Objective To investigate the role and mechanism of miR-203a-3p in myocardial hypoxia/reoxygenation injury.
Methods The hypoxia/reoxygenation cells (H/R) and ischemia/reperfusion animal models (I/R) were constructed, and the expression level of miR-203a-3p in each group was detected by quantitative real-time PCR (RT-PCR). The cells were divided into the control group, H/R groupH9c2 cell line treated with (H/R), H/R + miR-NC group (H/R processing H9c2 cell line transfected with miR-NC) and H/R + miR-203a-3p inhibitor group (H/R processing H9c2 cell line transfected with miR-203a-3p- inhibitor). The flow cytometry and TUNEL method were used to detect the apoptosis rate of cells in each group, and the levels of CK and LDH in extracellular fluid in four groups were biochemically detected. Western blotting was used to detect the expression levels of Hspb8 in H9c2 and HCM cells after hypoxia/reoxygenation treatment. The potential target of miR-203a-3p, Hspb8, was predicted by TargetScan online tool, and verified by double luciferase reporting experiment. The miR-203a-3p-mimic was transfected into H9c2 cells, and the expression level of Hspb8 was detected by Western blotting. To further verify the function of Hspb8, the cells were divided into the Control + miR-NC + shRNA-Con group, H/R + miR-NC + shRDNA-Con group, H/R + miR-203a-3p-inhibitor + shRNA-Con group and H/R + miR-203a-3p-inhibitor + shRNA-Hspb8 group, and the apoptosis rate of cells in each group was detected by flow cytometry, and the levels of CK and LDH in extracellular fluid of each group were biochemically detected.
Results The expression levels of miR-203a-3p significantly increased in the H/R-treated H9c2 cell lines and I/R-treated rat myocardial tissues (P ＜ 0.01). Down-regulating the expression of miR-203a-3p could inhibit the H/R-induced apoptosis in H9c2 cells (P ＜ 0.01) and decrease the CK and LDH activities in cell cultures (P ＜ 0.01). The relative luciferase activity of Hspb8 transfected with miR-203a-3p-mimic was significantly reduced in WT group (wild-type) H9c2 and HCM cells (P ＜ 0.01). The overexpression of miR-203a-3p could improve the Hspb8 expression in cells (P ＜ 0.01). Compared with the H/R + miR-203a-3p-inhibitor + shRNA-Con group, the apoptosis rate of H9c2 cells in the H/R + miR-203a-3p-inhibitor + shRNA-Hspb8 group significantly increased (P ＜ 0.05), and the activities of CK and LDH in the cell culture medium significantly increased (P ＜ 0.01).
Conclusions MiR-203a-3p inhibits hypoxia/reoxygenation injury of cardiomyocytes by targeting Hspb8.
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