Screening And Analysis Of Defferential Expressed Genes For Prognosis In EBV-related Gastric Cancer Based On RNA-seq And Methylation Profile Sequencing Databases

Objective: Gastric cancer is a common malignant tumor of digestive system.About 10% of gastric cancer is associated with EBV infection,which is called EBV associated gastric carcinoma(EBVa GC).EBV viral proteins could induce differential gene expressions or methylation of host cellular genes,which may play an important role in the development of EBVa GC.At present,few biomarkers have been widely used in the diagnosis and prognosis of EBVa GC.The purpose of this study is to explore new biomarkers of EBVa GC.Methods: The next generation sequencing dataset of EBVa GC GSE51575 were downloaded from the GEO database,and the sequencing dataset of gastric cancer TCGA-STAD were downloaded from the TCGA database.The differentially expressed genes(DEGs)were screened by limma and edge packages.The overlapped DEGs of the GSE51575 and TCGA-STAD datasets were obtained by using Vendiagram packages for further study.The GO(gene ontology)and KEGG(Kyoto Encyclopedia of genes and genomes)pathway analysis of DEGs were carried out by using cluster profiler packages.STRING database were subjected to establish the protein-protein interaction(PPI)network of DEGs,and hub genes were obtained by using cytohubba plug-in of Cytoscape software.The GO and KEGG pathway enrichment of hub genes were ayalyzed also.To screen the prognostic genes from 11 hub genes,the survival packages were used to evaluate the relationships between the genes expression levels and the survival time of patients.Wilcoxon signed rank test were used to analyze the gene expression levels between EBVa GC and EBV-negative gastric cancer in the GSE51575 dataset,and between the gastric cancer tissues and the adjacent normal tissues in the TCGA-STAD dataset.Furthermore,another EBVa GC dataset GSE66629 and gastric cancer dataset GSE33335 were downloaded to verify the expression levels of the prognostic genes.Kruskal-Wallis test were used to analyze the gene expression levels of prognostic genes in different stages of gastric cancer in the TCGA-STAD dataset.The relationship of the DNA methylation of prognostic genes and survival rates in EBVa GC and EBV-negative gastric cancer were analyzed by using survival and surviviner packages.Results: A total of 181 overlapped DEGs in both of the GSE51575 and TCGA-STAD datasets were screened including 89 up-regulated DEGs and 92 downregulated DEGs.The results of GO and KEGG pathway analysis showed that these DEGs were mainly enriched in the regulation of other biological defense responses and leukocyte activation,extracellular membrane,cytokine receptor binding and other biological functions,chemokine signaling pathway and toll like receptor signaling pathway.In 181 DEGs,11 hub genes were obtained,which were cytotoxic T-lymphocyte associated protein 4(CTLA4),C-X-C motif chemokine ligand 9(CXCL9),Guanylate binding protein 5(GBP5),2'-5'-Oligoadenylate synthetase 2(OAS2),Signal transducer and activator of transcription 1(STAT1),Interferon gamma(IFNG),C-X-C motif chemokine ligand 11,(CXCL11),Indoleamine 2,3-dioxygenase1(IDO1),Interleukin 2 receptor subunit alpha(IL2RA),Integrin subunit alpha X(ITGAX)and C-X-C motif chemokine ligand(CXCL10).The pathway enrichment analysis of hub genes showed that they were mainly enriched in other biological defense responses,extracellular membrane,cytokine receptor binding,cytokine cytokine receptor interaction,chemokine signaling pathway and toll like receptor signaling pathway.Survival analysis showed that CTLA4,OAS2,CXCL9,GBP5 and STAT1 were related to the prognosis of EBVa GC.In addition,the expression levels of CTLA4,OAS2,CXCL9,GBP5 and STAT1 in EBVa GC were higher than those in EBV-negative gastric cancer.Also,the gene expression levels in gastric cancer tissues were significantly higher than those in adjacent normal tissues,as shown in the TCGA-STAD and GSE33335 gastric cancer datasets.It was shown that CTLA4 and STAT1 were hypomethylated genes in gastric cancer,which may be used as biomarkers related to methylation level in the EBV associated gastric carcinoma.Conclusions: CTLA4,OAS2,CXCL9,GBP5 and STAT1 may be involved in the development of EBVa GC.Among them,CTLA4 and STAT1 genes were hypomethylated,which could be used as new prognostic biomarkers for the diagnosis and prognosis of EBVa GC and may help to develop new strategies for gastric cancer treatment.