We tested the hypothesis that Let-7d-3p contributes to cardiac cell protection during hypoxic challenge. Myoblast H9c2 cells and primary neonatal rat ventricular cardiomyocytes (NRVM) were transfected with five selected miRNA mimics. Both cell lines were subjected to 0.2% oxygen hypoxia. The protective effects of these miRNAs were determined by assessment of cell metabolic activity by CCK8 assay and measurement of lactate dehydrogenase (LDH) release as a marker of cell injury. Apoptosis and autophagy flux were assessed by Annexin V/7-AAD double staining and the ratio of LC3 II/I with Baf-A1 treatment, an autophagy flux inhibitor, respectively. Luciferase-reporter assay, RT-qPCR and Western blots were performed to identify the changes of relevant gene targets. Among five miRNA mimic transfections, Let-7d-3p increased CCK8 activity, and decreased LDH release in both H9c2 and NRVM during hypoxia. Apoptosis was significantly reduced in H9c2 cells transfected with Let-7d-3p mimic. Autophagy and autophagy flux were not affected. In silico, mRNAs of HMGA2, YY1, KLF9, KLF12, and MEX3C are predicted targets for Let-7d-3p. Luciferase-reporter assay confirmed that Let-7d-3p bound directly to the 3'-UTR region of HMGA2, MEX3C, and YY1, the down-regulations of these mRNAs were verified in both H9c2 and NRVM. The protein expression of HMGA2, but not others, was downregulated in H9c2 and NRVM. It is known that HMGA2 is a strong apoptosis trigger through the blocking of DNA repair. Thus, we speculate that the anti-apoptotic effects of Let-7d-3p mimic during hypoxia challenge are due to direct targeting of HMGA2.
Keywords: HMGA2; Let-7d-3p; apoptosis; cardiac protection; microRNA.