Mechanism of microRNA-1 regulating H9c2 cardiomyocyte apoptosis after hypoxia/reoxygenation

Abstract

To investigate the changes of cardiomyocyte apoptosis after hypoxia/reoxygenation (H/R) regulated by microRNA-1 (miR-1). Cardiomyocyte strain H9c2 derived from rat embryonic heart tissue were cultured in vitro. The cells in logarithmic growth phase were divided into blank control group, H/R group, miR-1 mimics+H/R group, miR-1 inhibitor antisense oligonucleotide (ASO)+H/R group and microRNA negative control fragment (miRNA NC)+H/R group. The low sugar DMEM medium containing low concentration of fetal bovine serum (FBS) was used as the medium under anoxic condition. After being cultured in a closed anaerobic incubator at 37 centigrade (95% N2 and 5% CO2) for 12 hours, the cells were cultured with the fresh high sugar DMEM medium containing 5% FBS in a closed incubator at 37 centigrade for reproducing cardiomyocyte H/R model. The blank control group was cultured in high glucose DMEM medium containing 10% FBS in 37 centigrade and 5% CO2 incubator. In miR-1 mimics+H/R group, miR-1 ASO+H/R group and miRNA NC+H/R group, the corresponding transfectants were mixed in high glucose DMEM medium and transfected into cells before H/R model was established, and the final concentration was 50 nmol/L. The blank control group and H/R group were added with DMEM medium at the same time. After the establishment of the model, the expression level of miR-1 was detected by real-time fluorescence quantitative polymerase chain reaction (qPCR). The expression levels of apoptosis-related proteins caspase-9, Bcl-2 and Bax were detected by Western blotting, and cardiomyocyte apoptosis was detected by flow cytometry. Compared with the blank control group, the expression levels of miR-1, caspase-9 and Bax protein and the apoptosis rate of cardiomyocytes were significantly increased, while the expression level of Bcl-2 was significantly decreased, which indicated that the expression of miR-1 and the level of apoptosis were increased in H/R group. Compared with H/R group, the expressions of miR-1, caspase-9 and Bax and the apoptosis rate of cardiomyocytes in miR-1 mimics+H/R group were further increased [miR-1 (2-ΔΔCt): 11.59±1.48 vs. 2.57±0.38, caspase-9 protein (caspase-9/β-actin): 2.59±0.12 vs. 1.56±0.20, Bax protein (Bax/β-actin): 4.09±0.38 vs. 1.97±0.13, apoptosis rate: (25.23±0.87)% vs. (17.86±0.73)%, all P < 0.01], while the expression of Bcl-2 was decreased (Bcl-2/β-actin: 0.37±0.02 vs. 0.49±0.03, P < 0.01). The expressions of miR-1, caspase-9 and Bax and the apoptosis rate were significantly decreased in miR-1 ASO+H/R group [miR-1 (2-ΔΔCt): 1.16±0.06 vs. 2.57±0.38, caspase-9 protein (caspase-9/β-actin): 1.05±0.24 vs. 1.56±0.20, Bax protein (Bax/β-actin): 0.93±0.11 vs. 1.97±0.13, apoptosis rate: (11.19±0.85)% vs. (17.86±0.73)%, all P < 0.05], while the expression of Bcl-2 was increased (Bcl-2/β-actin: 0.84±0.17 vs. 0.49±0.03, P < 0.05). There was no significant difference in miR-1 expression, caspase-9, Bax and Bcl-2 protein expressions, and apoptosis rate between H/R+miRNA NC group and H/R group. The expression of miR-1 and level of apoptosis were increased in H/R cells, and miR-1 could aggravate cardiomyocyte apoptosis.