Tang Yan, Wang Meng-hong
Department of Cardiology, First Affiliated Hospital of Nanchang University, Medical College of Nanchang University, Nanchang 330006, China.
Zhonghua Xin Xue Guan Bing Za Zhi. 2013 Feb;41(2):135-42.
To explore the effect of microRNA-21 on tumor necrosis factor (TNF)-α induced cardiomyocytes apoptosis and the association with PTEN/AKT/FOXO3a signaling pathway.
Neonatal cardiomyocytes were isolated and cultured in vitro. Cardiomyocytes apoptosis was induced by TNF-α (10 ng/ml for 24 h) and examined by the cardiomyocytes apoptotic index. Eukaryotic expression vector for lenti-microRNA-21 was established and then transferred into the cardiomyocytes. MicroRNA-21 and PTEN mRNA were examined by qRT-PCR. Intracellular signal molecules, such as the expression of PTEN, phosphorylated PTEN, AKT, phosphorylated AKT (pAKTser473, pAKTThr308), FOXO3a, phosphorylated FOXO3a and FasL were detected by Western blot.
MicroRNA-21 reduced TNF-α induced cardiomyocytes apoptosis [(23.42 ± 1.98)% vs. (78.37 ± 2.03)%, P < 0.05]. TNF-α downregulated the expression of microRNA-21 and upregulated the mRNA and protein expressions of PTEN. Phosphorylation of PTEN, AKT and FOXO3a was enhanced in cardiomyocytes transfected with lenti-microRNA-21 (P < 0.05). TNF-α also significantly activated the phosphorylation of PTEN, AKT and FOXO3a (P < 0.05). Compared with cardiomyocytes treated with TNF-α (10 ng/ml), the phosphorylation of PTEN, AKT and FOXO3a as well as expression of pPTEN, pAKTser473, pFOXO3a and FasL were significantly suppressed in cardiomyocytes treated with lenti-microRNA-21 and TNF-α (P < 0.05). Total AKT and FOXO3a were similar among all groups (P > 0.05).
MicroRNA-21 could protect cardiomyocytes from TNF-α- induced apoptosis through PTEN/AKT/FOXO3a pathway, which might serve as a new therapy option for various cardiovascular diseases in the future.
探讨微小RNA-21对肿瘤坏死因子（TNF）-α诱导的心肌细胞凋亡的影响及其与PTEN/AKT/FOXO3a信号通路的关系。
分离新生大鼠心肌细胞并进行体外培养。用TNF-α（10 ng/ml，作用24小时）诱导心肌细胞凋亡，通过心肌细胞凋亡指数检测凋亡情况。构建慢病毒微小RNA-21真核表达载体，然后将其转入心肌细胞。采用qRT-PCR检测微小RNA-21和PTEN mRNA。通过蛋白质免疫印迹法检测细胞内信号分子，如PTEN、磷酸化PTEN、AKT、磷酸化AKT（pAKTser473、pAKTThr308）、FOXO3a、磷酸化FOXO3a和FasL的表达。
微小RNA-21减少了TNF-α诱导的心肌细胞凋亡[（23.42±1.98）%对（78.37±2.03）%，P<0.05]。TNF-α下调微小RNA-21的表达，上调PTEN的mRNA和蛋白表达。在转染慢病毒微小RNA-21的心肌细胞中，PTEN、AKT和FOXO3a的磷酸化增强（P<0.05）。TNF-α也显著激活了PTEN、AKT和FOXO3a的磷酸化（P<0.05）。与用TNF-α（10 ng/ml）处理的心肌细胞相比，用慢病毒微小RNA-21和TNF-α处理的心肌细胞中，PTEN、AKT和FOXO3a的磷酸化以及pPTEN、pAKTser473、pFOXO3a和FasL的表达均显著受到抑制（P<0.05）。各组间总AKT和FOXO3a相似（P>0.05）。
微小RNA-21可通过PTEN/AKT/FOXO3a途径保护心肌细胞免受TNF-α诱导的凋亡，这可能为未来各种心血管疾病提供一种新的治疗选择。
