He Fang, Liu Huibin, Guo Jing, Yang Di, Yu Yang, Yu Jie, Yan Xiuqing, Hu Juan, Du Zhimin
Institute of Clinical Pharmacy, the Second Affiliated Hospital of Harbin Medical University, The University Key Laboratory of Drug Research, Heilongjiang Province, Harbin, China.
Institute of Clinical Pharmacy, the Second Affiliated Hospital of Harbin Medical University, The University Key Laboratory of Drug Research, Heilongjiang Province, Harbin,
Cell Physiol Biochem. 2018;51(1):186-200. doi: 10.1159/000495173. Epub 2018 Nov 15.
BACKGROUND/AIMS: MicroRNAs play an important role in regulating myocardial infarction (MI)-induced cardiac injury. MicroRNA-124 (miR-124) plays a vital role in regulating cellular proliferation, differentiation and apoptosis. Although the alteration of miR-124 was confirmed in peripheral blood of MI patients, little is known regarding the biological functions of miR-124 in cardiomyocytes. This study was designed to explore the role of miR-124 in MI and its underlying mechanisms.
Real-time PCR was used to quantify the microRNAs levels. TUNEL and Flow cytometry were performed to measure cell apoptosis. Western blot analysis was employed to detect expression of Bcl-2, Bax, Caspase-3 and STAT3 proteins.
We revealed that miR-124 was significantly up-regulated in a mice model of MI and in neonatal rat ventricular myocytes (NRVMs) with H2O2 treatment. H2O2 treatment induced cardiomyocyte injury with reduced cell viability and enhanced apoptotic cell death, whereas silencing expression of miR-124 by AMO-124 (antisense inhibitor oligodeoxyribonucleotides) alleviated these deleterious changes. AMO-124 decreased the expression of Bax and cleaved-caspase-3 and upregulated the expression of Bcl-2 in H2O2-treated NRVMs. Besides, AMO-124 improved mitochondrial dysfunction of NRVMs induced by H2O2 treatment. Moreover, antagomir-124 markedly decreased the infarct area and apoptotic cardiomyocytes and improved cardiac function in MI mice. Furthermore, we identified STAT3 as a direct target of miR-124, and downregulation of miR-124 ameliorated the diminished levels of STAT3 and p-STAT3 (Tyr705) in response to H2O2 or MI. STAT3 inhibitor, stattic, was shown to attenuate the elevation of p-STAT3 in NRVMs with AMO-124 transfection. Inhibiting of STAT3 activity by stattic abrogated protective effects of AMO-124 on H2O2-induced cardiomyocytes apoptosis.
Taken together, our data demonstrate that downregulation of miR-124 inhibits MI-induced apoptosis through upregulating STAT3, which suggests the therapeutic potential of miR-124 for myocardial infarction.
背景/目的：微小RNA在调节心肌梗死（MI）诱导的心脏损伤中发挥重要作用。微小RNA-124（miR-124）在调节细胞增殖、分化和凋亡中起关键作用。虽然在MI患者的外周血中已证实miR-124发生改变，但关于miR-124在心肌细胞中的生物学功能知之甚少。本研究旨在探讨miR-124在MI中的作用及其潜在机制。
采用实时定量PCR检测微小RNA水平。采用TUNEL法和流式细胞术检测细胞凋亡。采用蛋白质免疫印迹分析检测Bcl-2、Bax、Caspase-3和STAT3蛋白的表达。
我们发现，在MI小鼠模型和经H2O2处理的新生大鼠心室肌细胞（NRVMs）中，miR-124显著上调。H2O2处理诱导心肌细胞损伤，细胞活力降低，凋亡细胞死亡增加，而通过AMO-124（反义抑制剂寡脱氧核糖核苷酸）沉默miR-124的表达可减轻这些有害变化。AMO-124降低了H2O2处理的NRVMs中Bax和裂解的Caspase-3的表达，并上调了Bcl-2的表达。此外，AMO-124改善了H2O2处理诱导的NRVMs的线粒体功能障碍。此外，抗miR-124显著减小了MI小鼠的梗死面积和凋亡心肌细胞数量，并改善了心脏功能。此外，我们确定STAT3是miR-124的直接靶点，miR-124的下调改善了H2O2或MI刺激后STAT3和p-STAT3（Tyr705）水平的降低。STAT3抑制剂stattic可减弱AMO-124转染的NRVMs中p-STAT3的升高。stattic抑制STAT3活性可消除AMO-124对H2O2诱导的心肌细胞凋亡的保护作用。
综上所述，我们的数据表明，miR-124的下调通过上调STAT3抑制MI诱导的凋亡，这提示miR-124在心肌梗死治疗中的潜在应用价值。
