Transcriptional And Functional Analysis Of Plasma Exosomal MicroRNAs In Acute Viral Myocarditis

Background:There is still a lack of non-invasive diagnostic methods with high specificity and sensitivity for acute viral myocarditis(AVMC).In addition,the underlying mechanisms of AVMC have not been fully elaborated.Recently,mounting evidences demonstrated that exosomes and micro RNA(miRNA)were involved in the development in various cardiovascular disease.Nevertheless,no relevant studies were performed to reveal the diagnostic value and biological function of serum exosomal miRNAs in AVMC.Objective:To assess the differential expression profiles of plasma exosome-derived miRNAs between AVMC patients and healthy control(HC)and perform function analysis to reveal their diagnostic value and potential molecular and biological functions in AVMC.Methods:1.Peripheral blood samples and medical records were collected from 9 patients diagnosed with AVMC in the Affiliated Hospital of Qingdao University from July 2021 to March 2023 as well as 9 HC.The diagnostic criteria of AVMC were referred to the diagnostic criteria in1999 in China.2.After centrifugation of plasma from peripheral blood,plasma exosomes were separated by ultrafast centrifugation,and miRNAs in exosomes were extracted.3.4 AVMC patients with typical clinical symptom and 4 HC were selected.RNA highthroughput sequencing was used to detect the expression of plasma exosomal miRNAs in patients with AVMC and the control group.Limma package in R language was used to analyze the expression of exosome miRNA.P < 0.05 and log2(Fold Change)absolute value > 2 were considered as cut-off value to screen miRNAs that were differently expressed.Volcanic maps and heat maps were also drawn.4.GO and KEGG functional analysis were used to predict the potential molecular,biological functions and related signaling pathways of differentially expressed miRNAs in exosomes.Target genes of exosomal miRNAs were predicted and miRNA-target gene network was mapped using gene databases such as Target Scan,miRTar Base,miRWalk and miRDB.5.3 up-regulated and 3 down-regulated miRNA were selected and the expression levels of plasma exosomal miRNA in 9 cases of AVMC and HC were detected by quantitative realtime polymerase chain reaction(qRT-PCR)to verify the sequencing results.Results:1.Transcription analysis showed that compared with HC,there were 28 miRNAs differentially expressed in plasma exosomes in AVMC patients,of which 14 were up-regulated and 14 were down-regulated.2.GO analysis showed that differentially expressed miRNAs may be related to β-catenin binding,DNA transcription activities,RNA polymerase,ubiquitin ligase,histone binding,cell connectivity and other molecular or biological functions.KEGG analysis showed that these differentially expressed miRNAs were mainly involved in PI3K-Akt,Fox O,P53,MAPK,Wnt,and other signaling pathways.3.The target genes of miRNA were predicted according to the gene database.METTL7 A,NRIP1,CACNB2,RAB5 C,SUGT1,and UTP18 were the common targets of many miRNAs.4.qRT-PCR verification results showed that the expressions of hsa-miR-548a-3p were up-regulated,while the expressions of hsa-miR-500b-5p were down-regulated,which was consistent with the sequencing results.Conclusions:1.Plasma exosomal hsa-miR-548a-3p and hsa-miR-500b-5p may be potential biomarkers for the diagnosis of AVMC.2.Exosomal miRNAs may affect gene expression through binding with target genes,directly or indirectly affect biological functions or signaling pathways including β-catenin,DNA transcription activities,ubiquitin ligase,epigenetic inheritance,PI3K-Akt,Fox O,P53,MAPK,Wnt,etc.,and thus participate in the pathogenesis of AVMC.