Cardiac shock wave therapy-induced exosome derived from OGD/R-treated H9c2 cells carrying miR-98-5p promote HUVECs angiogenesis

Figures

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  Fig. 1. Identification of exosomes derived from H9c2 cells. (A) The exosome morphology was visualized by transmission electron microscopy. Scale bar, 100 nm. (B) The particle size distribution and particle concentration of exosomes were analyzed by nanoparticle tracking analysis. (C) The expression of exosomal protein markers CD9, CD63 and TSG 101 was tested by western blot.

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  Fig. 2. The exosomal miRNA expression profile changes in H9c2-OGD/R-Exo and CSWT-H9c2-OGD/R-Exo. (A) The differentially expressed miRNAs between H9c2-OGD/R-Exo and CSWT-H9c2-OGD/R-Exo were presented with volcano plot. (B) Heatmap diagram showed the expression level of the Top 10 differentially expressed miRNAs between H9c2-OGD/R-Exo and CSWT-H9c2-OGD/R-Exo. (C) The Top 10 differentially expressed miRNAs expression in CSWT-H9c2-OGD/R-Exo compared to H9c2-OGD/R-Exo was assessed by qRT-PCR. (D) The KEGG functional enrichment analysis of target genes of miR-98-5p was displayed in a scatter plot.

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  Fig. 3. CSWT-H9c2-OGD/R-Exo carrying miR-98-5p inhibited OGD/R-induced cardiomyocyte injury. H9c2 cells after OGD/R injury were treated with CSWT-H9c2-OGD/R-Exo and transfected with miR-98-5p inhibitor, (A) the expression of miR-98-5p was assessed by qRT-PCR. (B) CCK-8 assay was utilized for testing cell viability. (C) LDH release was analyzed by LDH cytotoxicity assay kit. (D) Flow cytometry was performed for evaluating cell apoptosis. (E-F) Western blot was performed to evaluate Bcl-2, Bax, cleaved caspase-3, HIF-1α, p-GSK-3β and GSK-3β protein levels. ***P < 0.001 vs. Ctrl group; ##P < 0.01 and ###P < 0.001 vs. OGD/R + PBS group; &P < 0.05, &&P < 0.01 and &&&P < 0.001 vs. OGD/R + Exo group.

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  Fig. 4. The uptake of CSWT-H9c2-OGD/R-Exo by HUVECs.

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  Fig. 5. exosomal miR-98-5p released by CSWT-H9c2-OGD/R-Exo promoted HUVECs proliferation, angiogenesis and metastasis. HUVECs were treated with CSWT-H9c2-OGD/R-Exo and transfected with miR-98-5p inhibitor, (A) miR-98-5p level was tested by qRT-PCR. (B) cell proliferation was detected by CCK-8. (C) Tube formation assay was conducted to measure the angiogenesis. (E-F) Transwell assay was applied to estimate cell migration and invasion. **P < 0.01 and ***P < 0.001 vs. PBS group; #P < 0.05, ##P < 0.01 and ###P < 0.001 vs. Exo + miR-NC group.

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  Fig. 6. miR-98-5p target and inhibited the expression of FOXN3. (A) The potential binding sites for miR-98-5p on FOXN3 3′UTR. (B) miR-98-5p and FOXN3 binding relationship was confirmed by luciferase report assay. (C-D) The expression of miR-98-5p and FOXN3 in miR-98-5p mimics-transfected HUVECs was assessed by qRT-PCR. (E) The protein level of FOXN3 in miR-98-5p mimics-transfected HUVECs was verified by western blot. **P < 0.01 and ***P < 0.001.

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  Fig. 7. FOXN3 reversed the promoting effect of miR-98-5p on HUVECs angiogenesis. HUVECs were co-transfected with si-FOXN3 and miR-98-5p inhibitor, (A) miR-98-5p level was tested by qRT-PCR. (B) FOXN3 protein level was assessed by western blot. (C) HUVECs proliferation was detected by CCK-8. (D) Tube formation assay was utilized to detect HUVECs angiogenesis. (E-F) HUVECs migration and invasion were assessed by Transwell assay. **P < 0.01 and ***P < 0.001 vs. Ctrl group; #P < 0.05, ##P < 0.01 and ###P < 0.001 vs. miR-inhibitor group.