| BackgroundMyocarditis is one of the common cardiovascular diseases in children,which is caused by infection,immunity,poisoning and other factors.Fulminant myocarditis(FM)is the most serious type of myocarditis in children.It has acute onset and severe condition,and often leads to death due to highly destructive heart failure,severe hemodynamic changes,cardiogenic shock and concurrent multiple organ failure.It is reported that FM accounts for up to 38%of acute myocarditis in children,and the mortality rate is as high as 48%,which seriously endangers children ’s health.It has been found that immune response disorders,inflammatory factor storms,and direct invasion of pathogenic microorganisms into cardiomyocytes play important roles in the occurrence and development of fulminant myocarditis,but its specific molecular mechanism has not been fully elucidated.Messager RNA(mRNA)is a kind of single-stranded RNAs that guide protein synthesis.It is the only coding RNA in organisms.It can link genetic information in DNA with protein translation and expression,and plays an important role in many life activities.Long non-coding RNA(lncRNA)is a class of non-coding RNA molecules with a length of more than 200 nucleotide sequences in eukaryotes.Because of its complex secondary and tertiary structures,it can bind to RNA,protein and other substances to exert rich biological functions.Studies have found that abnormal expression of mRNA and lncRNA is closely related to the pathophysiological processes of various cardiovascular diseases such as proliferation and differentiation of cardiomyocytes,cardiac remodeling,cardiac stress and immune regulation.In addition to regulating intracellular functions,mRNA and lncRNA can also be released into the extracellular environment,acting on adjacent cells or distal cells or released into the body fluid circulation as potential diagnostic markers of diseases.Circulating lncRNAs are closely related to small extracellular vesicles(sEVs).sEVs are a class of lipid bilayer membrane vesicles with a diameter of less than 200 nm produced by a variety of cells,which play an important role in cell communication.Studies have shown that long-chain RNA in small extracellular vesicles can play an important role in cardiovascular diseases such as atherosclerosis and myocardial infarction,but its research in children myocarditis has not been reported.ObjectiveTo find the differentially expressed small extracellular vesicle mRNAs(sEVmRNAs)and lncRNAs(sEV-lncRNAs)in plasma of children with FM and healthy controls,and to perform functional enrichment analysis of differentially expressed sEVmRNAs and sEV-lncRNAs and further analyze their potential mechanisms in the development of FM.Starting from the mRNA and lncRNA in plasma small extracellular vesicles,it provides new research ideas for the molecular mechanism of FM pathogenesis.Methods1.In this study,peripheral blood plasma was collected from 15 children with fulminant myocarditis and 15 healthy volunteers of similar age and sex in Shandong Provincial Hospital from January 15,2021 to May 20,2022.sEVs were extracted by differential centrifugation,and sEVs were characterized by transmission electron microscopy(TEM),nanoparticle tracking analysis(NTA)and Western blot.2.The plasma sEVs of 5 children with FM and 5 healthy controls were selected,and their RNA was extracted.RNA sequencing was performed using Illumina NovaSeq to find differentially expressed sEV-mRNAs and sEV-lncRNAs and perform functional enrichment analysis.3.String was used to analyze the protein-protein interaction(PPI)network of differentially expressed mRNAs and Cytoscape 3.9.1 was used to visualize the network.Five algorithms(DMNC,MCC,MNC,EPC and Degree)in the CytoHubba plug-in were used to calculate the top 10 genes,and the intersection genes were taken as the core genes.The GeneMANIA database was used to predict the genes interacting with the core genes,and the Metascape database was used to annotate the function of the interacting genes.4.The lncRNAs related to immune regulation were screened and their potential immune regulation functions were predicted.Then the hub lncRNAs molecules were selected and verified by qRT-PCR and clinical correlation analysis.Results1.A total of 194 differentially expressed sEV-mRNAs(152 up-regulated and 42 down-regulated)and 79 differentially expressed sEVs-lncRNAs(68 up-regulated and 11 down-regulated)were detected by whole transcriptome sequencing analysis according to the criteria of more than 2-fold difference and P<0.05.2.The results of enrichment analysis showed that the differentially expressed mRNA and lncRNA-related target genes in plasma sEVs of FM and NC groups were related to changes in related signaling pathways such as immunity,apoptosis and protein efflux,and were also closely related to FM-related immune dysfunction.3.The interaction network of differentially expressed sEV-mRNAs was analyzed by String database,and three mRNAs,YWHAH,AXIN1 and CSNK1A1,which play a central role in the interaction,were screened by various algorithms.Functional enrichment analysis showed that these three core genes and their interacting genes were mainly enriched in the biological processes closely related to FM,such as Wnt signaling pathway-related processes,phosphoserine residue binding,protein ubiquitination regulation,membrane potential regulation and positive regulation of cell migration.4.Further screening and analysis of immune-related lncRNA target genes showed that they were mainly enriched in immune system activation,macrophage chemotaxis and polarization-related signaling pathways and biological processes,which were closely related to the immune regulation mechanism in FM.5.The results of qRT-PCR showed that the expression level of NONHSAT144744.2 was consistent with the sequencing results and the difference was statistically significant.The expression level of NONHSAT144744.2 was positively correlated with cardiac troponin T(Hs-TnT)and B-type natriuretic peptide(BNP),and negatively correlated with left ventricular ejection fraction(LVEF).Conclusion1.The expression of mRNA and lncRNA in plasma small extracellular vesicles of children with fulminant myocarditis was significantly different from that of healthy children.Functional enrichment analysis showed that these differentially expressed lncRNA molecules may be involved in the immune regulation process in the progression of fulminant myocarditis.2.In the interaction network of differentially expressed sEVs-mRNAs in plasma,YWHAH,AXIN1 and CSNK1A1 play a central role.They can play a role in many biological processes related to fulminant myocarditis,such as immunity,inflammation and viral infection,and may become potential diagnostic markers and therapeutic targets for FM.3.Plasma sEVs-lncRNA may be involved in regulating the occurrence and development of FM by regulating the migration and differentiation of immune cells4.The expression of NONHSAT144744.2 in plasma small extracellular vesicles is significantly increased in children with fulminant myocarditis,which may regulate the antiviral response of target cells by regulating the expression of MAVS gene. |
PDF Full Text Request