Department of Internal Medicine-Cardiovascular, Jinan Fourth People's Hospital, Jinan, China.
Eur Rev Med Pharmacol Sci. 2020 Dec;24(24):12904-12911. doi: 10.26355/eurrev_202012_24194.
The aim of this study was to explore the influence of micro ribonucleic acid (miR)-145-5p on myocardial cell apoptosis in rats with myocardial infarction (MI) through the phosphatidylinositol 3-hydroxy kinase/protein kinase B (PI3K/Akt) pathway.
In this study, Sprague-Dawley rats were used as research objects to establish the acute MI model in vivo. Infarction tissues and non-infarction tissues were both collected from rats. The expression level of miR-145-5p was determined using quantitative Polymerase Chain Reaction (qPCR), and the pathological changes in myocardial tissues of rats were observed through hematoxylin-eosin (HE) staining. In addition, H9c2 rat myocardial cells were cultured under hypoxia or normal oxygen concentration to simulate hypoxia in MI tissues. The changes in the expression of miR-145-5p in H9c2 cells in normal oxygen and hypoxia were determined. Meanwhile, the ratio of apoptotic cells to viable cells, and the changes in the expressions of proteins B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax), Caspase-3 and Caspase-9 were evaluated through flow cytometry assay and Western blotting, respectively. The expression levels of crucial proteins in the PI3K/Akt pathway were measured as well. Additionally, H9c2 cells were transfected with miR-145-5p control and miR-145-5p mimic to evaluate cell apoptosis.
QPCR results revealed that the expression level of miR-145-5p was substantially elevated in MI tissues (p<0.05). HE results indicated that the soma exhibited deformation after MI, suggesting that there were more necrotic and apoptotic cells. Compared with those cultured under normal oxygen concentration, H9c2 cells cultured in hypoxia environment exhibited significantly upregulated expression level of miR-145-5p, downregulated expression level of anti-apoptosis protein Bcl-2, upregulated level of pro-apoptosis protein Bax, activated Caspase-3 and Caspase-9, and downregulated expression level of functional proteins in the PI3K/Akt pathway (p<0.05). Furthermore, the expression levels of apoptosis-associated proteins significantly rose in H9c2 cells transfected with miR-145-5p mimic compared with those transfected with miR-145-5p control, showing statistically significant differences (p<0.05).
MiR-145-5p is notably raised in MI tissues of rats. After infarction, there are evidently more apoptotic myocardial cells. The expression of miR-145-5p is markedly elevated in H9c2 rat myocardial cells in hypoxia. Compared with those cultured in normal oxygen, H9c2 cells cultured in hypoxia showed increased apoptosis. The apoptosis of myocardial cells transfected with miR-145-5p mimic is notable higher than that of myocardial cells transfected with miR-145-5p control. Moreover, the expressions of active Akt and PI3K proteins decrease remarkably. The results of this study demonstrate that miR-145-5p inactivates the PI3K/Akt pathway to promote the apoptosis of MI cells.
本研究旨在通过磷脂酰肌醇 3-激酶/蛋白激酶 B（PI3K/Akt）通路探讨微小 RNA-145-5p 对心肌梗死（MI）大鼠心肌细胞凋亡的影响。
本研究采用 Sprague-Dawley 大鼠作为研究对象，建立体内急性 MI 模型。收集大鼠的梗死组织和非梗死组织。采用实时定量聚合酶链反应（qPCR）检测 miR-145-5p 的表达水平，通过苏木精-伊红（HE）染色观察大鼠心肌组织的病理变化。此外，在缺氧或正常氧浓度下培养 H9c2 大鼠心肌细胞，模拟 MI 组织中的缺氧。检测正常氧和缺氧条件下 H9c2 细胞中 miR-145-5p 的表达变化。同时，通过流式细胞术检测细胞凋亡率，Western blot 检测 B 细胞淋巴瘤 2（Bcl-2）、Bcl-2 相关 X 蛋白（Bax）、Caspase-3 和 Caspase-9 蛋白的表达变化。还测量了 PI3K/Akt 通路中关键蛋白的表达水平。此外，用 miR-145-5p 对照和 miR-145-5p 模拟物转染 H9c2 细胞，评估细胞凋亡。
qPCR 结果显示，MI 组织中 miR-145-5p 的表达水平显著升高（p<0.05）。HE 结果表明，MI 后细胞体变形，提示坏死和凋亡细胞增多。与正常氧浓度培养的细胞相比，缺氧环境培养的 H9c2 细胞中 miR-145-5p 的表达水平显著上调，抗凋亡蛋白 Bcl-2 的表达水平下调，促凋亡蛋白 Bax 的表达水平上调，Caspase-3 和 Caspase-9 被激活，PI3K/Akt 通路中的功能蛋白表达水平下调（p<0.05）。此外，与转染 miR-145-5p 对照的细胞相比，转染 miR-145-5p 模拟物的 H9c2 细胞中凋亡相关蛋白的表达水平显著升高，差异具有统计学意义（p<0.05）。
大鼠 MI 组织中 miR-145-5p 显著升高。梗死后，心肌细胞凋亡明显增多。H9c2 大鼠心肌细胞在缺氧时 miR-145-5p 的表达明显升高。与正常氧培养相比，缺氧培养的 H9c2 细胞凋亡增加。转染 miR-145-5p 模拟物的心肌细胞凋亡明显高于转染 miR-145-5p 对照的心肌细胞。此外，活性 Akt 和 PI3K 蛋白的表达明显下降。本研究结果表明，miR-145-5p 通过抑制 PI3K/Akt 通路促进 MI 细胞凋亡。
