Construction Of Atrial Fibrillation-related CircRNA/lncRNA-miRNA-mRNA Regulatory Network And Analysis Of Potential Biomarkers

BackgroundAF(atrial fibrillation)is the most common clinical arrhythmia.In recent years,more and more studies have shown that ncRNA(non-coding RNA)is involved in the development of AF,but the specific pathogenesis of ncRNA involvement in AF remains unclear.In this study,we detected the differential expression of mRNAs(messenger RNA),circRNAs(circular RNA),and lncRNAs(long non-coding RNA)in peripheral blood mononuclear cells of AF patients by high-throughput sequencing technology,and initially established the pathogenesis of AF based on To establish an AF-related ceRNA(competing endogenous RNA)regulatory network based on circRNA/lncRNA-miRNA-mRNA,and to further explore the pathogenesis of AF and screen for potential diagnostic biomarkers.MethodsFrom May 1 to December 1,2021,24 patients with AF hospitalized in Taizhou People’s Hospital(AF group)and 24 healthy physical examiners were selected as controls(control group),peripheral blood was collected,monocytes were extracted and frozen,total RNA was extracted from monocytes using Trizol reagent,and qualified total RNA was selected for further synthesis of cDNA for high-throughput sequencing as well as The cDNA was further synthesized for high-throughput sequencing and qRT-PCR(quantitative real-time polymerase chain reaction)validation.High-throughput sequencing was first used to detect differential expression of mRNAs,circRNAs and lncRNAs in peripheral blood mononuclear cells from four AF patients and four matched healthy subjects.Then 20 pairs of peripheral blood from AF patients and healthy checkups were selected for qRT-PCR validation of differentially expressed mRNAs,circRNAs and lncRNAs.The miRanda database,startbase database and miRWalk database were used to predict the interaction between differentially expressed RNAs and miRNAs(micro RNAs).The GO(Gene Ontology analysis)and KEGG(Kyoto Encyclopedia of Genes and Genomes)analyses were also performed,and finally the AF-related circRNA/lncRNA-miRNA-mRNA ceRNA network.Results1.In peripheral blood mononuclear cells of people with AF compared to healthy patients:(1)There were 445 up-regulated and 368 down-regulated mRNAs out of 813 differently expressed mRNAs.ADGRE3,MME,TRPM6,TCTEX1D4,ALOX15,CCNJL,CSF1,DDIT3,JUN,and CYP4F3 were the top 10 mRNAs that were up-regulated,while ZCWPW2,AKAP5,ABCA13,OLFM4,KIR2DS4,PCDH1C1orf21,KRT73,and KIR3DL1 were the top 10 mRNAs that were down-regulated;(2)There were 120 circRNAs that were differently expressed,of which 55 were down-regulated and 65 were up-regulated.the top 10 up-regulated circRNAs were hsa＿circ＿0039161,circRNA＿0161,hsa＿circ＿0001947,hsa＿circ＿0003916,circRNA＿6991,hsa＿circ＿0008021,hsa＿circ＿0001394,circRNA＿7571,hsa＿circ＿0008699,hsa＿circ＿0072697,and the top 10 downregulated circRNAs are circRNA＿1834,circRNA＿4184,circRNA＿9064,hsa＿circ＿0004096,circRNA＿8108,circRNA＿4624,hsa＿circ＿0085438,hs a＿circ＿0006208,circRNA＿3830,circRNA＿1800;(3)912 differentially expressed lncRNAs,531 up-regulated,381 down-regulated.the top 10 up-regulated lncRNAs are ENST00000648905,TCONS＿00001709,ENST00000609281,XR＿001754622.1,TCONS＿00038119,ENST00000649467,XR＿947046.2,TCONS＿00019391,XR＿001739616.1,T CONS＿00019687,the top 10 downregulated lncRNAs are TCONS＿00026213,XR＿942786.2,TCONS＿00015252,XR＿001738325.1,XR＿001740454.1,TCONS＿00026209,NR＿037650.1,TCONS＿00026208,XR＿944963.2,TCONS＿00026211.2.GO analysis of differentially expressed RNAs was mainly enriched in inflammatory response,neutrophil degranulation,antimicrobial humoral response,antigen binding,G protein-coupled receptor activity,sphingosine-1-phosphate receptor activity,etc.The top 20 gene pathways in the KEGG enrichment screen corresponded to Pathway entries with differential gene numbers greater than 2(p<0.05),mainly involving PI3K-Akt signaling pathway,MAPK signaling pathway,EBV,natural killer cell-mediated cytotoxicity and other related pathways.3.Based on high-throughput sequencing data and predicted miRNAs,a ceRNA network containing 34 mRNAs,212 circRNAs,108 lncRNAs and 38 miRNAs was constructed,in which XIST/circRNA＿2773-miR-486-5p-CADM1 was selected for further study.qRT-PCR experiments showed consistent results with high-throughput sequencing results,in addition to analyzing the correlation of expression levels.Conclusion1.Differentially expressed circRNAs,lncRNAs,and mRNAs expression profiles were present in peripheral blood of AF patients and healthy subjects.2.Construction of circRNA/lncRNA-miRNA-mRNA regulatory network.We identified a novel ceRNA network in the AF,suggesting that XIST/circRNA＿2773-miR-486-5p-CADM1 is involved in AF.3.Build a PPI network and discover that JUN is the most important key hub gene in the PPI network,which is AF.The diagnosis reveals potential biomarkers.