The role of microRNA in cardiac cell death

描述(申请人提供)：microRNA是主要的转录后调节分子，主要通过结合它们的3‘UTR来抑制蛋白质翻译。MicroRNA-21(miR-21)在肥大或癌细胞生长过程中高度上调。相反，我们发现，当心肌细胞暴露在长期缺氧中时，它会下降。因此，我们的主要目标是研究调控miR-21的信号通路及其靶点，以及它们在缺氧或缺血时心肌细胞存活中的作用。我们的初步结果表明，miR-21不仅调控10号染色体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)。在缺氧过程中，miR-21的下调是增强这两种蛋白表达的必要条件和充分条件。因此，在细胞缺氧时补充外源性miR-21是一种有效的细胞凋亡抑制方法。我们还观察到，激活的AKT抑制PTEN和FasL在心肌细胞中的表达，并诱导miR-21上调。为了探讨miR-21在活体心脏中的功能，我们建立了心脏特异的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，与野生型小鼠相比，转基因miR-21小鼠PTEN和FasL的表达完全受到抑制，心肌梗死面积较小，纤维化和心腔扩张较少。因此，心功能得到了更好的保护。因此，我们的假设是：1)AKT被短暂的低氧[低氧预适应(HPC)]激活，并诱导心肌细胞或心脏miR-21表达上调。相反，长期低氧与AKT的抑制有关，AKT的抑制导致miR-21的下调。2)AKT磷酸化RNA结合蛋白CUGBP1，与初级miR-21的环区结合，增强其加工能力，从而增加成熟miR-21的水平。3)MIR-21直接靶向并调控PTEN和FasL的翻译。因此，在缺氧期间miR-21的下调是必要的，并且足以增强它们的翻译。4)AKT-miR-21通路对PTEN水平的调节会反向调节AKT的活性，从而产生一个反馈环，使该通路的信号持续存在。5)FasL严格定位于心肌细胞之间的界面，传递细胞间的凋亡信号。因此，miR-21对FasL的抑制限制了细胞凋亡的扩散。6)外源性miR-21可抑制这些靶基因的表达，减少缺氧或缺血时心肌细胞的凋亡。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.