Department of Cardiovascular Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei 230022, China.
Department of Cardiovascular Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei 230022, China.
Biomed Pharmacother. 2017 Dec;96:812-817. doi: 10.1016/j.biopha.2017.09.148. Epub 2017 Nov 6.
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.
本研究旨在探讨 miR-23b 在低氧心肌细胞中的作用及其潜在机制。
收集青紫型和非青紫型先天性心脏病（CHD）患者的心肌样本，评估 miR-23b 的表达。将 miR-23b 的激动剂或拮抗剂转染到 H9C2 细胞中。在低氧条件下，通过 MTT、LDH 测定和 TUNEL 染色检测细胞增殖和凋亡。此外，通过 Western blot 和 qRT-PCR 分别测定低氧 H9C2 细胞中 cleaved-caspase-3、cleaved-PARP、Bad、Bcl-2 和 Bax 的表达水平。
与非青紫型 CHD 患者相比，青紫型 CHD 患者的 miR-23b 表达水平更高。此外，在 H9C2 细胞中，随着缺氧时间的延长，miR-23b 的表达逐渐上调。通过 MTT 和 LDH 测定，agomir 组的细胞生长明显低于 agomir-NC 组，而 antagomir 则增加了细胞生长。通过 TUNEL 染色和流式细胞术分析，miR-23b 促进了低氧诱导的细胞凋亡。在 agomir 组中，促凋亡蛋白如 cleaved-caspase-3、cleaved-PARP 和 Bad 的表达水平显著增加，而 Bcl-2 水平和 Bcl-2/Bax 比值降低。相反，在 antagomir 组中观察到相反的趋势。双荧光素酶报告基因检测和 Western blot 分析证实 Smad3 是 miR-23b 的直接靶标。
miR-23b 的过表达可能会增加低氧条件下心肌细胞的凋亡并减少细胞生长。
