Phenotypic Characteristics Of Gastric Cancer With Different Fusobacterium Sp. Infection Status And The Association Between Fusobacterium Nucleatum Infection And Risk/Prognosis/Proliferation/Metabolic Phenotype Combined With Effect And Mechanisms

Objective:As an important composition of the mucosal environment of the gastrointestinal tract,the microbiota could not only cause DNA damage and produce toxic metabolites,but also promote the occurrence of tumors.In recent years,as an important part of the microbiota in digestive tract,Fusobacterium sp.played an important role in the occurrence and development of digestive tract tumors.In esophageal cancer,the abundance of Fusobacterium DNA was related to the depth of tumor infiltration.In colon cancer,Fusobacterium sp.could be isolated from cancer tissue.A study of GC patients in Taiwan showed that Fusobacterium sp.,Clostridium sp.and Lactobacillus sp.were abundantly distributed in the gastric mucosa of GC.The combination diagnosis of enriched Fusobacterium sp.and Clostridium sp.had a 100%sensitivity and 70%specificity.Fusobacterium nucleatum is a symbolic strain of Fusobacterium sp.,and is also involved in the occurrence of gastrointestinal tumors.Fusobacterium nucleatum is a gram-negative obligate anaerobic bacterium,which can be detected by PCR amplification of F.nucleatum DNA in esophageal cancer and colorectal cancer.In colorectal cancer,Fusobacterium nucleatum was considered to be the pathogenic microorganism of cancer.It was closely related to cell proliferation,T cell,inflammatory cytokine expression,microRNA,Cp G island methylation phenotype,and microsatellite instability.At present,as for Fusobacterium nucleatum and GC,only a small sample experiment suggests that there were Fusobacterium and Fusobacterium nucleatum in GC,but the molecular mechanism of their infection is not yet clear.In our previous researches,through 16S rRNA sequencing technology,we found that the distribution and characteristics of flora in GC had a difference with its distant normal paracancerous tissue.We found the distribution of Fusobacterium sp.in GC and paracancerous tissues was different:the abundance of Fusobacterium sp.in GC tissues was higher than paracancerous tissues.In recent years,the research of Fusobacterium sp.and Fusobacterium nucleatum infection in GC is still in its infancy and most studies prefer to focus on the changes in microbiology distribution and diversity.However,the relationship between Fusobacterium sp.and GC phenotype has not yet been determined.The effect and molecular mechanism of Fusobacterium nucleatum on the occurrence and development of GC are still unknown.Purpose:To investigate the phenotypic characteristics of GC with different Fusobacterium sp.infection states.To clarify the relationship,effect and mechanism between Fusobacterium nucleatum and the risk,prognosis,proliferation and metabolic phenotype of GC.Methods:The First Part:Phenotypic characteristics of GC with different Fusobacterium sp.infection status1.Selection of research cases:61 patients with GC who underwent subtotal gastrectomy from June 2012 to June 2014 in the Department of Anorectal Surgery,the First Affiliated Hospital of China Medical University was obtained and approved by the Ethics Committee of the First Affiliated Hospital of China Medical University,who met the inclusion and exclusion criteria and signed an informed consent.General information(including age and sex),clinicopathological variables information(TNM stage,tumor size,depth of invasion,Lauren's classification,tumor lymphocyte infiltration,vascular cancer embolus,lymphatic metastasis,differentiation and H.pylori infection)and immunohistochemical information of tumor biomarkers(Ki67,p53,CEA,C-erb B)were collected from the patients'medical records and pathological reports.The prognostic information including survival state,distant metastasis,overall survival(OS),and date of death were collected through telephone follow-up every six months with a follow-up period until November 13,2019.The fresh gastric mucosal tissue collected from the lesion site for pathological diagnosis was frozen immediately after the operation and stored in a-80°C refrigerator for later use.2.16s rRNA sequencing related microbial analysissOTU data of GC tissues was obtained through Qiime2 16s denoising by 16s rRNA sequencing data and was compared with the Greengene 18.3 database.The differences of?diversity indexes(Richness,Chao1,ACE,Shannon,Simpson,phylogenetic diversity(PD)whole tree indices)were analyzed through R language vegan package.The differences of?diversity(weighted Uni Frac,unweighted Uni Frac,Jaccard distance and Bray-Curtis dissimilarity)indicators were compared by PERMANOVA through q2-diversity.Differential bacteria phyla and bacterial genera were screened by linear discriminant analysis of LEf Se.The correlation among genus was performed by Spearman correlation analysis.3.Non-targeted metabolomics sequencing related metabolic analysisMetabolites in tissues were extracted according to conventional standards and then were sequenced by Vanvosh ultra-high performance liquid chromatography-mass spectrometry(UHPLC-MS/MS)system and Orbitrap Q Exactive TM HF mass spectrometer combined with Hypesil Gold chromatographic column.The offline data was searched,compared and matched with the mzCloud?mzVault and Mass List database to obtain the qualitative and relative quantitative results of metabolites.HMDB database was applied to annotate metabolites.Then principal component analysis was performed.The analysis of differential metabolites was performed by Mann–Whitney U test.The analysis of metabolic function was performed by KEGG database through Metabo Analyst 5.0 website.4.Integrated analysis of microbe and metabolite in GCThe overall correlation analysis of microbes and metabolites was performed in M²IA online analysis process through PA and O2PLS analysis,the correlation between differential microbes and metabolites was performed by Spearman correlation analysis,and the coexistence relationship was analyzed through mmvec neural network algorithm in Qiime2 platform.The Second Part:Study on the relationship between F.nucleatum infection and the risk,clinicopathological parameters and prognosis of GC1.Selection of research cases:53 patients with GC who underwent subtotal gastrectomy from June 2012 to June 2014 in the Department of Anorectal Surgery,the First Affiliated Hospital of China Medical University was obtained.78 cases of superficial gastritis and 59 cases of atrophic gastritis from 2004 Zhuanghe GC Screening and Early Diagnosis and Treatment Project was obtained.The sample storage method,informed consent,medical record data collection and follow-up methods were the same as the first part of the paper.2.For the tissues with a mass of no more than 10 mg,genomic DNA was extracted by TIANamp Micro DNA Kit.For the tissues with a mass of more than 10 mg,genomic DNA was extracted by conventional DNA extraction methods.The abundance of F.nucleatum DNA in the genomic DNA was detected by Real-time fluorescence quantitative PCR.The relative abundance was calculated according to the relative quantitative 2^-?Ct method.3.The difference in the abundance of F.nucleatum between GC and its distant normal tissue was analyzed by paired rank sum test.Logistic regression analysis was used for the correlation between F.nucleatum infection and the risk of GC.The correlation analysis of F.nucleatum infection and clinicopathological parameters was applied by Mann-Whitney U test after grouping by pathological parameters.The X-tile software was used to distinguish the high and low abundance cutoff values of F.nucleatum DNA related to the prognosis of GC,Kaplan-Meier survival analysis and COX univariant and multivariate analysis were performed based on the high and low abundance.The Third Part:Study on the effect and mechanism of F.nucleatum on the biological behavior of gastric mucosal epithelial cells and GC cells1.Selection of cell lines and bacteria strains:GES-1 immortalized human gastric mucosal epithelial cell line was purchased from Hangzhou Meisen Cell Technology Co.,Ltd.AGS GC cell line and HGC27 GC cell line were purchased from the Cell Resource Center of the Institute of Basic Medicine,Chinese Academy of Medical Sciences.All three types of cells have STR identification certificates.The strain of F.nucleatum(ATCC 25586)was presented as a gift from the Periodontal Teaching and Research Office of the Affiliated Stomatological Hospital of China Medical University.2.The adhesion and invasion ability of F.nucleatum to gastric mucosal epithelial cells and GC cells was detected by Gram staining and agar plate method of co-culture cell lysate solution,as well as laser confocal microscope.3.CCK-8 experiment was performed to detect changes in cell viability and cell proliferation behavior after F.nucleatum infection.Flow cytometry was performed to detect the proportion of early and late apoptotic cells after F.nucleatum infection.4.Total cellRNA was extracted by Trizol method.Mon Script TMRTIII All-in-One Mix with ds DNase was used for reverse transcription.Then fluorescence quantitative Realtime-PCR was performed to detect the changes of the expression of GSS and GPX4mRNA in cells after F.nucleatum infection.5.Protein in the experimental and control group was extracted.Then after detecting protein concentration through BCA protein quantification kit,cell protein was used for western to detect the changes in the expression of GSS and GPX4 protein after F.nucleatum infection.6.In accordance with the operating procedure of the micro-reduced glutathione determination kit,use a microplate reader to detect the content of reduced glutathione in the cells of the experimental and control group.7.In accordance with the operating procedure of the kit for determination of intracellular reactive oxygen species,use a fluorescent microplate reader to detect the content of ROS in the cells of the experimental and control group.Results:The First Part:Phenotypic characteristics of GC with different Fusobacterium sp.infection status1.The infection status of Fusobacterium sp.in GC tissues.The absolute abundance of microbial s OTU was obtained through analyzing 16s rRNA sequencing data by Qiime2 16s Deblur.The GC tissues were divided into positive and negative groups by absolute abundance of Fusobacterium sp.Nearly half of GC tissues had Fusobacterium sp.infection.2.Differences in clinicopathological parameters of GC with different Fusobacterium sp.infection status.After the correlation analysis between Fusobacterium sp.infection status and clinicopathological parameters of GC,elderly patients with GC were more likely to have Fusobacterium sp.infection(P=0.041).The presence or absence of Fusobacterium sp.infection in GC was related to the pericarcinoma lymphocyte infiltration(P=0.040),but were not related to other general clinicopathological parameters:gender,tumor size,degree of differentiation,Lauren classification,tumor invasion depth,lymph node metastasis,vascular tumor thrombus,TNM staging(P>0.05).In terms of the expression of tumor biomarkers,the infection status of Fusobacterium sp.was related to the positive expression of mutant p53(P=0.016),but not related to the expression of CEA,Ki67,and C-er B-2(P>0.05).3.Differences in the prognosis of GC with different Fusobacterium sp.infection status.In the Kaplan-Meier survival analysis,there was no significant difference in OS between Fusobacterium sp.positive and negative GC patients(P=0.899).In COX univariate analysis,indicators with P<0.1(tumor size,degree of differentiation,depth of infiltration,vascular tumor thrombus,lymph node metastasis,TNM staging)were included into the multivariate analysis.But,in each clinicopathological parameter index group,Fusobacterium sp.infection could not be used as an independent risk factor for prognosis(P>0.05).4.Differences in the distribution of the microbiota of GC with different Fusobacterium sp.infection status.The alpha diversity(Richness,Chao1 index,ACE index,faith-PD?whole?tree)in Fusobacterium sp.positive GC tissues was higher than the negative ones.As for microbial community distribution,the three indexes of?diversity were different between two groups(P<0.05).Differential genera were screened by LEf Se and the related bacterial network was constructed,showing a more relationship in the Fusobacterium sp.positive GC tissues.5.Differences in the metabolite enrichment,microbial-related metabolic pathways of GC with different Fusobacterium sp.infection status and microbial-metabolite correlation,coexistence analysis.Metabolites in GC tissue were qualitatively quantified through non-targeted metabonomic sequencing.11 metabolites were differently enriched between two groups by U test(P<0.05).Using PICRUST2 to predict microbial-related metabolic pathways,three metabolic pathways of lysine,peptidoglycan and aminoacyl-tRNA biosynthesis showed different between two groups(P<0.05).The PA and O2PLS analysis showed an overall correlation between microbe and metabolites.Through microbial-metabolite Spearman correlation analysis and mmvec coexistence analysis,three metabolites(glutathione,uric acid,pyrophosphate)coexist and negatively correlate with Fusobacterium sp.The Second Part:Study on the relationship between F.nucleatum infection and the risk,clinicopathological parameters and prognosis of GC1.Correlation analysis between F.nucleatum infection and the risk of GC.Real-Time PCR method was used to detect the abundance of F.nucleatum in tissues.The paired rank sum test results of GC and its distant normal tissue in the same individual suggest that the abundance of F.nucleatum in GC was higher than that in the distal normal tissues(P<0.001).Logistic regression analysis was performed on superficial gastritis,atrophic gastritis and GC respectively,showing that compared with superficial gastritis,the risk of GC with higher abundance of F.nucleatum was 5.189times than that of lower ones(P<0.01);compared with atrophic gastritis,the risk of GC with higher abundance of F.nucleatum was 11.250 times t than that of lower ones(P<0.01).2.Correlation analysis between F.nucleatum infection and clinicopathological parameters of GC.Among all clinicopathological parameters,only the tumor size was correlated with the abundance of F.nucleatum:GC patients with a tumor diameter greater than 6 cm had a higher abundance of F.nucleatum(P<0.05),while the other parameters had no significant correlation(P>0.05).However,the abundance trend was consistent with the results of the first part in terms of age,pericarcinoma lymphocyte infiltration and p53expression.3.Correlation analysis between F.nucleatum infection and prognosis of GC.Using X-tile software to analyze the best cut-off value for judging prognosis.Based on this,the GC tissues are divided into two groups with higher and lower abundance of F.nucleatum.In Kaplan-Meier survival analysis,patients with higher abundance of F.nucleatum had a shorter OS(P=0.148).In COX univariate analysis,patients with higher abundance of F.nucleatum in GC tended to have poorer prognosis with 1.893times death risk than lower ones(P>0.05).4.Correlation analysis between F.nucleatum infection and metabolites of GC.The glutathione content in higher abundance tissues of F.nucleatum showed a decreasing trend(P>0.05).In Spearman correlation analysis,the 2-^?Ct value expressed by F.nucleatum DNA was negatively correlated to glutathione(r=-0.277,P>0.05).The Third Part:Study on the effect and mechanism of F.nucleatum on the biological behavior of gastric mucosal epithelial cells and GC cells1.The adhesion and invasion of F.nucleatum to gastric mucosal epithelial cells and GC cells.In the co-cultured cell lysate,F.nucleatum could be detected as Gram negative.After plating on an agar plate,the growing colony of F.nucleatum could be observed.Under the laser confocal microscope,F.nucleatum could adhere to and invade into gastric mucosal epithelial cells and GC cells.In the detection of mRNA expression related to invasion and adhesion,F.nucleatum had a tendency to promote the expression of E-cadherin mRNA in GES-1 cells and inhibit the expression of E-cadherin mRNA in AGS and HGC27 cells.2.F.nucleatum could affect the proliferation viability of gastric mucosal epithelial cells and GC cells.In CCK-8 experiment,at MOI=1,F.nucleatum could increase the viability of AGS cells(P<0.05),slightly inhibited HGC27 cells(P<0.05)and had a tendency to increase GES-1 cells.At MOI=10,F.nucleatum could inhibit the viability of gastric mucosal epithelial cells and GC cells(P<0.05).In flow cytometry,F.nucleatum could decrease the ratio of normal cells/total cells and increase the ratio of early and late apoptotic cells/total cells(P<0.05).3.F.nucleatum could affect the GSH and ROS content in gastric mucosal epithelial cells and GC cells.At MOI=10,F.nucleatum could inhibit the production of GSH in GC cells and promotes the production of GSH in gastric mucosal epithelial cells(P<0.05).At MOI=1,the GSH content was not statistically significant between groups(P>0.05).As for intracellular ROS detection,with the increase in the content of F.nucleatum,ROS in gastric mucosal epithelial cells and GC cells increased first and then decreased.AGS cells showed the highest state at MOI=1,while at MOI=1 and 10,both HGC27 cells and GES-1 cells showed a decrease in ROS.4.F.nucleatum could affect the expression of GSS in gastric mucosal epithelial cells and GC cells.At MOI=1,the effect of F.nucleatum on the expression of GSS mRNA in the three cells showed no statistically significant(P>0.05),but it increased GSS protein expression in HGC27 cells(P<0.05)and had a tendency to promote the expression of GSS protein in AGS and GES-1 cells.At MOI=10,F.nucleatum inhibited the expression of GSS mRNA in AGS cells(P<0.05),and the effect on GSS mRNA and protein expression in GES-1and HGC27 cells was not statistically significant(P>0.05),but it did have the trend to promote GSS mRNA and protein expression in GES-1 cells and to inhibit the GSS mRNA and protein expression in HGC27 cells.5.F.nucleatum could affect the expression of GPX4 in gastric mucosal epithelial cells and GC cells.At MOI=1,F.nucleatum inhibited the expression of GPX4 mRNA in AGS cells(P<0.05)and had a tendency to inhibit its protein expression.F.nucleatum also had a trend to promote the expression of GPX4 mRNA and protein(P<0.05),but had no effect on HGC27.At MOI=10,F.nucleatum inhibited the expression of GPX4 mRNA in AGS cells(P<0.05)and showed the same protein expression trend as mRNA expression.In HGC27 cells and GES-1 cells,it could promote the expression of GPX4 protein(P<0.05)with same trend of mRNA expression.Conclusions:1.The phenotypic characteristics of GC with or without Fusobacterium sp.infection showed a difference.The infection status of Fusobacterium sp.in GC was related to the pericarcinoma lymphocyte infiltration,the positive expression of mutant p53,the diversity distribution of the microbial community,and the enrichment of three metabolites.2.F.nucleatum was abundant in GC tissues,could increase the risk of GC and had a tendency to lead to a poor prognosis.3.F.nucleatum had the ability to adhere to and invade gastric mucosal epithelium and GC cells.It could affect the production of GSH and ROS in gastric mucosa and GC cells by regulating the expression of GSS and GPX4,resisting the death of gastric mucosal epithelial cells and promoting the proliferation of GC cells.