Changes And Significance Of DNA Methylation Of Lgr5-positive Gastric Epithelial Stem Cells Induced By Helicobacter Pylori Infection During Chronic Gastritis

Background and Purpose Helicobacter pylori(H.pylori)is the major cause of chronic gastritis,peptic ulcers and gastric cancer,and it is also closely associated with many extragastrointestinal diseases,imposing significant social and economic burdens worldwide.However,its pathogenic mechanism is not yet fully understood.Gastric epithelial stem cells,including those labeled by leucine-rich repeat-containing G-protein-coupled receptor 5(Lgr5)in gastric gland,are responsible for generating all lineages of gastric epithelial cells and maintenance of gastric tissue homeostasis.Recent studies suggest that the effect of H.pylori on gastric epithelial stem cells may play an important role in the pathogenesis of gastric mucosal lesions.DNA methylation is one of the most common epigenetic modifications.Abnormal DNA methylation can lead to dysfunction of related tumor suppressor genes or oncogenes,activation of proto-oncogenes,genome instability,etc,which are important for tumorigenesis mechanism.H.pylori infection can lead to an increase in the DNA methylation level of the gastric mucosa.After H.pylori eradication,the methylation level decreases,indicating that the effect is caused by H.pylori infection.However,relatively few studies are available regarding the effects of H.pylori induced gastric epithelial stem cells DNA methylation in gastric mucosal pathogenesis.This work used Illumina Infinium Methylation EPIC Bead Chip(850K)methylation chip detection technology to detect DNA methylation modification levels in Lgr5-positive gastric epithelial stem cells from patients with H.pylori infection,to explore whether H.pylori infection can cause abnormal methylation modification levels,so as to screen specific genes and metabolic pathways in order to provide a basis for subsequent research on the pathogenesis of H.pylori infection.From the perspective of molecular genetics,to explore the abnormal DNA methylation caused by H.pylori infection and the abnormal gene expression,which leads to the occurrence of disease,deepen the research on the pathogenic process of H.pylori infection,and provide new avenue for the clinical prevention and treatment.Materials and Methods In this study,we collected the gastric mucosal tissues of patients who attended the Department of Gastroenterology,People’s Hospital of Zhengzhou University from December 2019 to October 2020 with H.pylori testing as the research samples.A total of 16 samples were included,of which 6 cases were negative and 10 cases were positive for H.pylori infection.Whole-genome DNA methylation chip sequencing detection technology was used to detect and analyze whole-genome methylation levels in the included samples of Lgr5-positive gastric epithelial stem cells,and screen out differential methylation sites(DMS)and analyze the gene regions and chromosomal locations of related genes.Then,Gene Ontology(GO)enrichment analysis,Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis and Copy number variation(CNV)were performed for DMS.Result The results of 850 K methylation microarray show that all autosomes of patients with chronic gastritis infected by H.pylori have changes in the level of methylation modification,and most of DMS is located in the intergenic and genomic regions.A total of 406 DMS are identified,including 113 hypermethylated and 293 hypomethylated sites.The results show that the most significant difference between H.pylori-positive chronic gastritis and H.pylori-negative control group DMS corresponding gene is RCN3,followed by WDR86,F2 R,ADK,etc.GO enrichment analysis of DMS showed that 96 GO terms are significantly enriched.Among them,the top 10 GO terms with the most significant difference are voltage-gated sodium channel complex,postsynaptic density,skeletal system development,SH3 domain binding,neural crest cell migration,GTPase activator activity,voltage-gated sodium channel activity,plasma membrane,muscle tissue morphogenesis,membrane depolarization during action potential.Four of the GO entries are related to cell signal transduction,suggesting that related genes mainly affect signal transduction.KEGG analysis is performed to identify enriched pathways of DMS,19 KEGG pathways are significantly enriched.The top 10 KEGG pathways with the most significant difference are dopaminergic synapse,amphetamine addiction,c GMP-PKG signaling pathway,Hedgehog signaling pathway,calcium signaling pathway,cocaine addiction,amyotrophic lateral sclerosis,gastric cancer,endometrial cancer,Rap1 signaling pathway.The differential genes of gastric cancer pathway include LEF1,MLH1,FGF1,SHH,ABCB1.Copy number variation analysis shows that there are 25 variation segments in H.pylori positive samples.The 7 genes affected by 5 variant segments are OR4N4,OR4M2,LOC642131,GOLGA6L6,POTEB2,POTEB,LOC101926972;the 3 genes affected by 3 variant segments are LOC100996713,LOC101929561,LOC645262.Conclusion 1.H.pylori infection can cause changes in the level of DNA methylation modification in gastric epithelial stem cells,DMS exists in all autosomes.2.Go analysis showed that differential gene mainly affect signal transduction.KEGG analysis suggested that multiple pathways are affected.This analysis and screening can provide a basis for further study on the pathogenesis of abnormal DNA methylation in gastric mucosa caused by H.pylori infection.