miR-34a 通过 Wnt/β-catenin 信号通路调节大鼠心肌梗死后细胞凋亡。
MiR-34a regulates cell apoptosis after myocardial infarction in rats through the Wnt/β-catenin signaling pathway.
机构信息
Department of Cardiac Surgery, Beijing Anzhen Hospital, Capital Medical University, Beijing, China.
出版信息
Eur Rev Med Pharmacol Sci. 2019 Mar;23(6):2555-2562. doi: 10.26355/eurrev_201903_17404.
OBJECTIVE
To explore the molecular mechanism of micro ribonucleic acid-34a (miR-34a) in promoting the apoptosis of myocardial cells in the rat model of myocardial infarction (MI).
MATERIALS AND METHODS
Sprague-Dawley (SD) rats were ligated with a left anterior descending branch to construct the MI model. The rats were randomly divided into four groups: sham operation group (Sham group), MI group, MI + miR-34a inhibitor group (MI + miR-34a antagomir group) and MI + miR-34a inhibitor negative control group (MI + antagomir NC group). Echocardiography (ECG) and magnetic resonance imaging (MRI) were adopted to detect the ejection fraction [EF (%)] and fraction shortening [FS (%)] of SD rats. Polymerase chain reaction (PCR) and Western blotting were used to detect expression levels of the apoptotic marker Caspase-3 and genes in Wnt/β-catenin signaling pathway. Hematoxylin and eosin (H&E) staining was applied to detect cardiac injury. In in vitro experiments, the rat-derived myocardial cell line H9C2 was selected to simulate myocardial ischemia and hypoxia at the time of MI with an anoxic and serum-free injury model. C59, the Wnt/β-catenin signaling pathway inhibitor was applied in MI + miR-34a antagomir + C59 group, and the effect of miR-34a on the apoptosis of myocardial cells through regulating the Wnt/β-catenin pathway was measured with Real-Time quantitative PCR (qPCR) and 3-(4,5)-dimethylthiazol(-z-y1)-3,5-diphenyltetrazolium bromide (MTT) cell activity detection kits, respectively.
RESULTS
It was found that after miR-34a antagomir reversed FS (%) and EF (%) in MI rats, the messenger RNA (mRNA) and protein levels of Caspase-3 in Sham group and MI + miR-34a antagomir group were significantly lower than those in the MI group (p < 0.05), indicating that the addition of miR-34a antagomir inhibited myocardial cell apoptosis after infarction, while the mRNA and protein levels of Wnt/β-catenin were both higher than those in the MI group. Besides, H&E staining proved that miR-34a reversed the myocardial injury after MI. Similarly, in vitro experiments showed that, compared with those in Hypoxia group, the level of Caspase-3 decreased in Hypoxia + miR-34a inhibitor group and Sham group, while the apoptosis level in Hypoxia + miR-34a inhibitor + C59 group increased (p < 0.05). The results of the MTT assay were consistent with those of PCR. MiR-34a affects myocardial cell apoptosis by regulating the activation and inactivation of the Wnt/β-catenin signaling pathway.
目的
探讨微小 RNA-34a（miR-34a）在促进心肌梗死（MI）大鼠模型中心肌细胞凋亡中的分子机制。
材料与方法
结扎 Sprague-Dawley（SD）大鼠左前降支构建 MI 模型。将大鼠随机分为四组：假手术组（Sham 组）、MI 组、MI+miR-34a 抑制剂组（MI+miR-34a 反义寡核苷酸组）和 MI+miR-34a 抑制剂阴性对照组（MI+反义寡核苷酸 NC 组）。采用超声心动图（ECG）和磁共振成像（MRI）检测 SD 大鼠的射血分数[EF（%）]和短轴缩短率[FS（%）]。聚合酶链反应（PCR）和 Western blot 检测凋亡标志物 Caspase-3 和 Wnt/β-catenin 信号通路基因的表达水平。苏木精和伊红（H&E）染色检测心脏损伤。在体外实验中，选择大鼠源性心肌细胞系 H9C2 模拟 MI 时的心肌缺血缺氧，采用缺氧无血清损伤模型。在 MI+miR-34a 反义寡核苷酸+C59 组中应用 Wnt/β-catenin 信号通路抑制剂 C59，通过实时定量 PCR（qPCR）和 3-（4,5）-二甲基噻唑（-z-y1）-3,5-二苯基四唑溴盐（MTT）细胞活性检测试剂盒分别测量 miR-34a 通过调节 Wnt/β-catenin 通路对心肌细胞凋亡的影响。
结果
发现 miR-34a 反义寡核苷酸逆转 MI 大鼠 FS（%）和 EF（%）后，Sham 组和 MI+miR-34a 反义寡核苷酸组的 Caspase-3 的信使 RNA（mRNA）和蛋白水平均明显低于 MI 组（p<0.05），表明 miR-34a 反义寡核苷酸抑制梗死后心肌细胞凋亡，而 Wnt/β-catenin 的 mRNA 和蛋白水平均高于 MI 组。此外，H&E 染色证实 miR-34a 逆转了 MI 后的心肌损伤。同样，在体外实验中，与缺氧组相比，缺氧+miR-34a 抑制剂组和 Sham 组 Caspase-3 水平降低，而缺氧+miR-34a 抑制剂+C59 组的凋亡水平升高（p<0.05）。MTT 检测结果与 PCR 结果一致。miR-34a 通过调节 Wnt/β-catenin 信号通路的激活和失活来影响心肌细胞凋亡。
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