Overexpression of miR-142-3p improves mitochondrial function in cardiac hypertrophy
Overexpression of miR-142-3p improves mitochondrial function in cardiac hypertrophy
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miR-142-3p 的过表达可改善心脏肥大中的线粒体功能
DOI：
10.1016/j.biopha.2018.09.146
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发表时间：
2018-12-01
影响因子：
7.5
通讯作者：
Wu, Gang
中科院分区：
文献类型：
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作者：
Liu, Bei-lei;Cheng, Mian;Wu, Gang
背景和目的：我们前期的研究表明Src同源2（SH 2）B衔接蛋白1（SH 2 B1）在心肌肥厚中起重要作用，但具体机制尚待进一步研究。通过生物信息学及相关研究发现，miR-14 2-3 p与SH 2B 1密切相关。探讨miR-14 2-3 p与SH 2B 1基因表达的关系，对心肌肥厚的治疗具有重要意义。SH 2B 1是调节能量代谢的关键因子，线粒体是能量代谢的主要细胞器，心肌肥厚与线粒体功能障碍密切相关。因此，探讨miR-14 2-3 p和SH 2B 1与心肌线粒体功能的关系尤为重要。本研究旨在探讨过表达miR-14 2-3 p是否能抑制SH 2B 1基因的表达，改善心肌线粒体功能障碍和心肌肥厚。造模4周后，超声心动图检查显示模型组心脏体积变大，HE染色显示心脏组织横截面积变大。实时荧光定量PCR检测心钠素（ANP）、脑钠素（BNP）、β-肌球蛋白重链（β-MHC）mRNA表达增加，证明心肌肥厚模型构建成功。然后，分别于2周和4周大鼠尾静脉注射miR-14 2-3 pagomir。经PCR检测，miR-4 2-3 p mRNA的表达量增加，提示miR-14 2-3 p质粒已成功转染。造模4周后，超声心动图检测心功能。采用HE染色及心钠素（ANP）、脑钠素（BNP）、β-MHC mRNA表达检测心肌肥厚。流式细胞术检测线粒体膜电位的变化。其次，通过体外培养乳鼠心肌细胞，观察miR-14 2-3 p对心肌细胞肥大和线粒体功能的影响。然后，使用血管紧张素（Ang）II-、miRNA模拟物-和miRNA模拟物nc-处理心肌细胞给定时间。α-肌动蛋白染色发现心肌细胞变大，PCR检测ANP、BNP、β-MHC mRNA表达增加，证明AngII诱导的心肌肥大已成功构建。然后，通过共聚焦显微镜使用线粒体Mito-Red染色测量线粒体密度，通过流式细胞术评估线粒体膜电位，通过Seahorse Extracellular Flux Analyzer XF 96测量线粒体呼吸耗氧率（OCR），以及miR-14 2-3 p、ANP、BNP、β-MHC mRNA、RT-PCR和Western blotting检测各组心肌细胞中SH 2B 1的表达。最后采用荧光素酶法检测转染miR-14 2-3 p agomir的大鼠心肌细胞，转染miR-14 2-3 p mimic的心肌细胞，发现心肌SH 2B 1 mRNA和蛋白表达均降低。建立压力负荷性心肌肥厚模型4周后，超声心动图显示心脏容积、左室舒张末期内径（LVIDd）、AB组左室收缩末期内径（LVID）、左室后壁厚度（LVPWd）、收缩末期左室后壁厚度（LVPWs）、左室质量（LV）增加，射血分数（EF）%降低，但转染miR-14 2-3 pagomir的AB组，这些增加不显著，EF%降低不明显。HE染色显示AB组心肌横截面积明显增加，而miR-14 2-3 p agomir处理的AB组心肌横截面积无明显增加。PCR分析显示AB组ANP、BNP、β-MHC mRNA的表达明显增加，而miR-14 2-3 pagomir处理AB组的表达无明显增加。流式细胞术显示AB组线粒体膜电位明显降低，AB组经miR-14 2-3 p agomir处理后无明显降低。在AngII诱导的心肌细胞肥大过程中，ANP、BNP、β-MHC mRNA表达增加，而miR-14 2-3 p模拟物处理组这些因子的表达没有显著增加; AngII处理组线粒体膜电位、线粒体密度和OCR显著降低，而miR-14 2-3 p模拟物处理组这些因子的表达没有显著降低。miR-14 2-3 p不仅通过直接抑制SH 2B 1基因的表达减轻心肌肥厚，而且在体内外心肌肥厚中具有保护线粒体功能的作用。
Background and Purpose: Our previous studies have shown that Src homology 2 (SH2) B adaptor protein 1 (SH2B1) plays an important role in cardiac hypertrophy, but the specific mechanism remains to be studied. Through bioinformatics and related research, it is found that miR-14 2-3 p is closely related to SH2B1. Exploring the relationship between miR-14 2-3 p and gene SH2B1 expression is beneficial for the treatment of cardiac hypertrophy. SH2B1 is a key factor regulating energy metabolism, mitochondria are the main organelles of energy metabolism and cardiac hypertrophy are closely related to mitochondrial dysfunction. So it is particularly important to explore the relationship between miR-14 2-3 p and SH2B1 and myocardial mitochondrial function. In this study, we investigated whether overexpression of miR-14 2-3 p can inhibit the expression of gene SH2B1, ameliorate cardiac mitochondrial dysfunction and cardiac hypertrophy.Methods: We first constructed a pressure overload myocardial hypertrophy model by ligation of the abdominal aorta(AB) of rats. After 4 weeks of modeling, echocardiographic examination showed that the heart volume of the model group became larger, and Hematoxylin and Eosin Staining Kit (HE) staining showed that the cross-sectional area of the heart tissue became larger. The expression of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), beta-Myosin Heavy Chain (beta-MHC) messenger RNA (mRNA) increased by real-time polymerase chain reaction (PCR), which proved that the model of cardiac hypertrophy was successfully constructed. Then, miR-14 2-3 p agomir was injected into the tail vein of rats 2 weeks and 4 weeks respectively. The expression of miR-4 2-3 p mRNA was increased by PCR, suggesting that the miR-14 2-3 p plasmid was successfully transfected. At 4 weeks of pressure overload myocardial hypertrophy model, echocardiography was used to detect cardiac function. HE staining of heart tissue and the expression of ANP, BNP, beta-MHC mRNA were used to detect cardiac hypertrophy. Flow cytometry was used to detect changes in mitochondrial membrane potential. Secondly, we observed the effect of miR-14 2-3 p on cardiomyocyte hypertrophy and mitochondrial function in vitro by culture neonatal rat cardiomyocytes. Afterwards, using angiotensin (Ang) II-, miRNA mimic-and miRNA mimic nc-treated cardiomyocytes for a given time. a-actin staining found that the myocardial cells became larger, The expression of ANP, BNP, beta-MHC mRNA increased by PCR, which proved that AngII-induced cardiac hypertrophy was successfully constructed. Then, the mitochondrial density was measured using mitochondrial Mito-Red staining by Confocal microscope, the mitochondrial membrane potential was evaluated using flow cytometry, Mitochondrial respiration oxygen consumption rate (OCR) was measured by a Seahorse Extracellular Flux Analyzer XF96, and the expression levels of miR-14 2-3 p, ANP, BNP, beta-MHC mRNA, SH2B1 in the cardiomyocytes of different groups were measured by RT-PCR and Western blotting. Finally, we used luciferase assay and transfected miR-14 2-3 p agomir in rats, transfected miR-14 2-3 p mimic in Cardiomyocytes, it is found that myocardial SH2B1 mRNA and protein expression both were reduced.Results: When the pressure overload myocardial hypertrophy model was constructed for four weeks, echocardiography revealed that the heart volume, Left ventricular end diastolic diameter(LVIDd), Left ventricular end systolic diameter (LVIDs), Left ventricular posterior wall thickness (LVPWd), Systolic left ventricular posterior wall (LVPWs), Left ventricle (LV) Mass increased, Ejection fraction (EF) % decreased of AB group increased, but transfected with miR-14 2-3 p agomir of AB, these increase was not significant, EF% reduction was not obvious. HE staining showed that the myocardial cross-sectional area of AB group increased significantly, but the miR-14 2-3 p agomir treatment of AB group did not increase significantly. PCR analysis showed that the expression of ANP, BNP, beta-MHC mRNA was significantly increased in AB group, but the miR-14 2-3 p agomir treatment of AB group was not significantly increased. Flow cytometry showed that the mitochondrial membrane potential of AB group was significantly reduced, and the miR-14 2-3 p agomir treatment of AB group was not significantly decreased. During AngII-induced cardiomyocyte hypertrophy, ANP, BNP, beta-MHC mRNA expression was increased, while these factors was not significantly increased in miR-14 2-3 p mimic treatment group; mitochondrial membrane potential, mitochondrial density and OCR was significantly decreased in AngII treated group, and these were not significantly reduced in miR-14 2-3 p mimic treatment group;Conclusions: miR-14 2-3 p not only mitigate cardiac hypertrophy by directly inhibit the expression of gene SH2B1, but also can protect mitochondrial function in cardiac hypertrophy of vitro and vivo.