Knockdown of miR-665 Protects Against Cardiomyocyte Ischemia/Reperfusion Injury-Induced ROS Accumulation and Apoptosis Through the Activation of Pak1/Akt Signaling in Myocardial Infarction
Knockdown of miR-665 Protects Against Cardiomyocyte Ischemia/Reperfusion Injury-Induced ROS Accumulation and Apoptosis Through the Activation of Pak1/Akt Signaling in Myocardial Infarction
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DOI：
10.1536/ihj.19-416
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发表时间：
2020-03-01
影响因子：
1.5
通讯作者：
Cao, Guangqing
中科院分区：
文献类型：
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作者：
Liu, Chuanzhen;Tang, Mengmeng;Cao, Guangqing
心肌梗死(MI)是世界范围内的主要死亡原因之一，其治疗策略仍然有限。本研究探讨miR-665在心肌梗死中的作用。本研究成功地建立了大鼠缺血/再灌注(I/R)模型和缺氧/复氧(H/R)诱导的H9c2细胞模型，以模拟MI进行体内和体外研究。测定乳酸脱氢酶(LDH)、肌酸激酶同工酶(CK-MB)、肿瘤坏死因子-α(TNF-α)、白介素6(IL-6)和活性氧(ROS)的含量。采用四甲基偶氮唑盐比色法、TdT介导的dUTP缺口末端标记法和PI/FITC-Annexin V法检测细胞存活率和凋亡率。荧光素酶分析法检测miR-665与pak-1的结合。MIR-665在I/R大鼠心肌细胞中表达上调，miR-665过表达显著增加LDH、CK-MB、TNF-α、IL-6和ROS浓度并诱导细胞凋亡，而miR-665过表达则相反。与体内结果一致，miR-665诱导H/R处理的H9c2细胞凋亡和ROS产生。更重要的是，pak1是miR-665的靶基因，miR-665的敲除通过靶向pak1和促进Akt的磷酸化来抑制ROS的积聚和细胞的凋亡，而pak1的敲除可以减弱miR-665抑制剂对H/R处理的H9c2细胞的保护作用。因此，miR-665基因敲除对心肌缺血/再灌注损伤所致的ROS积聚和细胞凋亡具有保护作用，其机制可能是通过激活心肌细胞内的Ak1/Akt信号通路来实现的。总体而言，了解miR-665的生物学和调控可能有可能抵消MI的发展。
Myocardial infarction (MI) is one of the major causes of death worldwide, and the therapeutic strategies of MI are still limited. In this study, we investigated the function of miR-665 in MI. In the present study, an ischemia/reperfusion (I/R) rat model and a hypoxia/reoxygenation (H/R)-induced H9c2 cell model were successfully established to mimic the MI for in vivo and in vitro studies. The concentrations of lactate dehydrogenase (LDH), creatine kinase-MB (CK-MB), tumor necrosis factor alpha (TNF-alpha), IL-6, and reactive oxygen species (ROS) were then measured. Moreover, cell viability and apoptosis were detected by MTT assay, TdT-mediated dUTP nick end labeling (TUNEL), and PI/FITC-annexin V assay. The binding of miR-665 and Pak1 was determined by luciferase assay. miR-665 was upregulated in I/R rats, and the overexpression of miR-665 significantly increased LDH, CK-MB, TNF-alpha, IL-6, and ROS concentrations and induced cell apoptosis, while knockdown of miR-665 had opposite results. Consistent with in vivo results, miR-665 induced cell apoptosis and ROS generation in H/R-treated H9c2 cells. More importantly, Pak1 was the target gene of miR-665, and knockdown of miR-665 depressed the accumulation of ROS and cell apoptosis by targeting Pak1 and promoting the phosphorylation of Akt, whereas knockdown of Pak1 could attenuate the protection of miR-665 inhibitor in H/R-treated H9c2 cells. Therefore, knockdown of miR-665 protects against cardiomyocyte ischemia/reperfusion injury-induced ROS accumulation and apoptosis through activating Pak1/Akt signaling in MI. In general, understanding the biology and modulation of miR-665 may have the potential to counteract the development of MI.