MiR-19家族促进心肌再生修复的调控机制与心梗后心衰治疗的策略研究

Cardiomyocytes in adult hearts are considered terminally differentiated that have exited from the cell cycle. Recent studies revealed that mature cardiomyocytes are able to proliferate through de-differentiation and re-entering cell cycle. However the intrinsic renew rate of cardiomyocyte in adult heart is low and is insufficient to reverse cardiomyocyte loss and restore cardiac function under pathophysiological conditions. Therefore, exploring new molecules, which can induce cardiomyocyte proliferation, is very promising for cardiac regeneration and repairing in ischemia-induced cardiac damage. We previously reported that miR-17-92 cluster is required and sufficient for cardiomyocytes proliferation and preserved cardiac function after myocardial infarction. The miR-17-92 cluster have 6 members which are co-transcripted. Our pilot experiments indicated that the members of miR-17-92 cluster, miR-19a/19b in particular, are required for and sufficient to induce cardiomyocyte proliferation in vitro through PTEN. In current study, (1)we will generate miR-19a/19b cardiac specific over-expression knock in mouse. (2) We will test the therapeutic potential of miR-19a/19b mimic in the heart using a mouse myocardial infarction model.(3) In addition, we will engineer a adeno-associated viral vector (AAV) carrying the precursors of hsa-miR-19a and hsa-miR-19b plus upstream and downstream flanking sequences (rAAV-miR-19a/19b) to test the long-term effects of cardiac specific overexpression of miR-19a/19b in heart attack. we will define the molecular mechanism by which miR-19a/19b targets and regulates the cell cycle events when adult cardiomyocyte proliferate. Overall, this study will highlight miRNA as a previously unrecognized class of post-transcriptional regulators for cardiomyocyte proliferation and cardiac regeneration and will develop microRNA therapies for so far untreatable heart attack and heart failure.

成年心肌细胞一直被认为终末分化细胞,退出细胞周期而不能增殖再生。最新研究表明,哺乳动物心肌细胞可以去分化重新回到细胞周期增殖再生,但内源性的增殖能力较弱。因此促进心肌细胞的增殖再生从而改善心脏疾病后心功能成为当前心衰治疗的重要方向。我们之前研究发现miR-17-92基因簇可以促进各时期心肌细胞增殖再生并保护心梗后的心功能。但此基因簇共转录6个成员miR-17,miR-20a,miR-18a,miR-19a,miR-19b,miR-92a,各成员对心肌再生的作用与机制并不清楚。本课题前期实验发现miR-19家族通过PTEN促进心肌细胞的增殖。因此拟通过小鼠遗传模型,腺相关病毒介导和miR-19 mimic输入三种方式过表达miR-19深入研究其促进心肌增殖的分子机制和心梗后再生修复治疗效果。研究结果将揭示miRNA促进心肌增殖修复疾病心脏的新机制并发展基于RNA治疗心力衰竭的新策略。

心肌梗塞等缺血性心脏病导致大量的心肌细胞死亡，纤维瘢痕形成是导致心力衰竭的主要原因。包括人类在内的哺乳动物成年心肌细胞成熟过程后退出细胞周期，仅保留有限的增殖能力。如何在心肌损伤后再生修复成为治疗缺血性心脏病的关键。我们之前研究发现miR-17-92 基因簇能够促进从胚胎到成年心脏的心肌增殖与再生。在此项目中，我们系统地研究了miR-17-92 基因簇成员中miR-19家族（miR-19a/19b）促进心肌再生修复的作用和调控机制以及心梗后保护心功能的RNA治疗策略。miR-19在成年心肌细胞富集表达，并在小鼠心梗后心脏中高表达，而在压力负荷引起的肥大模型中并无异常，提示miR-19可能参与病理损伤后的再生修复。在心梗后的RNA治疗上，我们应用新型高效脂质体包埋miR-19 mimic，发现心梗后心肌内注射（开胸局部方式）与鼠尾静脉注射（类似临床治疗的系统方式），均有效保护心梗后心脏功能，减少心脏损伤产生的梗死面积与纤维化，维持心脏收缩能力。荧光染色与遗传谱系示踪系统证实了miR-19在心梗后促进成年心肌细胞的增殖与再生，改善心脏功能。miR-19 mimic的注射后所引起的过表达虽然会在1周内消失，但生存曲线结果证明miR-19对心梗后的保护作用具有短期10天内以及至心梗后一年的长期心脏保护功能。通过转录组学测序及基因富集分析，我们发现miR-19主要通过改善免疫炎症反应，抑制细胞凋亡，下调纤维化基因表达，来保护心脏功能。并且miR-19通过调控靶基因SOCS以及下游STAT3信号轴调控早期免疫炎症反应，通过调控靶基因Bim、Pten等抑制凋亡促进再生保护心脏功能。我们进一步利用基因治疗手段证明在心梗后通过AAV系统长时效地心肌特异地过表达miR-19同样有效地保护了心脏功能，减少了梗死面积。对于miR-19的靶向基因Pten,我们利用遗传模型及谱系示踪技术证明条件敲除Pten或PTEN 抑制剂均能改善心梗后心脏功能，促进心肌细胞的增殖与再生修复。综上，此项目我们证明了miR-19家族 (2019发表在 Nature Communications)以及其靶标PTEN (2020发表在Circulation) 在心肌增殖与心脏再生中的重要作用，为缺血性心脏病提供新的RNA治疗理论基础和策略。