合的方法可以从菌种甚至菌株的水平上寻找与胃癌发生、发展相关的微生物。

Objective We aim to profile the stomach microbiota of gastric adenocarcinoma patients based on multiple displacement amplification( MDA). Methods Four individuals with gastric adenocarcinoma( GA) and four health controls( HC) were recruited from September 2017 to December 2017. We collected the stomach fluid samples of the participants,then performed the procedures of sample pretreatment,MDA of the extracted DNA,sequencing library preparation,highthroughput sequencing,and bioinformatic analysis to profile the compositional features of microbiota in these samples. Results The genomic DNA extracted from the samples was amplified remarkably by the MDA procedure( 207. 27 ± 33. 17,fold change). This amount of DNA is sufficient for the preparation of high-quality sequencing libraries. The bacterial communities varied among GA and HC group. In general,totally 131 bacterial species were identified in the gastric fluid of all the participants. Significantly decreased microbial diversity was observed in GA group( P < 0. 01,t =4. 189). The bacterial composition was dominated by phyla Proteobacteria and Bacteroidetes,followed by Firmicutes and Fusobacteria in both groups. We compared the dominant species among two groups and found that Porphyromonas endodontalis,Streptococcus oralis and Neisseria sicca were significantly enriched in GA group( P<0. 05). Conclusion MDA can be used to amplify the trace amounts of DNA extracted from human stomach fluid for metagenomic sequencing. The gastric microbiota varies between GA and HC groups at species level. Some opportunistic pathogens colonized in oral cavity and upper respiratory tract were strongly associated with GA and were promising to act as potential biomarkers for GA. The method combining high-throughput sequencing and whole genome amplification method is promising to identify biomarkers of gastric carcinogenesis at species or strains level.