Extracellular vesicles from hypoxia-preconditioned mesenchymal stem cells alleviates myocardial injury by targeting thioredoxin-interacting protein-mediated hypoxia-inducible factor-1α pathway

Figure 7 Extracellular vesicle-associated miR-224-5p inhibits hypoxia-induced apoptosis in cardiomyocytes by downregulating thioredoxin-interacting protein. A: MicroRNA (miRNA) sequencing analysis in adipose-derived stem cell-extracellular vesicles (ADSC-EVs). A total of 88 miRNAs were upregulated in hypoxia preconditioned EVs (HP-EVs) compared to normoxic EVs (NC-EVs), of which 10 (shown in the heatmap) were predicted to associate with thioredoxin-interacting protein (TXNIP). Validation of differential EV-associated miRNA expression through quantitative PCR. U6 small nuclear RNA served as the internal reference; B: Dual luciferase reporter assay. HEK 293 T cells were co-transfected with miR-224-5p mimics and PGL3 Luciferase reporter plasmids containing wild-type (WT) or mutated TXNIP 3′-untranslated region (3’-UTR). mutated-TXNIP 3′-UTR served as the control. Dual luciferase reporter assay (All data are expressed as the mean ± SD , ^kP < 0.05, ^lP < 0.01, miR-224-5p + TXNIP WT 3′-UTR vs miR-224-negative + 3’-UTR-mutant groups; n = 3); C: Neonatal mouse CMs subjected to hypoxia were pre-treated with EVs derived from ADSCs (ADSCs group), ADSCs overexpressing miR-224 (ADSCs-AAV-miR-224 group), ADSCs overexpressing miR-224-NC (ADSCs-AAV-miR-224NC group) and ADSCs with miR-224 knocked-out (ADSCs-cas9-miR-224 group). Western blotting was used to detect the expression of hypoxia-inducible factor-1 alpha and TXNIP (n = 5); D: Neonatal mouse CMs subjected to hypoxia were pre-treated with EVs derived from ADSCs (ADSCs group), ADSCs overexpressing miR-224 (ADSCs-AAV-miR-224 group), ADSCs overexpressing miR-224-NC (ADSCs-AAV-miR-224NC group) and ADSCs with miR-224 knocked-out (ADSCs-cas9-miR-224 group). Annexin V/PI assay was used to assess the degree of apoptosis in four groups (n = 5). All data are expressed as the mean ± SD, ⁱP < 0.05, ^jP < 0.01, compared with CMs subjected to hypoxia were pre-treated with EVs derived from ADSCs group(ADSCs group). HP-EV: Hypoxia-preconditioned extracellular vesicles; NC-EV: Normoxic extracellular vesicle; ADSC: Adipose-derived mesenchymal stem cell; WT: Wild-type.