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miR-21 reduces hydrogen peroxide- (H2O2-) induced apoptosis in c-kit+ CSC and estimated the contribution of PTEN/PI3K/Akt signaling to this oxidative circumstance.
CSCs were isolated from rat atrial appendage and purified using anti-rabbit secondary antibody conjugated magnetic beads. Harvested CSCs were treated with H2O2 100 uM for 2 h. miR-21 mimics, miR-21 inhibitor, and their control scrambles with transfection reagent Lipofectamine 2000,Early apoptosis and necrosis of CSCs were determined by flow cytometry using Annexin V-FITC/PI staining assay . miR-21 and PTEN mRNA levels were determined by using quantitative RT-PCR. Western blot analysis of apoptosis-related protein from c-kit+ cell.
miR-21 mimics efficiently reducedH2O2-induced apoptosis in c-kit+ CSC, as evidenced by the downregulation of the proapoptosis proteins caspase-3 and Bax and upregulation of the antiapoptotic Bcl-2. In addition, the gain of function of miR-21 in c-kit+ CSC downregulated the protein level of PTEN although its mRNA level changed slightly; in the meantime, miR-21 overexpression also increased phospho-Akt (p-Akt).The antiapoptotic effects of miR-21 were comparable with Phen (bpV), the selective inhibitor of PTEN, while miR-21 inhibitor or PI3K’s inhibitor LY294002 efficiently attenuated the antiapoptotic effect of miR-21.
these results indicate that the anti-H2O2-induced apoptosis effect of miR-21 in c-kit+ CSC is contributed by PTEN/PI3K/Akt signaling. miR-21 could be a potential molecule to facilitate the c-kit+ CSC therapy in ischemic myocardium.