The Role And Mechanism Of PMEPA1 In Helicobacter Pylori-Induced Diseases

Background and objectives:Gastric cancer(GC)is one of the most common malignant tumors with poor prognosis.The occurrence and development of GC has gone through a long process of evolution,in which H.pylori infection is the most important initiating factor.H.pylori regulates the biological behavior of gastric epithelial cells through a variety of virulence factors such as Cag A,Oip A,Vac A and so on,but its pathogenic mechanism is not clear.In the preliminary experiments,we used H.pylori 7.13 and CCS9803wild strains to infect GES-1 gastric epithelial cells and constructed a bacterial cell co-culture model in vitro.After performing transcriptional sequence analysis for the model and experimental verification,we found the transcription and protein expression levels of prostate transmembrane protein androgen inducible gene1(PMEPA1)were upregulated by H.pylori stimulated GES-1.The results indicated that PMEPA1 may be involved in the biological process of gastric mucosal changes induced by H.pylori infection.PMEPA1 is a new androgen-induced gene found in prostate tissue in 2000.It is mainly expressed on cell membrane and subcellular membrane of subcellular organelles such as lysosome and Golgi apparatus.It is known that PMEPA1 is a TGF-?induced gene that could negatively regulate TGF-?signal pathway.It has been found that PMEPA1 is highly expressed in a variety of tumors and participates in various biological processes such as cell proliferation,invasion and migration.However,the study of PMEPA1 in GC is not clear.The role of PMEPA1 in gastric mucosal changes caused by H.pylori infection has not been studied.Therefore,this paper studied the role and mechanism of PMEPA1 in H.pylori infected gastric mucosal lesions by bioinformatics,PMEPA1 gene intervention,cell transcriptome and other methods,so as to further reveal the new molecular mechanism of H.pylori induced GC and to provide a new theoretical basis for the targeted regulation of H.pylori related GC.Materials and Methods:I.Exporing the expression,prognosis of PMEPA1 in GC by bioinformatics analysis.1.Expression analysis of PMEPA1 in GC:(1)using the GC sequencing data from Oncomine database,GEO database and TCGA database,the expression of PMEPA1 in GC and normal gastric mucosa and the expression of PMEPA1 in different Lauren type GC were analyzed;(2)Eighteen pairs of fresh GC and adjacent normal gastric tissues were collected from the operating room of the first affiliated Hospital of Nanchang University,and the expression of PMEPA1 in GC and adjacent gastric tissues was detected by real-time fluorescence quantitative PCR and Western blotting.2.Prognosis analysis of PMEPA1 in GC patients:using Kaplan-Meier Plotter online analysis platform,we evaluated the relationship between PMEPA1 and overall survival time(OS),progression-free survival time(PFS)and progression-free survival time(PPS)of GC patients,so as to determine the relationship between PMEPA1 expression and prognosis of GC patients.3.Diagnostic value of PMEPA1 in GC:we used p ROC package to analyze the receiver working curve(ROC)of 375 GC and 32 normal gastric mucosal tissue sequencing samples from TCGA database,and to calculate the area under the curve(AUC),so as to evaluate the diagnostic value of PMEPA1 in GC.4.Biological process of PMEPA1 in GC predicted by bioinformatics:(1)according to the median expression of PMEPA1,the 375 GC samples of TCGA database were divided into PMEPA1 high and low expression groups.Gene set enrichment analysis(GSEA)was used to analyze the potential biological process of two different gene sets;(2)Correlation analysis of was performed between PMEPA1and other genes in 375 GC samples from TCGA database in R software and KEGG pathway enrichment analysis was conducted among the PMEPA1 related genes.II.Effect of H.pylori infection on the expression of PMEPA1 in gastric mucosal epithelial cells.1.Effects of H.pylori and its virulence factors on PMEPA1 expression in gastric epithelial cells:(1)we constructed oip A virulence gene knockout strains of Hp7.13and Hp PMSS1(Hp7.13?oip A and Hp PMSS1?oip A),and constructed anti-Oip A antibodies to verify the knockout effect of oip A gene at gene and protein level.(2)the wild strain of H.pylori PMSS1 was co-cultured with gastric epithelial cell GES-1with different infection plural(MOI)for different time(1h,3h,6h,12h,24h),the expression level of PMEPA1 protein was detected,and the m RNA expression level of PMEPA1 was detected at 6 hours after infection.(3)AGS cells were infected with H.pylori 7.13 and PMSS1 wild strains(Hp7.13wt and Hp PMSS1wt)with different MOI for 6 hours,and the expression level of PMEPA1 protein was detected.(4)H.pylori7.13 and PMSS1 wild strains(Hp7.13wt and Hp PMSS1wt),cag A gene knockout strains(Hp7.13?cag A and Hp PMSS1?cag A)and oip A gene knockout strains(Hp7.13?oip A and H PMSS1?oip A)were co-cultured with gastric epithelial cells GES-1 cells and AGS cells for 6 hours,and the protein and m RNA expression levels of PMEPA1 were detected.2.Expression of PMEPA1 in different stages of gastric mucosal diseases and its relationship with H.pylori infection:paraffin sections of endoscopic specimens from different pathological stages of gastric mucosa infected by H.pylori were collected from the Department of Pathology of the first affiliated Hospital of Nanchang University,and the expression of PMEPA1 was detected by immunohistochemical method.III.Effects and mechanism of PMEPA1 on the biological behavior of H.pylori infected gastric epithelial cells.1.Construction of stable gastric cells with PMEPA1 overexpression and knockout by lentivirus transfection:the GES-1 cell with overexpression of PMEPA1gene(PMEPA1^highGES-1)and its control cell(NC-PMEPA1^highGES-1),the GES-1cell with knockout of PMEPA1 gene(PMEPA1^lowGES-1)and its control cell(NC-PMEPA1^low GES-1),the BGC-823 cell with overexpression of PMEPA1 gene(PMEPA1^highBGC-823)and its control cell(NC-PMEPA1^highBGC-823)were constructed.2.Effect and mechanism of PMEPA1 on the biological behavior of gastric epithelial cells and gastric cancer cells:(1)the effects of PMEPA1 on the proliferation of gastric epithelial cells and gastric cancer cells were detected by plate clone formation assay,real-time unlabeled cell analysis(RTCA)and CCK8 assay;(2)the effects of PMEPA1 on the invasion and migration of gastric epithelial cells and gastric cancer cells were detected by transwell invasion and migration assay and scratch repair assay.(3)the correlation between PMEPA1 and EMT transcription factors in GC samples from TCGA database was analyzed by bioinformatics,and the protein expression level of related EMT molecules was detected by Western Blot.3.Effect of PMEPA1 on biological behavior of H.pylori infected gastric epithelial and gastric cancer cells:(1)Hp PMSS1wt,Hp PMSS1?cag A and Hp PMSS1?oip A strains were used to infect with PMEPA1^highBGC-823 and its control cells,and RTCA technique,transwell invasion and migration assay were used to detect the effects of PMEPA1 on the proliferation,invasion and migration of H.pylori-infected gastric cancer cells.(2)Hp7.13wt was used to infect with PMEPA1^lowGES-1 and its control cells and transwell assay were used to detect the effects of PMEPA1 on the invasion and migration of H.pylori-infected GES-1cells.IV.Transcriptome analysis of PMEPA1 in H.pylori-infected gastric epithelial cells.1.Transcriptome sample preparation:the PMEPA1^lowGES-1 cell and its control cell(NC-PMEPA1^lowGES-1)that transfected with empty vector virus were respectively infected with Hp7.13wt and Hp PMSS1wt strains(MOI=100),the PMEPA1^highGES-1 cell and its control cells(NC-PMEPA1^highGES-1)were respectively infected with Hp7.13wt?Hp7.13?cag A and Hp7.13?oip A strains(MOI=100);Total cell RNA of above cells as well as PMEPA1^highBGC823 and NC-PMEPA1^highBGC823 cells were collected.Three biological repeats were set in each group,and a total of 48 RNA samples were collected for transcriptome analysis.2.Transcriptional sequencing analysis:(1)the transcriptional sequencing was based on Illumina Novaseq 6000 sequencing platform in Shanghai Meiji Biopharmaceutical Technology Co.,Ltd.The gene expression data was obtained through a series of progress including c DNA library construction,computer sequencing,sequencing data quality control,gene annotation and other steps;(2)the differential expression analysis was carried out using DESeq2 software,and the screening standard was:the fold of differential gene expression(Fold Change)>1.5,adjusted P<0.05;(3)the GO enrichment analysis of differential genes was carried out using Goatools software(adjusted P<0.05 was significant enriched)and the KEGG pathway enrichment was performed using R software(P<0.05 was significant enriched).Results:I.Expression,prognosis value of PMEPA1 in GC by the bioinformatics analysis.1.PMEPA1 was overexpressed in GC:(1)Oncomine database showed that PMEPA1 was significantly overexpressed in a variety of cancer tissues(P<0.05),including breast cancer,cervical cancer,head and neck cancer,lung cancer and multiple digestive system tumors such as esophageal,gastric,colon and pancreatic cancer.(2)408 TCGA GC samples and 211 normal gastric mucosal tissue samples including TCGA and GETx database were included in GEPIA database,and the results showed that PMEPA1 was significantly overexpressed in GC tissue samples(P<0.05).(3)Three GC sequencing data(GSE54129,GSE79973,GSE118916)were downloaded from the GEO database.The results showed that the transcription level of PMEPA1 in GC tissues was significantly higher than that in normal gastric mucosa tissues in the three GEO data sets.(4)The expression of PMEPA1 in different Lauren types of GC was analyzed by Oncomine database.The results showed that PMEPA1 was significantly high expressed in different Lauren types of GC,especially in intestinal type gastric cancer.(5)the q RT-PCR and Western Blotting method were used to detect the expression of PMEPA1 in 18 pairs of fresh GC and its adjacent tissues.2.High expression of PMEPA1 was associated with poor prognosis in GC patients:Kaplan-Meier Plotter database showed that PMEPA1 was a risk factor for GC(HR>1).The overall survival time(HR=1.52),first progressive survival time(HR=1.38)and progressive survival time were significantly shorter in PMEPA1 high expressed GC patients.3.The clinical diagnosis value of PMEPA1 in GC:the ROC of PMEPA1 gene was plotted based on TCGA-STAD dataset.The AUC of ROC was 0.893,indicating that PMEPA1 is a good diagnostic marker for GC.4.Biological process of PMEPA1 in GC predicted by bioinformatics:(1)GSEA results showed that high expression of PMEPA1 was positively correlated with TGF-?,epithelial mesenchymal transformation(EMT),angiogenesis,hypoxia,Wnt signal pathway and Hedgehog signal pathway(FDR q-value<0.05).(2)the KEGG results of PMEPA1 related genes showed that PMEPA1 and its related genes were mainly enriched in E2F target gene,mitotic spindle,MYC target gene,protein secretion,DNA repair,G2M cell cycle checkpoint,angiogenesis,Wnt signal pathway,TGF-?signal pathway,tight junction,EMT and so on,in which PMEPA was significantly positively correlated with the EMT pathway(adjusted P=0.005).II.Effect of H.pylori infection on the expression of PMEPA1 in gastric mucosal epithelial cells.1.The Hp7.13?oip A and Hp PMSS1?oip A strains as well as rabbit polyclonal antibodies against Oip A were successfully constructed.2.H.pylori infection upregulates the expression of PMEPA1 protein and m RNA in gastric epithelial cells:(1)Previous transcriptome results showed that the expression of PMEPA1 in GES-1 cells infected with Hp7.13 and Hp CCS9803 strains for 24 hours was 1.59 and 1.13 times higher than that in cells without H.pylori infection and the difference was statistically significant(P<0.05).(2)Gastric epithelial GES-1 cells were infected with different MOI for 1,3,6,12,24 h.At 6 h and 12 h,the expression level of PMEPA1 protein increased gradually with increased MOI.The MOI of the highest protein and m RNA expression level of PMEPA1 at 6h is 50 and 100(P<0.05).(3)The gastric cancer cell line AGS was infected with Hp7.13 and PMSS1 wild type with different MOI for 6h.With the increase of MOI,the protein expression level of PMEPA1 increased,and there was significant difference in MOI=50 between the two strains(P<0.05).3.The effects of Hp virulence factors Cag A and Oip A on the expression of PMEPA1 in gastric epithelial cells:(1)western blot results showed that the protein expression levels of PMEPA1 in gastric epithelial cells GES-1 infected with Hp7.13wt(or Hp7.13?cag A,Hp7.13?oip A)and Hp PMSS1wt(or Hp PMSS1?cag A,Hp PMSS1?oip A)were higher than that in the uninfected control group,while the expression levels of PMEPA1 protein in cag A and oip A gene knockout strains infected GES-1 cell were lower than that in the wild group.(2)Real-time fluorescence quantitative PCR results showed that the m RNA levels of PMEPA1 in GES-1 and AGS cell lines infected with Hp7.13wt and Hp PMSS1wt were significantly higher than that of uninfected cells(P<0.05),and also significantly higher than that of in cag A and oip A gene knockout infected cells(P<0.05).4.The expression of PMEPA1 in different stages of gastric mucosal diseases and its relationship with H.pylori infection:(1)PMEPA1 was expressed in both inflammatory cells and glandular cells,mainly located in the cytoplasm and nuclear membrane.(2)in glandular cells,the expression levels of PMEPA1 in mild chronic non-atrophic gastritis,intestinal metaplasia and dysplasia specimens infected by H.pylori were significantly higher than that in patients without H.pylori infection,while in inflammatory cells,the expression of PMEPA1 in severe non-atrophic inflammation,intestinal metaplasia and dysplasia specimens infected by H.pylori was significantly higher than that in patients without H.pylori infection.(3)in H.pylori infected gastric specimens,the expression of PMEPA1 in gland cells of mild and severe non-atrophic gastritis was significantly lower than that of intestinal metaplasia,dysplasia and gastric cancer(P<0.05),and in inflammatory cells of mild non-atrophic gastritis was significantly lower than that of other groups(P<0.05).In patients without H.pylori infection,the expression levels of PMEPA1 in both glandular cells and inflammatory cells in gastric cancer group were significantly higher than that in other groups(P<0.05).These results suggest that PMEPA1 may be involved in the process of gastric mucosal lesions caused by H.pylori infection.III.The effect and mechanism of PMEPA1 on the biological behavior of H.pylori infected gastric epithelial cells.1.PMEPA1 promoted the proliferation of gastric epithelial cells and gastric cancer cells:(1)Plate clone formation assay,RTCA assay and CCK8 proliferation assay showed that the proliferation ability of PMEPA1^highGES-1 was significantly higher than that of WT-type GES-1 cell(P<0.05),while the proliferation ability of PMEPA1^lowGES-1 was significantly lower than that of WT-type GES-1(P<0.05).(2)Plate clone formation assay,RTCA assay and CCK8 proliferation assay showed that the proliferation ability of PMEPA1^highBGC-823 was significantly higher than that of WT-type BGC-823 cell(P<0.05).2.PMEPA1 promotes the invasion and migration of gastric epithelial and gastric cancer cells:(1)transwell invasion and migration assay showed that the invasion and migration abilities of PMEPA1^highGES-1 and PMEPA1^highBGC-823 were significantly higher than that of the corresponding WT-type cells(P<0.05),while the invasion and migration abilities of PMEPA1^lowGES-1 cells were significantly lower than that of the corresponding WT-type cells(P<0.05).(2)Scratch repair assay showed that the migration abilities of PMEPA1^highGES-1 and PMEPA1^highBGC-823 were significantly higher than that of the corresponding WT-type cells(P<0.05).3.PMEPA1 is related to epithelial-mesenchymal transformation(EMT):(1)the correlation between PMEPA1 and EMT related genes was analyzed by Cbioportal database.The results showed that PMEPA1 was significantly positively correlated with mesenchymal cell markers N-cadherin coding gene CDH2,Vimentin coding gene VIM(P<0.05),as well as with EMT mesenchymal cell transcription factors ZEB1,TWIST1 and TWIST2(P<0.05),the Snail transcription factor coding gene SNAI1 and Slug transcription factor coding gene SNAI2 and matrix metalloproteinases encoding genes MMP11,MMP14,MMP7,MMP2,MMP1,MMP17,MMP13,MMP24(P<0.05).(2)Western Blot showed that the expression of E-cadherin protein in PMEPA1^lowGES-1 was significantly higher than that in NC cells,and the expression of N-cadherin protein in PMEPA1^lowGES-1 cell was significantly lower than that in NC cells(P<0.05).However,in PMEPA1^highGES-1cell,the results were oppositive(P<0.05).4.Effect of PMEPA1 on the proliferation of H.pylori infected BGC-823 cell:the PMEPA1^highBGC-823 and its control cells were infected with Hp PMSS1wt strain,and the RTCA results showed that H.pylori infection began to promote the proliferation of NC-PMEPA1^highBGC-823 cell after infected by Hp PMSS1wt for 18hours.Within 40-78 hours after infection,Hp PMSS1wt strain significantly promoted the proliferation of NC-PMEPA1^highBGC-823 cells(P<0.05).However,there was no significant difference in cell proliferation among Hp PMSS1?cag A infection group,Hp PMSS1?oip A infected group and Hp PMSS1wt infection group(P>0.05).In addition,there was also no significant difference in the proliferation of PMEPA1^highBGC-823 cells infected by Hp PMSS1wt strain and its cag A and oip A knockout strains(P>0.05).5.Effect of PMEPA1 on migration and invasion abilities of H.pylori infected gastric cancer cells and gastric epithelial cells:(1)the effect of overexpression of PMEPA1 on migration and invasion of H.pylori infected BGC-823 gastric cancer cells:in H.pylori uninfected group,overexpression of PMEPA1 significantly promoted the migration and invasion of BGC-823 cells.The migration and invasion ability of PMEPA1^highBGC-823 and control cells infected by Hp7.13wt was significantly higher than that of uninfected cells,and the migration and invasion ability of Hp7.13wt infection group was significantly higher than that of HP7.13?cag A and Hp7.13?oip A infection groups(P<0.05);the cell invasion ability in Hp PMSS1wt infection group was After Hp PMSS1wt infection of PMEPA1^highBGC-823 and control cells,the cell as the same as that of Hp7.13 strain group,but there was no significant difference in migration ability among Hp PMSS1infected PMEPA1^highBGC-823 cells.(2)the effect of PMEPA1 knockout on the migration and invasion of Hp7.13wt infected gastric epithelial GES-1 cells:The migration and invasion abilities of PMEPA1^lowGES-1 and its control cell were increased significantly(P<0.05)after Hp7.13wt infection.IV.Transcriptome analysis of the biological function of PMEPA1 in H.pylori-induced changes in gastric epithelial cells.1.Effect of PMEPA1 on transcriptional level of gastric epithelial cells and gastric cancer cells:(1)Effect of PMEPA1 overexpression on GES-1 transcription level of gastric epithelial cells:i.GES-1 overexpression of PMEPA1 gene,there were 1025differential genes(up-regulated 673,down-regulated 352).ii.According to the GO classification of differential gene enrichment,there were 26 cellular components(CC),111biological process(BP)and 19 molecular functional(MF),which mainly included extracellular matrix,extracellular matrix containing collagen,extracellular surface,Golgi apparatus,inherent components of plasma membrane,cell membrane and so on.It mainly involves biological processes related to cell movement,such as intercellular adhesion of plasma membrane adhesion molecules,chemotaxis,adhesion process,cell migration,cell movement,extracellular matrix components,regulation of cell movement,cell adhesion and so on.The differential genes have calcium binding,extracellular matrix structure,signal receptor binding,receptor ligand activity,receptor regulatory activity,collagen binding and tumor necrosis factor activated receptor activity.iii.A total of 24 KEGG pathways were enriched.There are mainly cytokine-cytokine receptor interaction,synthesis and secretion of cortisol,tryptophan metabolism,complement and coagulation cascade,tumor necrosis factor signal pathway,cell adhesion molecule(CAM),interaction of viral proteins with cytokines and their receptors,transforming growth factor-?signal pathway,CGMP-PKG signal pathway,IL-17 signal pathway,JAK-STAT signal pathway and so on.(2)The effect of PMEPA1 knockout on the GES-1 transcription level of gastric epithelial cells:i.A total of 1376 differential genes(up-regulated and down-regulated),ii.GO pathway was mainly enriched by 77 cellular components(CC),469 biological processes(BP)and 47 molecular functional(MF).There are mainly extracellular regions,extracellular vesicles,inherent components of plasma membrane,cytoskeleton,Golgi apparatus,cell surface,extracellular matrix containing collagen and so on.It is mainly related to cell movement,such as matrix-dependent cell migration,cell movement,cell response to stimulation,chemotaxis,cell migration,as well as the regulation of epithelial cell apoptosis,response to hypoxia,inflammatory response,vesicle-mediated transport and other biological processes.The main functions of differential genes are receptor ligand activity,signal receptor activation,cytoskeleton protein binding,protein binding,G protein coupled receptor binding,chemotaxis,CXCR chemokine receptor binding,collagen binding and so on.iii.A total of 40 KEGG pathways were enriched,including AGE-RAGE signaling pathway,tumor necrosis factor signaling pathway,viral protein interaction with cytokines and their receptors,cell adhesion molecules(CAM),IL-17 signaling pathways,cytokine-cytokine receptor interactions,chemokine signaling pathways and so on.(3)Effect of PMEPA1 overexpression on the transcription level of BGC823 in gastric cancer cell line:i.a total of 132 differential genes(70 up-regulated and 62down-regulated).ii.The GO pathway,including 20 cellular components(CC),201biological processes(BP)and 52 molecular functional(MF)were enriched.It is mainly located in extracellular region,collagen fiber XI,neurofilament cytoskeleton,extracellular matrix,plasma membrane region,extracellular matrix containing collagen,endoplasmic reticulum and so on.It is mainly involved in biological processes such as angiogenesis,cell differentiation,development,extracellular matrix composition,tissue morphogenesis,acute inflammation,secretion and regulation of TGF-?,metabolism and so on.It mainly has molecular functions such as extracellular matrix structural components,protein binding,NADPHX enzyme activity,extracellular matrix binding,and protease binding and so on.iii.A total of 7 KEGG pathways,including Yersinia infection,small cell lung cancer,endocytosis,ascorbic acid and uronic acid metabolism,actin cytoskeleton regulation,natural killer cell-mediated cytotoxicity and ECM-receptor interaction.To sum up,the overexpression or knockout of PMEPA1 mainly affects the processes of cell movement,migration,cell adhesion,receptor ligand related work and so on.This result verifies our experimental results in chapter 4 and provides a good theoretical basis for further mining the new molecular mechanism of PMEPA1.2.Effect of Hp infection on transcriptional level of gastric epithelial cells with knockdown or overexpression of PMEPA1.(1)Effect of Hp infection on the GES-1 transcription level of knockdown PMEPA1:1)Effect of Hp7.13 infection on PMEPA1^lowGES-1 cells:i.a total of 2315differential genes(up-regulated 1041,down-regulated 1274);ii.GO pathways enriched mainly include 61 cellular components(CC),102 biological processes(BP)and 604 molecular functional(MF).It mainly includes chromosome region,nuclear chromatin,cytoskeleton,centrosome,chromatin components,Golgi apparatus,cellular components and so on.It is mainly involved in the positive regulation of pri-mi RNA transcription by RNA polymerase II,the response of cells to hypoxia,the regulation of mitosis,the regulation of cell division,the regulation of vascular development,the negative regulation of phosphorylation,the response of cells to oxygen levels,the positive regulation of cell cycle and the positive regulation of cell movement.iii.A total of 51 KEGG pathways were enriched,including MAPK signal pathway,hypoxia inducible factor-1 signal pathway,tumor necrosis factor signal pathway,cytokine-cytokine receptor interaction,insulin resistance,IL-17 signal pathway,AGE-RAGE signal pathway,transforming growth factor-?signal pathway,JAK-STAT signal pathway,micro RNAs in tumor,estrogen signal pathway,p53 signal pathway and so on.2)Effect of Hp PMSS1 infection on PMEPA1^lowGES-1 cells:i.a total of 959differential genes(up-regulated 668,down-regulated 271);ii.GO terems were enriched in 27 cellular components(CC),590 biological process(BP)and 61molecular functional(MF).The enrichment entry of GO is similar to that of 7.13.iii.A total of 104 KEGG pathways were enriched.There are mainly tumor necrosis factor signal pathway,MAPK signal pathway,IL-17 signal pathway,AGE-RAGE signal pathway,cytokine-cytokine receptor interaction,hypoxia-inducible factor-1signal pathway,NOD-like receptor signal pathway,viral protein interaction with cytokines and their receptors,NF-?B signal pathway,epithelial cell signal transduction in Helicobacter pylori infection and so on.(2)Effect of Hp infection on GES-1 transcription level of overexpressed PMEPA1.Effects of Hp7.13 infection on NC-PMEPA1high GES-1 cells:there were 1646genes(1094 up-regulated and 552 down-regulated),and 69 KEGG signaling pathways were enriched.There are mainly MAPK signal pathway,cytokine-cytokine receptor interaction,insulin resistance,tumor necrosis factor signal pathway,JAK-STAT signal pathway,AGE-RAGE signal pathway,hypoxia inducible factor-1signal pathway,AMPK signal pathway,IL-17 signal pathway,interaction of viral proteins with cytokines and their receptors,estrogen signal pathway and so on.Compared with Hp7.13 wild bacteria infection group,Hp7.13cag A gene knockout strains enriched 17 signal pathways in NC-PMEPA1high GES-1 cells,including micro RNAs in tumor,cell cycle,p53 signal pathway,cell senescence,FOXO signal pathway,abnormal transcriptional regulation in cancer,Wnt signal pathway,Hippo signal pathway,JAK-STAT signal pathway,Hedgehog signal pathway,indicating that cag A may play a role through these pathways.To sum up,Hp infection may mediate transcriptional changes in gastric epithelial cells that knock down or over-express PMEPA1 through the regulation of transcription factors,hypoxia-inducible factor-1 signal pathway,AMPK signal pathway,IL-17 signal pathway,Wnt signal pathway,Hippo signal pathway,JAK-STAT signal pathway and Hedgehog signal pathway,in which the virulence gene cag A plays an important role.Conclusion:1.PMEPA1 is highly expressed in GC,which is related to the poor prognosis of GC and is a good diagnostic marker of GC2.PMEPA1 promotes the proliferation,invasion and migration of gastric epithelial cells,which is related to EMT molecules.3.H.pylori regulates the expression of PMEPA1 and promotes cell proliferation,invasion and migration,which partly depends on the role of virulence factors Cag A and Oip A.4.H.pylori regulates a variety of biological processes of gastric epithelial cells,and PMEPA1 participates in the changes of H.pylori-mediated biological processes of gastric epithelial cells through many ways.