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  

MicroRNA-21 protects cardiomyocytes from tumor necrosis factor-α induced apoptosis in vitro via modulating PTEN/AKT/FOXO3a pathway

作　　者：唐艳[1] 王梦洪[1] 

机构地区：[1]南昌大学医学院第一附属医院心内科,330006

出　　处：《中华心血管病杂志》2013年第2期135-142,共8页Chinese Journal of Cardiology

基　　金：国家科技支撑计划（2008BA168BA02）

摘　　要：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／又头蛋白3a（PTEN／AKT／FOX03a）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 10ng／ml组（TNF—α组）以及microRNA-21＋TNF-α 10ng／ml组（miR．21＋TNF—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．d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PTENmRNA的表达水平，Westernblot检测VFEN、磷酸化PTEN（pPTEN）、AKT、磷酸化AKT（pAKTser473、pAKTThr308）、FOX03a、磷酸化FOX03a（pFOX03a）和FasL表达情况。结果miR-21＋TNF．伐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PTENmRNA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PTENmRNA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TNF-α（10ng／m1）诱导的心肌细胞凋亡的靶基因之一。Westernblot结果提示转染过表达microRNA-21的重组慢病毒载体人心肌细胞能激活PTEN、AKT、FOX03a的磷酸化，TNF-α能更显著地激活PTEN、AKT、FOX03a的磷酸化，而microRNA-21和TNF-α共同作用则可抑制PTEN、AKT、FOX03a的磷酸化，磷酸化PTEN、AKT、FOX03a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／FOX03a信号传导通路实现的。本实验为用microRNA-21诊断及治疗心血管Objective To explore the effect of mieroRNA-21 on tumor necrosis factor （TNF）-a induced eardiomyocytes apoptosis and the association with PTEN/AKT/FOXO3a signaling pathway. Methods Neonatal eardiomyoeytes were isolated and cultured in vitro. Cardiomyocytes apoptosis was induced by TNF-α （ 10 ng/m] for 24 h） and examined by the caxdiomyocytes apoptotic index. Eukaryotic expression vector for lenti-mieroRNA-21 was established and then transferred into the eardiomyocytes. MicroRNA-21 and PTEN mRNA were examined by qRT-PCR. Intracellular signal molecules, such as the expression of PTEN,phosphorylated PTEN, AKT, phosphorylated AKT （pAKTe473, pAKTThr30s ）, 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, pAKT473, pFOXO3a and FasL were significantly suppressed in cardiomyocytes treated with lenti-microRNA-21 and TNF-α （ P 〈 O. 05 ）. Total AKT and FOXO3a were silhilar among all groups （P 〉 O. 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.

关 键 词：肌细胞 心脏 细胞凋亡 微RNAS 信号传导 

分 类 号：R285.5[医药卫生—中药学]