Screening And Preliminary Verification Of Methylation Markers For Gastric Cancer Based On GEO And TCGA Database

Objective:DNA methylation data from GEO and TCGA databases were analyzed by bioinformatics to screen DNA methylation markers for diagnosis and prognosis of gastric cancer and to determine whether it can be used as a methylation marker for diagnosis and prognosis of gastric cancer.Methods:1.The DNA methylation microarray data of gastric cancer were selected from the Gene Expression Omnibus(GEO).The different methylated genes of gastric cancer were screened by GEO2 R and analyzed by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG).2.The DNA methylation data and clinical data of gastric cancer and paracancerous tissues were downloaded from The Cancer Genome Atlas(TCGA)database,and the difference of methylation data was analyzed by R software package Limma.Methylation markers related to prognosis were screened by univariate Cox regression analysis,Lasso regression analysis and multivariate Cox regression analysis.Cox proportional hazard regression model was used to identify DNA methylation biomarkers,and Receiver Operating Characteristic(ROC)curve was used to evaluate the performance of the model.Results:1.A total of 46 gastric cancer different methylated genes were screened,including 26 hypermethylated genes and 20 hypomethylated genes.Finally,five methylated genes,such as LRRC4?KCNA3?SLCO4C1?ADHFE1?LOC728392,were selected as potential diagnostic markers for gastric cancer.GO analysis showed that the differential genes of gastric cancer were mainly focused on cyclosporine A binding,G protein coupled receptor activity,olfactory receptor activity,peptide-prolyl cis-trans isomerase activity and so on.KEGG analysis showed that the differential genes of gastric cancer were mainly enriched in 19 functions,such as ganglioside biosynthesis series,glycosphingolipid biosynthesis sphere and isosphere series,glycosphingolipid biosynthesis lactose and neolactose series,olfactory conduction and so on.2.The R software package Limma was used to analyze the methylation data,and the Cox proportional hazard regression model was used to identify DNA methylation biomarkers.Finally,five methylation genes,including ATOH1,GLIPR1L1,IFIT3,TSGA14,and TUBB3,were selected as potential prognostic markers for gastric cancer.Conclusion:1.LRRC4?KCNA3?SLCO4C1?ADHFE1?LOC728392 may be potential methylation markers in the diagnosis of gastric cancer.2.ATOH1,GLIPR1L1,IFIT3,TSGA14 and TUBB3 may be potential methylation markers for the prognosis of gastric cancer.Objective:Methylation Specific PCR(MSP)was used to verify the methylation markers of circulating tumors in patients with gastric cancer.Methods:Methylation Specific PCR(MSP)was used to detect specific methylation markers in the blood of patients with gastric cancer,the blood of patients with gastric ulcer and the blood of healthy volunteers.SPSS25.0 was used to analyze the results,Fisher exact probability method and corrected chi-square test were used to analyze the data of gene methylation and clinicopathological characteristics in patients with gastric cancer.The sensitivity and specificity of methylation of each gene were calculated.Draw the ROC curve of methylation of each gene,and calculate the area under the ROC curve,namely Area Under ROC(AUC)Curve.Results:1.LRRC4,KCNA3,SLCO4C1,ADHFE1 and LOC728392 had no significant difference in different sex,age and tumor location,LOC728392 and SLCO4C1 had no significant difference in different histological grades and clinical stages,LRRC4 had significant differences in different histological grades and clinical stages,KCNA3 had significant differences in different clinical stages,but there was no significant difference in histological grades,while ADHFE1 had significant differences in different histological grades.But there was no significant difference in clinical stage.2.The sensitivity and specificity of LOC728392 are 0.82 and 0.925,respectively,and the AUC is 0.872;the sensitivity and specificity of SLCO4C1 are 0.68 and 0.725,respectively,and the AUC is 0.703;the sensitivity and specificity of LRRC4 are 0.6 and0.925,respectively,and the AUC is 0.763;The sensitivity and specificity of KCNA3 were 0.76 and 0.725,respectively,and the AUC was 0.742;the sensitivity and specificity of ADHFE1 were 0.82 and 0.95,respectively,and the AUC was 0.885.Conclusion:LRRC4,KCNA3,SLCO4C1,ADHFE1,and LOC728392 all have good diagnostic performance in the diagnosis of gastric cancer,among which ADHFE1>LOC728392>LRRC4>KCNA3>SLCO4C1.