Effects of miR-23b on hypoxia-induced cardiomyocytes apoptosis

The aim of this study was to investigate the role of miR-23b in hypoxic cardiomyocytes and the potential mechanism. Myocardial samples of patients with cyanotic or acyanotic congenital heart disease (CHD) were collected to evaluate miR-23b expression. Agomir or antagomir of miR-23b was transfected into H9C2 cells. MTT, LDH assay and TUNEL staining were used to determine the cell proliferation and apoptosis under hypoxic conditions. Besides, the expression levels of cleaved-caspase-3, cleaved-PARP, Bad, Bcl-2 and Bax in hypoxic H9C2 cells were determined by western blot and qRT-PCR, respectively. Higher miR-23b expression levels were found in the patients with cyanotic CHD compared with the patients with acyanotic CHD. In addition, the expression of miR-23b was gradually up-regulated with prolonged hypoxia time in the H9C2 cells. Using MTT and LDH assays, cell growth was significantly decreased in the agomir group than that in the agomir-negative control (NC) group, while antagomir increased the cell growth. Using TUNEL staining and flow cytometry analysis, miR-23b promoted hypoxia-induced apoptosis. The expression levels of pro-apoptotic proteins, such as cleaved-caspase-3, cleaved-PARP and Bad, were significantly increased in the agomir group, while the Bcl-2 levels and Bcl-2/Bax ratio were decreased. Opposite tendency was observed in the antagomir group. Dual luciferase reporter assay and western blot analysis confirmed that Smad3 was a direct target of miR-23b. Over-expression of miR-23b may increase cardiomyocyte apoptosis and reduce cell growth under hypoxic conditions.