The role of microRNA in cardiac cell death

描述（由申请人提供）：MicroRNA是主要的转录后调节分子，主要通过结合其3 'UTR来抑制蛋白质翻译。microRNA-21（miR-21）在肥大或癌细胞生长期间高度上调。与此相反，我们发现，它的下降后，暴露的心肌细胞长期缺氧。因此，我们的主要目标是研究调节miR-21的信号通路，其靶点，以及它们在缺氧或缺血期间在心肌细胞存活中的作用。我们的初步研究结果表明，miR-21不仅调节10号染色体上缺失的磷酸酶和张力蛋白同源物（PTEN），而且还直接靶向Fas配体（FasL）。在缺氧期间，下调miR-21对于增强两种蛋白质的表达是必要且充分的。因此，在缺氧期间用外源性miR-21补充细胞是细胞凋亡的有效抑制剂。我们还观察到激活的AKT抑制了心肌细胞中PTEN和FasL的表达，并诱导miR-21的上调。为了探索miR-21在体内心脏中的功能，我们建立了心脏特异性miR-21转基因小鼠模型。这些小鼠没有明显的心脏表型，然而，在慢性冠状动脉闭塞后，在miR-21转基因小鼠中与野生型小鼠相比，PTEN和FasL表达被完全抑制，梗死面积较小，纤维化和室扩张较少。因此，心脏功能得到了更好的保护。因此，我们的假设是：1）AKT通过短暂的缺氧事件[缺氧预处理（HPC）]激活，并诱导心肌细胞或心脏中miR-21的上调。相反，长时间缺氧与AKT的抑制有关，这导致miR-21的下调。2)AKT使RNA结合蛋白CUGBP 1磷酸化，CUGBP 1结合初级miR-21的环区并增强其加工，从而增加成熟miR-21水平。3)miR-21直接靶向并调节PTEN和FasL的翻译。因此，在缺氧期间下调miR-21是必需的，并且足以增强它们的翻译。4)AKT- miR-21途径对PTEN水平的调节反向调节AKT活性，并因此产生通过该途径使信号传导永久化的反馈环。5)FasL严格定位于肌细胞之间的界面，并在细胞之间传递凋亡信号。因此，miR-21对FasL的抑制限制了凋亡的扩散。6)在细胞中补充外源性miR-21可以抑制这些靶点的表达，并减少缺氧或缺血期间的心肌细胞凋亡。因此，本研究的目的是：1）阐明miR-21调控的上游途径和机制及其功能意义。2)研究miR-21及其靶基因FasL和PTEN在心肌细胞中的作用机制。3)研究miR-21的功能及其在体内的调控。

DESCRIPTION (provided by applicant): MicroRNA are major posttranscriptional regulatory molecules that mainly suppress protein translation through binding their 3'UTR. MicroRNA-21 (miR-21) is highly upregulated during hypertrophic or cancerous cell growth. In contrast, we found that it declines upon exposure of cardiac myocytes to prolonged hypoxia. Thus, our main objective is to investigate the signaling pathway that regulates miR-21, its targets, and their role in myocyte survival during hypoxia or ischemia. Our preliminary results show that miR-21 not only regulates phosphatase and tensin homologue deleted on chromosome 10 (PTEN), but also directly targets Fas Ligand (FasL). During hypoxia, downregulation of miR-21 is necessary and sufficient for enhancing the expression of both proteins. Consequently, supplementing the cells with exogenous miR-21 during hypoxia is an effective inhibitor of apoptosis. We also observed that activated AKT suppresses the expression of PTEN and FasL in myocytes and induces upregulation of miR-21. To explore the function of miR-21 in the heart in vivo, we generated a cardiac-specific miR-21 transgenic mouse model. These mice have no overt cardiac phenotype, however, following chronic coronary artery occlusion there was complete suppression of PTEN and FasL expression, smaller infarct size, and less fibrosis and chamber dilatation, in the miR-21 transgenic versus the wild type mice. Accordingly, cardiac functions were better preserved. Thus, our hypotheses are: 1) AKT is activated by brief hypoxic episodes [hypoxia preconditioning (HPC)] and induces upregulation of miR-21 in cardiac myocytes or the heart. Conversely, prolonged hypoxia is associated with inhibition of AKT, which results in downregulation of miR-21. 2) AKT phosphorylates the RNA-binding protein CUGBP1, which binds the loop region of primary miR-21 and enhances its processing, thus, increasing mature miR-21 levels. 3) MiR-21 directly targets and regulates translation of PTEN and FasL. Thus, downregulation of miR-21 during hypoxia is required and sufficient for enhancing their translation. 4) Modulation of PTEN levels by the AKT- miR-21 pathway inversely regulates AKT activity and, thus, creates a feedback loop that perpetuates signaling through this pathway. 5) FasL is strictly localized to the interface between myocytes and relays apoptosis signals between cells. Thus, suppression of FasL by miR-21 limits the spread of apoptosis. 6) Supplementing cells with exogenous miR-21 suppresses the expression of these targets and reduces myocyte apoptosis during hypoxia or ischemia. Thus, the aims are to: 1) Delineate the upstream pathways and mechanisms involved in the regulation of miR-21 and its functional signficance. 2) Examine the mechanisms of function of miR-21 and its target genes, FasL and PTEN, in cardiac myocytes. 3) Study the function of miR-21 and its regulation in vivo.