Department of Emergency Medicine, Sichuan Provincial People's Hospital, Chengdu, Sichuan, China.
Department of Ophthalmology and Altman Clinical and Translational Research Institute, University of California San Diego, La Jolla, CA, USA.
Oxid Med Cell Longev. 2018 Dec 2;2018:7042105. doi: 10.1155/2018/7042105. eCollection 2018.
In recent years, microRNAs (miRNAs) have received increasing attention for their role in ischemia/reperfusion injury (I/RI), and many miRNAs have been demonstrated to play a very important role in cardiac I/RI. The miRNA miR-24-3p is a tumor suppressor that regulates multiple tumors; however, it remains unclear whether the expression level of miR-24-3p is altered in cardiac cells under I/RI. In this study, we used mouse primary cardiomyocytes and the H9C2 cardiomyocyte cell line to perform stimulated ischemia/reperfusion (SI/R) and then detected miR-24-3p expression level using quantitative real-time PCR (qRT-PCR). We discovered that the expression of miR-24-3p was significantly increased in cardiomyocytes following SI/R, and that the miR-24-3p level was inversely correlated to the ischemia marker HIF-1a. Furthermore, we transfected cardiomyocytes with miR-24-3p mimic or inhibitor to explore the role of miR-24-3p in cardiomyocyte ischemia/reperfusion injury . We performed flow cytometry to detect the apoptotic rate of H9C2 cardiomyocytes and found that the transfection of miR-24-3p mimic resulted in the decrease of the apoptosis rate of cardiomyocytes after SI/R, whereas the transfection of miR-24-3p inhibitor increased the number of apoptotic cardiomyocytes. These data suggest that the overexpression of miR-24-3p could reduce myocardial cell apoptosis induced by I/R injury. Finally, we applied the dual luciferase reporter gene system to verify whether miR-24-3p targets the Keap1 gene, and found that the luciferase signal intensity from a vector carrying the Keap1 wild-type reporter gene was significantly reduced after transfection with miR-24-3p mimic. The Keap1 protein level was also reduced following the transfection of miR-24-3p. The results from this study suggest a novel function of miR-24-3p in protecting cardiomyocytes from ischemia/reperfusion injury by the activation of the Nrf2-Keap1 pathway.
近年来，微小 RNA（miRNA）因其在缺血/再灌注损伤（I/RI）中的作用而受到越来越多的关注，许多 miRNA 已被证明在心脏 I/RI 中发挥着非常重要的作用。miRNA miR-24-3p 是一种肿瘤抑制因子，可调节多种肿瘤；然而，目前尚不清楚在 I/RI 下心脏细胞中 miR-24-3p 的表达水平是否发生改变。在这项研究中，我们使用小鼠原代心肌细胞和 H9C2 心肌细胞系进行刺激缺血/再灌注（SI/R），然后使用定量实时 PCR（qRT-PCR）检测 miR-24-3p 的表达水平。我们发现，在 SI/R 后心肌细胞中 miR-24-3p 的表达显著增加，并且 miR-24-3p 的水平与缺血标志物 HIF-1a 呈负相关。此外，我们用 miR-24-3p 模拟物或抑制剂转染心肌细胞，以探讨 miR-24-3p 在心肌细胞缺血/再灌注损伤中的作用。我们通过流式细胞术检测 H9C2 心肌细胞的凋亡率，发现转染 miR-24-3p 模拟物可降低 SI/R 后心肌细胞的凋亡率，而转染 miR-24-3p 抑制剂则增加了凋亡心肌细胞的数量。这些数据表明，miR-24-3p 的过表达可减少 I/R 损伤引起的心肌细胞凋亡。最后，我们应用双荧光素酶报告基因系统来验证 miR-24-3p 是否靶向 Keap1 基因，发现转染 miR-24-3p 模拟物后携带 Keap1 野生型报告基因的载体的荧光素酶信号强度显著降低。转染 miR-24-3p 后，Keap1 蛋白水平也降低。这项研究的结果表明，miR-24-3p 通过激活 Nrf2-Keap1 通路在保护心肌细胞免受缺血/再灌注损伤方面具有新的功能。
