Identification and functional analysis of circulating small extracellular vesicle lncRNA signatures in children with fulminant myocarditis

FIGURE 1. Isolated sEVs‐enriched fractions from participants' plasma. (A) TEM images showed that sEVs were bowl‐shaped capsules without the nucleus, the left panel showed sEVs in the normal control group and the right panel shows sEVs in the FM group. (B) NTA results suggested that sEVs enriched from plasma were about 75‐200 nm in diameter. (C) Comparison of plasma sEVs concentration between FM group and NC group (p = 0.00460). (D) sEVs markers CD63, TSG101 and CD81 were all detected in the sEVs‐enriched fractions isolated from the plasma, and the negative marker of sEVs, calnexin, was absent in our isolated sEVs‐enriched fraction samples.

FIGURE 2. Identification of sEVs‐mRNA and sEVs‐lncRNA signatures between fulminant myocarditis and normal control. (A) Volcano plot showing differentially expressed sEVs‐mRNAs with various p‐values and fold changes. Horizontal line, p‐value = 0.05 (−log10 scaled); vertical line, fold change = 2 (log2 scaled). Red points, increased sEVs‐mRNAs; blue points, decreased sEVs‐mRNAs. (B) Hierarchical cluster analysis of sEVs‐mRNAs with altered levels (p < 0.05, fold change >2) between the two groups. Red strip, the level of mRNAs relative increased in the sEVs; blue strip, the level of mRNAs relative decreased; white strip, no change in gene content. Colour brightness reflects the degree of content increase or decrease. (C) Volcano plot of sEVs‐lncRNAs with different levels. (D) Hierarchical cluster analysis of sEVs‐lncRNAs.

FIGURE 3. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses of dysregulated sEVs‐mRNAs and sEVs‐lncRNAs in fulminant myocarditis. (A) Top 30 Gene ontology of dysregulated sEVs‐mRNAs. (B) Top 30 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of dysregulated sEVs‐mRNAs. (C) Ten most enriched gene ontology terms in biological process, cellular component and molecular function for sEVs‐lncRNAs with different levels. (D) Top 30 Kyoto Encyclopedia of Genes and Genomes pathways of dysregulated sEVs‐lncRNAs. In GO enrichment analysis, the rich factor = (number of differential genes in GO entries/number of all differential genes)/(total number of genes in GO entries in the database/total number of genes in the database); in KEGG enrichment analysis, rich factor = (Number of differential genes involved in the KEGG pathway/total number of differential genes)/(total number of genes in the KEGG pathway in the database/total number of genes in the database).

FIGURE 4. Screening and functional analysis of immune‐related target genes. (A) Venn diagram analysis, to identify the relationship between immune‐related genes and lncRNA target genes. (B) The distribution of identified immune‐related target genes across immune categories. (C) Ten most enriched gene ontology terms in biological process, cellular component and molecular function for immune‐related target genes. (D) Top 30 KEGG pathways of immune‐related target genes.

FIGURE 5. Identification and functional characterization of hub immune‐related target genes. (A) The STRING database is used to construct the PPI network of immune‐related target genes, with 75 nodes and 105 edges. (B) The hub genes of top 20 were screened using six topological methods of the cytoHubba plug‐in in Cytoscape. There were 10 genes in the six methods. (C) The interaction diagram between the ten hub genes.(D) GO enrichment analysis of the biological process of the hub immune‐related target genes. (E) Interaction networks of the enriched biological terms for the hub immune‐related target genes.

FIGURE 6. Verification and clinical correlation analysis of selected lncRNA molecules. (A–D) Verification of the four selected lncRNAs assessed by quantitative real time‐PCR (qRT‐PCR). FM: the fulminant myocarditis group; NC: normal control. Data are mean ± standard deviation. (E) The receiver operating characteristic (ROC) curve analysis for NONHSAT144744.2 in the diagnosis of FM. (F–I) Clinical correlation analysis of NONHSAT144744.2 in plasma sEVs.