Department of Cardiology, Renmin Hospital of Wuhan University, Cardiovascular Research Institute, Wuhan University, Hubei Key Laboratory of Cardiology, Wuhan, China.
Department of Geriatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
J Cell Mol Med. 2019 Apr;23(4):2505-2516. doi: 10.1111/jcmm.14135. Epub 2019 Jan 25.
MicroRNAs (miRNAs) have been shown to commonly contribute to cardiac hypertrophy (CH). The aim of this study was to test the hypothesis that miR-200c plays an important role in the progression of CH by targeting myosin light chain kinase (MLCK/MYLK).
Cardiac hypertrophy was induced by aortic banding (AB) in rats. Cellular hypertrophy in neonatal rat cardiomyocytes (NCMs) was induced by AngII treatment. Echocardiography, histology and molecular measurements were used to assess the results of the experiments. The levels of apoptosis and reactive oxygen species (ROS) were also measured. Quantitative real-time PCR (qRT-PCR) and Western blotting were used to measure mRNA and protein levels respectively. The present results showed that miR-200c expression was increased in response to CH both in vivo and in vitro. The down-regulation of miRNA-200c by a specific inhibitor markedly ameliorated CH resulting from AngII treatment, and the mRNA levels of atrial natriuretic peptide, brain natriuretic peptide and β-myosin heavy chain were simultaneously decreased. Notably, minimal apoptosis and ROS accumulation were identified in AngII-induced hypertrophic cardiomyocytes. Conversely, the up-regulation of miR-200c using specific mimics reversed these effects. Mechanistic investigations demonstrated that the MLCK gene is a direct target of miR-200c; an increase in miR-200c levels led to a decrease in the expression of MLCK and its downstream effector, p-MLC2, while miR-200c inhibition increased the expression of these proteins. Furthermore, inhibiting MLCK impaired the anti-hypertrophic effects contributions produced by the knockdown of miR-200c.
Our studies suggest that miR-200c may serve as a potential therapeutic target that could delay hypertrophy. We have also uncovered a relationship between miR-200c and MLCK, identifying MLCK as a direct mediator of miR-200c.
MicroRNAs (miRNAs) 已被证明在心肌肥厚 (CH) 中普遍发挥作用。本研究旨在通过靶向肌球蛋白轻链激酶 (MLCK/MYLK) 来验证 miR-200c 通过靶向肌球蛋白轻链激酶 (MLCK/MYLK) 在 CH 进展中发挥重要作用的假设。
通过主动脉缩窄 (AB) 在大鼠中诱导心肌肥厚。用 AngII 处理诱导新生大鼠心肌细胞 (NCMs) 的细胞肥大。使用超声心动图、组织学和分子测量来评估实验结果。还测量了细胞凋亡和活性氧物种 (ROS) 的水平。使用定量实时 PCR (qRT-PCR) 和 Western blot 分别测量 mRNA 和蛋白水平。结果显示，miR-200c 在体内和体外均因 CH 而升高。miR-200c 的特异性抑制剂下调可显著改善 AngII 处理引起的 CH，同时降低心房利钠肽、脑利钠肽和β-肌球蛋白重链的 mRNA 水平。值得注意的是，在 AngII 诱导的肥大心肌细胞中，细胞凋亡和 ROS 积累很少。相反，用特异性模拟物上调 miR-200c 可逆转这些作用。机制研究表明，MLCK 基因是 miR-200c 的直接靶标；miR-200c 水平的升高导致 MLCK 及其下游效应物 p-MLC2 的表达降低，而 miR-200c 抑制增加了这些蛋白的表达。此外，抑制 MLCK 损害了 miR-200c 敲低产生的抗肥大作用。
我们的研究表明，miR-200c 可能是一种潜在的治疗靶点，可以延缓肥大。我们还发现了 miR-200c 与 MLCK 之间的关系，确定 MLCK 是 miR-200c 的直接调节剂。
