MicroRNA-766 contributes to high glucose-induced cardiomyocyte apoptosis by targeting exchange protein directly activated by cyclic adenylic acid isoform 1

Objective To investigate the expression levels and functional role of microRNA 766 (miR-766) in cardiomyocytes treated with high glucose conditions. Method Cardiomyocytes were cultured with different concentrations of glucose levels at 5.0 mmol/L (Controls), 15, 25 or 35 mmol/L, respectively. The expression of miR-766 was detected by real-time PCR, apoptosis was detected by flow cytometry, and the expressions of exchange protein directly activated by cyclic adenylic acid isoform 1 (Epac-1), Bax and cleaved cysteinyl aspartate specific proteinase-3 (caspase-3) were determined by Western blot. The target gene of miR-766 calculated by bioinformatics was further determined by using dual luciferase system. T test was used for comparison between groups. Results Compared with the controls, the expressions of miR-766 were significantly upregulated in different high glucose (HG)-stimulated cardiomyocytes (3.40±0.38, 4.61±0.34, 6.17±0.42 vs 0.97±0.04, t=11.131-21.493, all P<0.05). Inhibition of miR-766 in cardiomyocytes treated with HG resulted in reduced apoptosis [(16.43±0.31)% vs (26.45±0.31)%, t=-39.566, P<0.05]. Overexpression of miR-766 decreased the protein levels of Epac-1 (0.48±0.07 vs 1.00±0.12, t=-6.695, P<0.05), and inhibition of miR-766 increased the protein levels of Epac-1 (1.23±0.12 vs 0.70±0.10, t=5.884, P<0.05). Luciferase reporter assays showed that overexpression of miR-766 significantly decreased the luciferase activity of wild-type reporter, and inhibition of miR-766 significantly increased the luciferase activity of wild-type reporter (3.22±0.07 vs 5.01±0.96; 6.98±0.61 vs 5.01±0.96, t=-3.239--3.008, P<0.05). Moreover, transfection of miR-766 inhibitor could partly reverse the suppression of Epac-1 by HG in H9c2 cells (0.80±0.05 vs 0.53±0.05, t=6.810, P<0.05). Restoration of Epac-1 in miR-766-treated H9c2 cells reversed the effects of miR-766 on cleaved caspase-3 expression (0.67±0.07 vs 1.07±0.12, t=-5.050, P<0.05). Conclusion miR-766 plays an essential role in high glucose-induced cardiomyocyte apoptosis through regulating Epac-1 expression. Key words: Diabetic cardiomyopathies; Apoptosis; MicroRNAs, miR-766; Exchange protein directly activated by cyclic adenylic acid isoform 1