微小RNA-21对乳鼠心肌细胞凋亡及磷酸脂酶-张力蛋白同源物/丝氨酸/苏氨酸激酶/叉头蛋白3a信号传导通路的影响

摘要

Objective 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.Methods 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.Results 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-o (P ＜ 0.05).Total AKT and FOXO3a were similar among all groups (P ＞ 0.05).Conclusions 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.%目的 探讨微小(micro) RNA-21对肿瘤坏死因子(TNF)-α诱导的心肌细胞凋亡的影响,以及磷酸脂酶-张力蛋白同源物/丝氨酸/苏氨酸激酶/叉头蛋白3a (PTEN/AKT/FOXO3a)信号传导通路在其中的作用.方法 体外分离培养乳鼠心肌细胞,利用TNF-α诱导建立心肌细胞凋亡模型,以心肌细胞凋亡率作为心肌细胞凋亡的观察指标.实验分为正常心肌细胞组(CM组),microRNA-21组(miR-21组),TNF-α 10 ng/ml组(TNF-α组)以及microRNA-21+ TNF-α 10 ng/ml组(miR-21+NF-α组).通过建立过表达microRNA-21重组慢病毒载体并将其转染入心肌细胞中,观察microRNA-21对TNF-α诱导的心肌细胞凋亡的影响,应用实时定量(qRT)-PCR法检测各组心肌细胞中microRNA-21及PTEN mRNA的表达水平,Western blot检测PTEN、磷酸化PTEN(pPTEN)、AKT、磷酸化AKT(pAKTser473、pAKTThr308)、FOXO3a、磷酸化FOXO3a (pFOXO3a)和FasL表达情况.结果 miR-21+ TNF-α组心肌细胞凋亡率显著低于TNF-α组[(23.42±1.98)％比(78.37±2.03)％,P＜0.05].TNF-α组microRNA-21的相对表达量为CM组的0.5倍(P＜0.05),TNF-α组PTEN mRNA的相对表达量约为CM组1.8倍(P＜0.05),且PTEN蛋白表达情况与PTEN mRNA表达的情况一致,提示在基因和蛋白水平上,PTEN可能为microRNA-21作用于NF-α(10 ng/ml)诱导的心肌细胞凋亡的靶基因之一.Western blot结果提示转染过表达microRNA-21的重组慢病毒载体入心肌细胞能激活PTEN、AKT、FOXO3a的磷酸化,TNF-α能更显著地激活PTEN、AKT、FOXO3a的磷酸化,而microRNA-21和NF-α共同作用则可抑制PTEN、AKT、FOXO3a的磷酸化,磷酸化PTEN、AKT、FOXO3a的表达变化趋势具有一致性.结论 microRNA-21能够抑制TNF-α诱导的心肌细胞凋亡,该作用可能是通过介导PTEN/AKT/FOXO3a信号传导通路实现的.本实验为用microRNA-21诊断及治疗心血管疾病提供了新的靶点.