Department of Pharmacology and Toxicology, Sun Yat-Sen University, Guangdong, Guangzhou, China.
Cell Biol Int. 2022 Feb;46(2):288-299. doi: 10.1002/cbin.11731. Epub 2021 Dec 19.
MicroRNAs (miRNAs) regulate multiple biological processes and participate in various cardiovascular diseases. This study aims to investigate the role of miR-339-5p in cardiomyocyte hypertrophy and the involved mechanism. Neonatal rat cardiomyocytes (NRCMs) were cultured and stimulated with isoproterenol (ISO). The hypertrophic responses were monitored by measuring the cell surface area and expression of hypertrophic markers including β-myosin heavy chain (β-MHC) and atrial natriuretic factor (ANF). Bioinformatic prediction tools and dual-luciferase reporter assay were performed to identify the target gene of miR-339-5p. Quantitative real-time polymerase chain reaction and western blot analysis were used to determine the levels of miR-339-5p and its downstream effectors. Our data showed that miR-339-5p was upregulated during cardiomyocyte hypertrophy triggered by ISO. MiR-339-5p overexpression resulted in enlargement of cell size and increased the levels of hypertrophic markers. In contrast, inhibition of miR-339-5p significantly attenuated ISO-induced hypertrophic responses of NRCMs. Valosin-containing protein (VCP), a suppressor of cardiac hypertrophy via inhibiting mechanistic target of rapamycin (mTOR) signaling, was validated as a target of miR-339-5p. MiR-339-5p suppressed VCP protein expression, leading to elevated phosphorylation of mTOR and ribosomal protein S6 kinase (S6K). VCP depletion activated the mTOR/S6K cascade and could compromise the anti-hypertrophic effects of miR-339-5p inhibitor. Additionally, the hypertrophic responses caused by miR-339-5p was alleviated in the presence of mTOR inhibitor rapamycin. In conclusion, our research revealed that miR-339-5p promoted ISO-induced cardiomyocyte hypertrophy by targeting VCP to activate the mTOR signaling, suggesting a promising therapeutic intervention by interfering miR-339-5p.
微小 RNA（miRNAs）调节多种生物学过程，并参与多种心血管疾病。本研究旨在探讨 miR-339-5p 在心肌细胞肥大中的作用及其相关机制。培养新生大鼠心肌细胞（NRCMs）并用异丙肾上腺素（ISO）刺激。通过测量细胞表面积和肥大标志物（包括β-肌球蛋白重链（β-MHC）和心钠素因子（ANF））的表达来监测肥大反应。利用生物信息学预测工具和双荧光素酶报告基因检测来鉴定 miR-339-5p 的靶基因。采用定量实时聚合酶链反应和 Western blot 分析来确定 miR-339-5p 及其下游效应物的水平。我们的数据显示，在 ISO 诱导的心肌细胞肥大过程中，miR-339-5p 上调。miR-339-5p 过表达导致细胞大小增大，并增加肥大标志物的水平。相反，抑制 miR-339-5p 显著减弱 ISO 诱导的 NRCMs 肥大反应。包含 valosin 的蛋白（VCP）通过抑制雷帕霉素靶蛋白（mTOR）信号通路抑制心肌肥大，被验证为 miR-339-5p 的靶基因。miR-339-5p 抑制 VCP 蛋白表达，导致 mTOR 和核糖体蛋白 S6 激酶（S6K）磷酸化升高。VCP 缺失激活 mTOR/S6K 级联，可削弱 miR-339-5p 抑制剂的抗肥大作用。此外，在 mTOR 抑制剂雷帕霉素存在的情况下，miR-339-5p 引起的肥大反应减轻。总之，我们的研究表明，miR-339-5p 通过靶向 VCP 激活 mTOR 信号促进 ISO 诱导的心肌细胞肥大，提示通过干扰 miR-339-5p 进行有希望的治疗干预。
