Background: MicroRNA-24 (miR-24) plays an important role in heart failure by reducing the efficiency of myocardial excitation-contraction coupling. Prolonged cardiac hypertrophy may lead to heart failure, but little is known about the role of miR-24 in cardiac hypertrophy. This study aimed to preliminarily investigate the function of miR-24 and its mechanisms in cardiac hypertrophy.
Methods: Twelve Sprague-Dawley rats with a body weight of 50 ± 5 g were recruited and randomly divided into two groups: a transverse aortic constriction (TAC) group and a sham surgery group. Hypertrophy index was measured and calculated by echocardiography and hematoxylin and eosin staining. TargetScans algorithm-based prediction was used to search for the targets of miR-24, which was subsequently confirmed by a real-time polymerase chain reaction and luciferase assay. Immunofluorescence labeling was used to measure the cell surface area, and 3H-leucine incorporation was used to detect the synthesis of total protein in neonatal rat cardiac myocytes (NRCMs) with the overexpression of miR-24. In addition, flow cytometry was performed to observe the alteration in the cell cycle. Statistical analysis was carried out with GraphPad Prism v5.0 and SPSS 19.0. A two-sided P < 0.05 was considered as the threshold for significance.
Results: The expression of miR-24 was abnormally increased in TAC rat cardiac tissue (t = -2.938, P < 0.05). TargetScans algorithm-based prediction demonstrated that CDKN1B (p27, Kip1), a cell cycle regulator, was a putative target of miR-24, and was confirmed by luciferase assay. The expression of p27 was decreased in TAC rat cardiac tissue (t = 2.896, P < 0.05). The overexpression of miR-24 in NRCMs led to the decreased expression of p27 (t = 4.400, P < 0.01), and decreased G0/G1 arrest in cell cycle and cardiomyocyte hypertrophy.
Conclusion: MiR-24 promotes cardiac hypertrophy partly by affecting the cell cycle through down-regulation of p27 expression.
microRNA-24促进大鼠心肌肥厚的发生摘要背景：MicroRNA-24通过下调心肌兴奋-收缩耦联效率从而在心力衰竭中发挥重要的调控作用。长期的心肌肥厚导致心力衰竭的发生，然而，miR-24在心肌肥厚中的作用尚未被广泛了解。本研究初步探究了miR-24在心肌肥厚中的作用及其作用机制。 方法：12只体重为50 ± 5 g 的SD大鼠随机分为两组，主动脉弓缩窄组（TAC）与对照组（sham）。超声心动图和苏木精-伊红染色用于评估心肌肥厚指标。通过TargetScans软件计算预测miR-24的靶基因，同时利用实时荧光定量PCR与荧光素酶报告基因实验进行验证。在乳大鼠心肌细胞中过表达miR-24后，使用免疫荧光实验测量细胞表面积的变化，利用3H亮氨酸掺入法检测心肌细胞总蛋白合成，以及采用流式细胞术分析观察细胞周期的变化。 结果： miR-24在主动脉宫缩窄的大鼠心肌组织中的表达异常升高（t = -2.938，P < 0.05）。TargetScans分析结果显示细胞周期调节因子CDKN1B （p27, Kip1）是miR-24的潜在靶基因，同时荧光素酶报告基因实验证实了miR-24与p27的靶效关系。同时，我们的结果显示p27在主动脉宫缩窄的大鼠心肌组织中的表达显著降低（t = 2.896，P < 0.05）。此外，在乳大鼠心肌细胞中过表达miR-24会导致p27表达水平的下降（t = 4.400，P < 0.01），造成细胞周期G0/G1期阻滞的降低，以及心肌细胞肥大表型的变化。 结论：miR-24通过下调p27的表达影响细胞周期进程，从而促进心肌肥厚的发生。.
Keywords: Cardiac Hypertrophy; Cell Cycle; MicroRNA; MicroRNA-24; p27.