Analysis Of MRNA?lncRNA And CircRNA Expression Profiles In Chronic Atrophic Gastritis Based On High-throughput Sequencing

Recently,lncRNAs and circRNAs play important role in the occurrence and development of diseases,because of development of high-throughput sequencing and have become a new research hotspot.More and more researchers have found that many lncRNAs and circRNAs can directly or indirectly regulate the expression of disease genes,suggesting that they have a very broad prospect as a tool for diseases diagnosis and treatment.CAG is a common type of gastritis,its clinical symptoms are relatively unremarkable and usually irreversible,so as a precancerous lesion of GC,early diagnosis and treatment is of great significance to reduce the occurrence of GC.At present,there are few studies about CAG and mRNA,lncRNA and circRNA.The object of this study is to analyze the expression profiles of mRNA,lncRNA and circRNA between CAG and CNAG by high-throughput sequencing,look for differentially expressed mRNA,lncRNA and circRNA in CAG,analyze the biological function of differentially expressed genes in CAG.This provides a basic research for finding CAG diagnostic biomarkers and potential therapeutic targets in the future.Part One Transcriptome analysis of lncRNA–mRNA interactions in chronic atrophic gastritisObjective:To explore the differences in the expression profiles of lncRNA and mRNA in CAG using high-throughput sequencing,GO/KEGG enrichment analysis was performed for differentially expressed genes,lncRNA-mRNA network analysis and protein-protein interaction(PPI)analysis were performed,prediction and validation of screened lncRNAs,thereby providing experimental basis for the diagnosis and treatment of CAG.Methods: In this study,gastric tissue samples of CAG and CNAG patients were collected by gastroscopy.The diagnosis based on the collected tissue samples was confirmed by histology.RNA integrity and g DNA contamination were measured by denaturing agarose gel electrophoresis.Removed the r RNA,constructed the RNA libraries and conducted principal component analysis.The differentially expressed mRNA and lncRNA in CAG and CNAG were analyzed by high-throughput sequencing and bioinformatics analysis.lncRNA-mRNA co-expression analysis and PPI analysis were performed.The quantitative reverse transcription polymerase chain reaction(qRT-PCR)assay was used to confirm the differentially expressed lncRNAs.Results:(1)This study included 20 patients with CAG(12 males,8 females,mean age of 59years)and 20 patients with CNAG(11 males,9 females,mean age of 43 years).Between the CAG and CNAG groups,the ratios of cigarette smoking and alcohol drinking were not statistically different.(2)A total of 97,286 lncRNAs and 20,308 mRNAs were obtained from all samples.We systematically analyzed the expression characteristics of these lncRNAs and mRNAs regarding their distribution in terms of length.Most of the lncRNAs ranged in length were200-3000 nt.Most of the mRNAs ranged in length were more than 6000 nt.Most of the circRNAs ranged in length were 200-3000 nt.The categories of 97,282 lncRNAs were mainly exon sense-overlapping,intronic antisense,and intergenic lncRNAs.(3)GO analysis showed that up-regulated lncRNAs were highly enriched in positive regulation of MAP kinase activity,cell surface and death receptor activity,respectively.In down-regulated lncRNAs: lung epithelial cell differentiation,lateral element and identical protein binding were the most enrichments.In up-regulated mRNAs: digestion,apical plasma membrane and transporter activity were the most enrichments.In down-regulated mRNAs: extracellular matrix organization,proteinaceous extracellular matrix and extracellular matrix structural constituent were the most enrichments.(4)KEGG pathway analysis showed that up-regulated lncRNAs and prolactin signaling pathway was the most enrichment.Down-regulated lncRNAs and homologous recombination was the most enrichment.Up-regulated mRNAs and mineral absorption was the most enrichment.Down-regulated mRNAs and cell adhesion molecules(CAMs)was the most enrichment.(5)Through the calculated pearson correlation values,we selected 24 differentially expressed lncRNAs and 101 differentially expressed mRNAs of CAG and CNAG to construct a lncRNA-mRNA co-expression network.Our analysis revealed that ENST00000488188,ENST00000583490,and NR₁21662 were the three lncRNAs with the most frequent interactions.(6)The PPI results showed that the upregulated mRNAs were mainly enriched in biological processes,such as digestion,epithelial cell differentiation,and response to an extracellular stimulus.We identified four significant modules via cluster analysis of the PPI network using MCODE,which is based on the degree of importance.We found that the 4modules were mainly enriched in cytochrome P450,arranged by substrate type,phase I-functionalization of compounds,etc.By venn diagram analysis using the Metascape database,8 mRNAs were found at the intersection of the 155 mRNAs,including DGKA,EIF6,HKDC1,DHRS11,CPS1,KRT15,TESPA1,and CDHR2 as well as 101 lncRNA-targeting genes.(7)The qRT-PCR was used to validate the expression levels of the differentially expressed lncRNAs that were identified by high-throughput sequencing and bioinformatics analysis.NR₁17090,ENST00000422847,ENST00000583490,ENST00000488188,and ENST00000459255 were selected for verification.The verification results by qRT-PCR were consistent with data from high-throughput sequencing and bioinformatics analysis.Summary: Differentially expressed lncRNAs and mRNAs in CAG and CNAG were screened,the sequencing results were analyzed by bioinformatics,and lncRNA-mRNA co-expression network was constructed.The expression levels of differentially expressed lncRNAs in CAG verified by qRT-PCR were consistent with the results of high-throughput sequencing and bioinformatics analysis.Part Two Analysis of immune-related mRNA and circRNA in CAGObjective:The level of immune cells in CAG was analyzed by flow cytometry.To explore the differences in the expression profiles of circRNA and mRNA in CAG using high-throughput sequencing,GO/KEGG enrichment analysis was performed for differentially expressed genes,circRNA-mRNA network analysis was performed,prediction and validation of immune-related mRNA and circRNA in CAG,to look for differentially expressed genes associated with CAG inflammation,thereby providing a new experimental basis for targeted therapy of CAG.Methods: The differentially expressed circRNA in CAG and CNAG were analyzed by high-throughput sequencing and bioinformatics analysis.The infiltration of immune cells in the local tissues of CAG and CNAG gastritis was analyzed by flow cytometry,the mRNA related to CAG immunity was analyzed by weighted gene co-expression network(WGCNA),PCC was calculated to construct circRNA-mRNA network.mRNAs and circRNAs related to CAG immunity were screened,mRNAs and circRNAs associated with CAG immunity were verified by qRT-PCR.Results:(1)Take the local tissue of gastritis,single cell suspension was prepared and stained,the levels of immune cells were analyzed by flow cytometry,the percentage of neutrophils in CAG was significantly higher than that in CNAG group.The percentages of NK cells,Th cells and Tc cells showed no statistical difference between CNAG group and CAG group.Analysis of immune cell infiltration in CAG versus CNAG using Immu Cell AI revealed decreased Tcm,NKT,CD8 T cell infiltration and increased neutrophil infiltration in CAG versus CNAG.(2)GO analysis showed that immune-related differentially expressed upregulated mRNAs were highly enriched in antimicrobial humoral response.In immune-related differentially expressed downregulated mRNAs:regulation of natural killer cell mediated cytotoxicity were the most enrichments.(3)A total of 6,245 circRNAs were obtained from all samples.We systematically analyzed the expression characteristics of these circRNAs regarding their distribution in terms of length.Most of the circRNAs ranged in length were 200-3000 nt.The categories of6,245 circRNAs were mainly exonic,sense overlapping,and intronic circRNAs.In total,19 upregulated circRNAs and 23 downregulated circRNAs in CAG were identified by the comparison of CAG and CNAG.(4)GO analysis showed that up-regulated circRNAs were highly enriched in carnitine metabolic process,clathrin coat of coated pit and protein domain specific binding.In down-regulated circRNAs: negative regulation of intrinsic apoptotic signaling pathway,actin cytoskeleton and calmodulin binding were the most enrichments.(5)KEGG pathway analysis showed that up-regulated circRNAs and lysine degradation was the most enrichment.Down-regulated circRNAs and regulation of autophagy was the most enrichment.(6)50 key immune-related mRNAs were screened by WGCNA analysis and venn diagram.PCC was calculated to screen 5 circRNAs associated with mRNA,including chr M:13298-13449+?hsa_circ₀005320?hsa_circ₀000339?chr7:100678797-100678973+?hsa_circ₀003664.(7)The expression levels of 8 mRNAs(AGPAT2?SLC7A9?OLFM4?EPCAM?ANXA2 ? PRSS3 ? MUC12 ? CD55)and 5 circRNAs(chr M:13298-13449+ ?hsa_circ₀005320?hsa_circ₀000339?hsa_circ₀003664?chr7:100678797-100678973+)associated with CAG immunity verified by qRT-PCR were consistent with the results of high-throughput sequencing and bioinformatics analysis.Summary: mRNAs and circRNAs associated with CAG immunity were screened,the sequencing results were analyzed by bioinformatics,and circRNA-mRNA co-expression network was constructed.The expression levels of 8 mRNAs and 5 circRNAs associated with CAG immunity verified by qRT-PCR were consistent with the results of high-throughput sequencing and bioinformatics analysis.Conclusion:In this study,the expression profiles of mRNA,lncRNA and circRNA in CAG and CNAG were analyzed by high-throughput sequencing,screened and verified the differentially expressed genes related to CAG,to find the key genes that may play an important role in the occurrence and development of CAG,this may provide a new idea for the early accurate diagnosis of CAG endoscopy,and it also provides a theoretical basis for finding new biomarkers and clinical therapeutic targets for diagnosis of CAG.