GATA4-overexpressing BMSCs-derived exosome regulation of myocardial infarction in mice by key miRNA and apoptosis gene CLU

Figures

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  Fig. 1. Bioinformatics analysis flowchart

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  Fig. 2. Effects of Exos from GATA4-overexpressing BMSCs on hypoxic cardiomyocytes. A Exosome morphology identified by TEM (scale bar = 100 nm); B NTA analysis; C Western blot was used to identify exosome markers; D IF detected PKH26-labelled exosomes (scale bar = 10 μm); E GATA4 detected by RT-qPCR (t-test); F cTnT, connexin-43, desmin, and α-actin expression detected by RT-qPCR (Two-way ANOVA); G cTnT, connexin-43, desmin, and α-actin fluorescence values detected by IF (scale bar = 20 μm); H cell apoptosis rate detected by flow cytometry (t-test); I protein expression detected by Western blot (two-way ANOVA). *P < 0.05, **P < 0.01, and ***P < 0.001 (n = 3)

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  Fig. 3. Analysis of the differentially expressed proteins in the Exos of mouse BMSCs. A Volcano plot of protein differential expression: the abscissa represents the differential expression multiple log2FC, the ordinate represents the significant − log10 (adj. p); each dot represents a protein; red indicates a significantly upregulated protein, green indicates a significantly downregulated protein; grey indicates a non-significant protein; B heat map of differential protein expression; sample in Abscissa direction and differential expression protein in ordinate order; red indicates high expression; blue indicates low expression; C KEGG enrichment results: top 5 differentially expressed proteins,); D GO enrichment results (differentially expressed proteins): the top 5 pathways (in their order of significance) and the proteins they contain; the circle size of the pathway indicates the number of proteins contained

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  Fig. 4. Key protein screening. A Venn diagram of the differentially expressed apoptosis proteins; B PPI network; C GO enrichment results (DEARPs) displaying the top 10 pathways of each classification (ranked by significance); the square size indicates the number of genes contained, and the colour indicates their significance

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  Fig. 5. Variance analysis of miRNA. A Volcano plot of miRNA differential expression. The abscissa represents the differential expression of multiple log2FC; the ordinate represents significant-log10 (adj. p); each point in the map represents a miRNA; red indicates significantly upregulated miRNAs, green indicates significantly downregulated miRNAs; gray indicates non-significant miRNAs; B Differential protein expression heat map. At the top is the miRNA distribution heat map; at the bottom is the miRNA expression heat map. The abscissa represents the sample, and the ordinate represents the differential expression of miRNA. Different colours indicate the standardised miRNA expression: red indicates high expression, and blue indicates low expression. C GO enrichment analysis results (GATA4 overexpression-related genes)

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  Fig. 6. Key protein/miRNAs. A Venn of common GO functions; B gene–protein–miRNA regulatory network: the purple circle represents the gene, the prism represents the key protein, red represents upregulation (G group is higher than N group), green represents downregulation (G group is lower than N group); rectangle represents key miRNA, red represents upregulation (higher in the G group than in the N group), green represents downregulation (lower in the G group than in the N group); the dotted frame represents the key proteins involved in GO functions (the top 10 ranked by significance)

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  Fig. 7. Gene set enrichment analysis (GSEA). A–D The GSEA enrichment trend map displays the enrichment pathways of the top 5 pathways (ranked by their significance). The upper part depicts the process of calculating the enrichment fraction (ES, enrichment score), value, calculated as an ES value and connected into a line. The particularly obvious peaks on the far left and the far right are the ES values for the phenotypes of the gene sets. Each line in the following section represents a gene in the gene set and its sequencing position in the gene list

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  Fig. 8. LncRNA/circRNA prediction. A mRNA–miRNA–lncRNA regulatory network. The red represents mRNAs, the yellow miRNAs, and the green lncRNAs; B mRNA–miRNA–circRNA regulatory network. The red represents mRNAs, the yellow miRNAs, and the blue circRNAs

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  Fig. 9. Ingenuity pathway analysis (IPA). A Key targets involved in the pathway; B regulatory relationships in the top 1 pathway; C intermolecular interactions network

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  Fig. 10. Expression of key proteins and miRNA. A The expression of key proteins vs the N group, *P < 0.05, **P < 0.01, and ***P < 0.001; B the expression of key proteins detected by RT-qPCR vs the Exos group (one-way ANOVA), *P < 0.05, **P < 0.01, and ***P < 0.001; C the expression of the key miRNAs vs the N group, *P < 0.05, **P < 0.01, and ***P < 0.001; D the expression of the key miRNAs by RT-qPCR vs the Exos group (one-way ANOVA), *P < 0.05, **P < 0.01, and ***P < 0.001

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  Fig. 11. Exos from GATA4-overexpressing BMSCs attenuated cardiomyocyte infarction through CLU. RT-qPCR detection of transfection efficiency (A) and the expression of CLU (B) (one-way ANOVA). C RT-qPCR detected cTnT, connexin-43, desmin, and α-actin expression (two-way ANOVA); D detection of cTnT, connexin-43, desmin, and α-actin fluorescence values by IF (scale bar = 20 μm); E detection of the cell apoptosis rate by flow cytometry (one-way ANOVA); F detection of protein expression by Western blot (two-way ANOVA). *P < 0.05, **P < 0.01, and ***P < 0.001 vs the Exos group. #P < 0.05, ##P < 0.01, and ###P < 0.001 vs the ExosGATA4 group

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  Fig. 12. Exos from GATA4-overexpressing BMSCs improve cardiac function in MI mice by CLU regulation. A Left ventricular FS and EF of MI mice (two-way ANOVA); B TTC staining; C HE staining (scale bar = 50 μm); D detection of c-kit expression by IHC assay (scale bar = 50 μm, one-way ANOVA); E detection of cell apoptosis by TUNEL staining (scale bar = 20 μm); RT-qPCR (F) and IHC (G) were used to detect the expression of CLU (scale bar = 50 μm) (one-way ANOVA). ***P < 0.001 vs the MI group, and ###P < 0.001 vs the Exos.GATA4 group (n = 6)