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.
MicroRNA are posttranscriptional regulators that have proven critical for organogenesis and pathogenesis. But until now we have very little knowledge of the their targets and mechanism of function. A single miRNA has the capacity to target multiple functionally- related genes, which is why we think they would be more effective therapeutic targets relative to a single gene approach. For example, miR-21 upregulation would not only target and inhibit PTEN, but would also inhibit FasL, which equally contributes to the demise of myocytes during ischemic injury. Our proposal involves investigating the full range of the antiapoptotic function of miR-21 in the heart. This would be the first step to exploit it for a therapeutic cardioprotective effect during ischemic heart disease.
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