| Background: In the digestive tract system,the stomach is a special ecological region.The high acid environment caused by gastric acid secretion has formed unique ecological environment.In recent years,with the development of high-throughput sequencing and bioinformatics analysis,the understanding of human relative microbial community has become more comprehensive,compensating for the limitations of the research heavily relied on the microbial cultivation technology.The study of microbiota has expanded from metagenomic to metabonomics.Microbial infection is one of the most important pathogenesis of gastric cancer(GC),and Helicobacter pylori(H.pylori)has always been the focus of research in the past decades.At present,it is generally believed that chronic H.pylori infection leads to inflammation,epithelial structure and dysfunction,which are closely related to atrophic gastritis,dysplasia and other precancerous gastric lesions.However,only 1-3% of patients infected with H.pylori developed into GC,and eradication of H.pylori could not completely prevent the occurrence of GC.In addition,colonization of H.pylori gradually decreases or even disappears in the process of superficial gastritis-atrophic gastritis-intestinal metaplasia-GC,so H.pylori may only play a limited role in the later stage of malignant tumor development.At present,studies have confirmed that there were a large number of microbial species other than H.pylori in the stomach,which are closely related to benign and malignant gastric lesions.Animal experiments have shown that non-H.pylori microbiota play an important role in the development of GC in mice.In human research,H.pylori chronic infection or long-term use of certain drugs,such as omeprazole,could decrease gastric acid secretion and increase gastric pH value,which would be kind for other kinds of microorganisms to colonize and grow.As a result,the overgrowth of gastric diseases related microbes broke the balance of local ecological environment of the gastric mucosa,and the accumulation of bacterial metabolites,such as nitrite and N-nitrosamines,were increased.All the studies above have confirmed that other microbial species other than H.pylori may play potentially pathogenic roles in the occurrence and development of GC.So far,our understanding of the complex gastric microbiota and gastric diseases is still limited.At present,a small number of studies have explored the differences in the composition and function of gastric microbiome between patients with gastric cancer and non-gastric cancer control group.However,few studies focus on microbiota in the tumor microenvironment,as well as its association with clinical features and clinical outcome of GC patients.Except for H.pylori,the gastric microbiota species that play important roles in the process of GC development have not been defined clearly yet.This study described the different distribution,fuction and ecological network of the mucosa-associated microbiota in gastric cancerous and non-cancerousthe tissues,and the correlations between mucosa-associated microbiota and gastric cancer risk factors,clinical pathological parameters,prognosis,and serum gastric function.Our work would help to explore the role of mucosa-associated microbiota in carcinogenesis and in the maintenance of the local microenvironment in GC patients.Methods: This study was approved by the Medical Ethics Review Committee of the First Affiliated Hospital of China Medical University and Informed consent was signed for patients with gastric cancer.According to strict inclusion and exclusion criteria,62 GC patients were selected from the cases receiving subtotal gastrectomy in the First Affiliated Hospital of China Medical University from June 2012 to June 2014.A total of 124 samples were collected from gastric mucosal tissues at tumor and non-tumor sites(at least 5cm away from the tumor site).Genomic DNA was extracted from gastric mucosal tissue samples,and 16 S rRNA gene was amplified using the universal primer V4-V5 regions.FLASH was used to combine paired readings from the original DNA fragment.Assign sequencing readings to each sample based on a unique barcode.UPARSE pipeline was used for sequence clustering analysis.Filter readings of at least 97% identity are clustered into the same operational classification unit(OTU).Analysis of α diversity included community richness and diversity.Among them,the community richness was assessed by Chao1 and ACE indices,diversity by Shannon index and PD-whole-tree.The Weighted Unifrac distance matrix and Bray-Curtis were used to evaluate the β diversity,which could be visualized by means of principal coordinate analysis(PCoA)and non-metric multidimensional scale(NMDS)diagrams.Similarity(ANOSIM)and PERMANOVA(ADONIS)were used to evaluate statistical differences in diversity.The ecological network was constructed through Spearman correlation analysis and Cytoscape was used to visualize.PICRUSt was used to predict the function of mucosa-associated microbiota.The accuracy of the predicted genome was assessed by the closest sequencing taxonomic group index(NSTI).Functional pathway enrichment was analyzed based on KEGG database.Furthermore,the predicted functional genes were classified as direct homologous group(COG)and compared between cancer and non-cancer groups by STAMP to identify the gene functions that distinguish bacterial communities in the two group comparisons.Non-parametric t test was used to determine the statistical differences between COG and Benjamini and Hochberg(BH)method for correction of error-discovery rate.Linear discriminant analysis(LEfSe)algorithm and Wilcoxon rank test were used to identify the differences in microbial taxonomic groups and functions between the two groups.Epidemiological information including age,sex,family history of GC,smoking and drinking status were collected from all the enrolled patients by questionnaire.Smoking was defined as having at least one cigarette a day for at least one year.A history of alcohol consumption was defined as consuming an average of at least 50 grams of alcohol per day for at least one year.Detailed clinicopathological information of patients with gastric cancer was collected according to medical records and surgical records,including tumor general classification(Borrmann classification),Lauren classification,tumor grade,infiltration depth,infiltration state of peritumoral lymphocytes,lymph node metastasis state,and tumor-lymph node-metastasis(TNM)system stage.Outcome variables monitored included total survival(OS)and date of death counted during the follow-up period ending August 25,2018.Enzyme-linked immunosorbent(ELISA)detection in the group of patients with gastric cancer serological gastric function indexes,including pepsinogen Ⅰ(PG Ⅰ),PG Ⅱ,PG Ⅰ/PG Ⅱ ratio(PGR)and gastrin 17(G17).The analysis of the correlation between microbiota and gastric cancer risk factors: categorical variables using Wilcoxon rank,continuous variables using Spearman correlation test.Wilcoxon signed rank was used for comparing microbiota diversity between groups with different clinical pathological parameters.The Mann-Whitney U test and Kruskal Wallis H test were used to analyze the associaotion between differential abundant microbiota and clinical pathological parameters.Kaplan-meier method was used to analyze patient survival time,and log-rank test was used to analyze the differences between groups.Canonical correlation analysis(CCA)and Pearson correlation analysis were used to evaluate the correlation between serum gastric function indicators and gastric microbiota.SPSS 20.0(SPSS Inc.Chicago IL)software was used for statistical analysis.P <0.05 was defined as statistically significant.Results: 1.Compared with non-cancerous tissues,the microbial richness and diversity in cancerous tissues increased,and the ecological network showed increased complexity.2.The bacterial taxa enriched in the cancer samples were predominantly represented by oral bacteria(such as Peptostreptococcus,Streptococcus,and Fusobacterium),while lactic acid-producing bacteria(such as Lactococcus lactis and Lactobacillus brevis)were more abundant in adjacent non-tumor tissues..3.The function of gastric microbiome were significantly different between the two groups.In cancer tissues,purine metabolism,carbohydrate metabolism and denitrification of the microbiota were enhanced,which were in balance with the increase of energy metabolism and nitrogenous compounds in the tumor microenvironment.4.The abundance of H.pylori in cancer tissues was lower than that in adjacent non-cancerous tissues,and H.pylori infection can affect the composition of the microbial community.5.The microbial diversity of gastric cancer tissues in patients over 60 years old was significantly increased.The relative abundance of mucosa-associated bacteria in gastric cancer tissues of male and female patients was different.6.Compared with the H.pylori-negative group,the positive group had a higher diversity of microbiota,with significant differences in the microbiota structure between the two groups.7.In cancer tissues and paracancinous tissues,the community richness of mucosa-associated microorganisms was correlated with lymphocyte infiltration and Borrmann typing,respectively,while the diversity was not significantly correlated with different clinicopathological parameters.8.The difference of microbial community structure in gastric cancer was related to tumor stage,lymphocyte infiltration and Borrmann type.There was no significant correlation between the microbiome structure and the clinicopathological parameters.9.There was no significant correlation between mucosal related bacteria and prognosis of GC patients.10.There were strong positive correlations between the differentially distributed microbiota and the gastric function indicators of GC patients,which was mainly found in the genus prevotella,Peptostreptococcus,Streptococcus and other oral bacteria.11.G17 and PGR were internal gastric mucosal environmental parameters closely related to mucosa-related gastric micromicrobiota in gastric cancer and adjacent non-cancer tissues.Conclusions: 1.The distribution characteristics of mucosa-associated microbiota in gastric cancerous and adjacent non-cancerous tissues were different.There were significant differences between the two groups in taxonomy,function and ecological network.2.Oral bacteria may play an important role in the occurrence and development of gastric cancer and the maintenance of tumor microenvironment.3.Colonization of H.pylori can change the diversity of mucosa-associated microbiota and affect the community structure of microbiota.4.The diversity of mucosa-associated microbiota in cancer tissues was correlated with the age of gastric cancer patients.5.The richness and community structure of mucosa-associated microorganisms were correlated with clinicopathological parameters,but had no significant correlation with prognosis.6.There were significant positive correlations between serum gastric function indexes G17 and PGR and mucosa-associated microbiota in patients with gastric cancer. |
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