Exosomes Derived from miR-214-Enriched Bone Marrow-Derived Mesenchymal Stem Cells Regulate Oxidative Damage in Cardiac Stem Cells by Targeting CaMKII

Figure 3

Effect of miR-214 expression on CSC apoptosis under oxidative stress. Cells were treated with miR-214 mimics, inhibitors, or negative control RNA for 48 h and/or pretreated with BMSC-exos (400 μg/ml) for 24 h and then cultured with 100 μM H₂O₂ for 2 h for subsequent analyses. (a) RT-qPCR was used to analyze miR-214 expression in exosomes after normoxic or hypoxic preconditioning. Compared that in Nor-exos, miR-214 expression was significantly upregulated in Hypoxic-exos. (b) RT-qPCR analysis of miR-214 expression in CSCs after different treatments. Compared with that in the H₂O₂ group, miR-214 significantly upregulated in the Hypoxic-exos or miR-214 mimics group. Compared with the Hypoxic-exos group, the inhibitor-exos group displayed a significantly decreased miR-214 expression. (c) Representative dot plots of cell apoptosis after Annexin V/PI dual staining are shown. The upper left quadrant (% gated) shows necrotic cells (Annexin V−/PI+); the upper right quadrant (% fated) shows late apoptotic cells (Annexin V+/PI+); the left lower quadrant (% gated) shows live cells (Annexin V−/PI−); and the right lower quadrant (% gated) shows early apoptotic cells (Annexin V+/PI−). These cells were measured for comparison. (d) The percentage of apoptotic cells represents both early and late apoptotic cells. Compared with H₂O₂ or miR-214 inhibitor, Hypoxic-exos or miR-214 mimics decreased the percentage of apoptotic cells. In addition, the Hypoxic-exo-induced protective effect against CSC apoptosis under oxidative stress was partially suppressed by miR-214 inhibitors. (e) Representative immunofluorescence staining for TUNEL (green), C-kit (red), DAPI (blue), and merged images. Photos were randomly captured using a fluorescence microscope. Scale bar = 20 μm. (f) The panel shows the percentage of TUNEL-positive cells. Compared with H₂O₂ or miR-214 inhibitors, Hypoxic-exos or miR-214 mimics could significantly decrease the percentage of TUNEL-positive cells. In addition, compared with Hypoxic-exos, inhibitors-exos could partially increase the percentage of TUNEL-positive cells. (g) The intracellular ROS level was determined by FCM. The P2 percentage indicates the proportion of cells with increased ROS production, with signals above background DCF fluorescence levels. (h) Compared with that in CSCs treated with H₂O₂ or miR-214 inhibitors, the fluorescence intensity of intracellular ROS was decreased in CSCs treated with Hypoxic-exos or miR-214 mimics. Inhibitor-exos showed higher ROS fluorescence intensity than Hypoxic-exos. (i and j) Graph represents the SOD and MDA levels in CSCs, compared with H₂O₂ group, Hypoxic-exos or miR-214 mimics inhibited MDA levels and increased SOD production, while miR-214 inhibitors or inhibitor-exos increased MDA levels and suppressed SOD production. (k and l) The expression of apoptosis-related proteins, such as procaspase-3, cleaved caspase-3, Bax, and Bcl-2 were detected using immunoblotting. Compared with H₂O₂-treated cells, the cells treated with Hypoxic-exos or miR-214 mimics displayed substantially decreased cleaved caspase-3 and Bax expression and increased Bcl-2 expression. However, compared with Hypoxic-exos, miR-214 inhibitors or inhibitor-exos significantly increased cleaved caspase-3 and Bax expression but decreased Bcl-2 expression, ; compared with the H₂O₂ group; compared with the Hypoxic-exos group.

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