Weilai He1, Hong Che2, Chaolong Jin3, Shenglin Ge2. 1. Department of Cardiovascular Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei 230022, China. Electronic address: weilaihe@yeah.net. 2. Department of Cardiovascular Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei 230022, China. 3. Department of Cardiology, The First Affiliated Hospital of Anhui Medical University, Hefei 230022, China.
Abstract
OBJECTIVES: The aim of this study was to investigate the role of miR-23b in hypoxic cardiomyocytes and the potential mechanism. METHODS: 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. RESULTS: 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. CONCLUSION: Over-expression of miR-23b may increase cardiomyocyte apoptosis and reduce cell growth under hypoxic conditions.
OBJECTIVES: The aim of this study was to investigate the role of miR-23b in hypoxic cardiomyocytes and the potential mechanism. METHODS: 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. RESULTS: 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. CONCLUSION: Over-expression of miR-23b may increase cardiomyocyte apoptosis and reduce cell growth under hypoxic conditions.