Differential Expression Of Plasma Exosome MiRNAsand Identification Of MiRNA Biomarkers For Pediatric Fulminant Myocarditis

BackgroundFulminant Myocarditis(FM)is a life-threatening clinical condition with abrupt onset,rapid progression,and high mortality rates in children.The precise pathogenesis of FM remains to be clarified further.microRNA(miRNA)is a class of single-stranded,small(1925 nucleotides),and endogenous non-coding RNA which regulate post-transcriptional expressions of its target genes.miRNA is proved to be involved in the pathophysiology of various cardiovascular diseases.In disease states,the expression of miRNA is altered in the circulation.Exosomes are small extracellular vesicles with a diameter of 30 to 150 nm that present in various body fluids,including plasma,tears,saliva,emulsion,as well as urine.Unlike circulatory miRNAs,exosome-encapsulated miRNAs are more stable and enriched in circulation.So exosome-packed miRNAs may have greater potential as diagnostic biomarkers.In the meantime,exossome-mediated intercellular communication may contribute to the development of various diseases through pathological changes in immune and inflammatory responses.ObjectiveThis study aimed to explore the expression profile of specific miRNA in plasma exosomes from children with FM.Bioinformatics analysis was performed to explore the possible role of miRNA in FM,while screening out miRNAs that might serve as specific biomarkers for FM.MethodsPeripheral blood specimens from fifteen pediatric FM patients and fifteen healthy controls(HCs)were collected at the Shandong Provincial Hospital between January 1,2021,and September 28,2022.Among them,samples from three patients and three healthy children were used for microarray assay to screen out differentially expressed miRNAs.Subsequetly bioinformatics analysis was performed,including prediction of miRNAs targeting genes,GO and KEGG pathway enrichment analysis,protein-protein interactions.Finally,the accuracy of the microarray data was verified by qRT-PCR in 15 FM and 15 HCs,and the diagnostic ability of miRNAs as a FM biomarker was assessed by ROC analysis.Results1.By small RNA microarray analysis,676 miRNAs were identified in 3 FM and 3 HCs.266 miRNAs showed differential expression(FC>2,p<0.05),of which 197 miRNAs were upregulated and 69 miRNAs were downregulated.2.GO enrichment analysis found that miRNAs with altered expression in the plasma exosomes of the children with FM were mainly involved in the regulation of transcription.KEGG pathway analysis found that the differentially expressed miRNAs was associated with inflammatory and immune-related pathways such as "PI3K-Akt signaling pathway" and"MAPK signaling pathway".3.Through the differentially expressed mRNA in peripheral blood mononuclear cells of pediatric FM patients from our previously reported study and miRNAs with differential expression related to inflammation and immunity according to bioinformatics analysis,the FM-specific miRNA-mRNA regulatory network and PPI network were constructed.7 hub genes were identified,and a functional network containing miRNAs,hub genes and related signaling pathways was established.4.The expression of hsa-miR-30e-5p,hsa-miR-146a-5p,hsa-miR-361-5p,and hsa-miR-532-5p was verified in 15 children with FM compared with HCs(p<0.05),and showed significant sensitivity and specificity for diagnosing FM.Conclusions1.The miRNAs with differential expression in the plasma exosomes of children with FM is involved in the development of FM,and plays a role in inflammation and immune mechanisms.2.The expression of hsa-miR-30e-5p,hsa-miR-146a-5p,hsa-miR-361-5p,and hsa-miR-532-5p was shown to be elevated in children with FM,with the potential to be a specific biomarker of FM in children.