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STUDY OF MIR-199a, ITS TARGETS, AND UPSTREAM REGULATORS IN CARDIAC MYOCYTES

by
Shweta Rane
Department of Cell Biology and Molecular Medicine

B.E. 2002, Mumbai University
M.S. 2005, Long Island University


Thesis Advisor: Maha Abdellatif, Ph.D., M.B.C.

Associate Professor

Department of Cell Biology and Molecular Medicine

Thursday, September 17, 2009
4:00 p.m., MSB G-609


Abstract

MicroRNA (miRNA) are small, single-stranded, non-coding post-transcriptional regulators that bind to 3’-untranslated region (UTR) of mRNA and abrogate their translation. Here we report for the first time that miR-199a is rapidly downregulated upon oxidative stress. Mir-199a targets Hif-1a, which is a “master-transcriptional regulator” that regulates, cell survival and apoptotic pathways. Overexpression of miR-199a during chronic hypoxia inhibits Hif-1a and its stabilization of p53 and in turn inhibiting hypoxia-induced apoptosis. On the other hand, knockdown of endogenous miR-199a during normoxia, results in upregulation of Hif-1a thus mimicking hypoxia preconditioning (HPC). The other novel finding in this report is the differential expression of Hif-1a. Hif-1a associates with mitochondria during HPC and protects it against hypoxic damage unlike severe hypoxia, where it becomes strictly nuclear and stabilizes p53. Apart from Hif-1a, miR-199a also targets Sirt1, a class III histone deacetylase. Downregulation of miR-199a during HPC results in Sirt1 upregulation, which degrades prolyl hydroxylase 2 (PHD2), a protein responsible for Hif-1a degradation, further enhancing the expression of Hif-1a. Thus, we show for the first time a unique functional link between HPC and longevity pathways. HPC is associated with the activation of AKT pathway. Interestingly, overexpression of constitutively active AKT results in downregulation of miR-199a and a subsequent upregulation of Hif-1a and Sirt1. Furthermore, receptor activation of PI3-AKT pathway by insulin also showed an increase in Hif-1a and Sirt1, which was completely abrogated upon miR-199a overexpression or inhibition of PI3K. Thus, it confirms that AKT activation and downregulation of miR-199a are both required for HPC-induced Hif-1a and Sirt1 expression. Conversely, others and we have reported that miR-199a is upregulated during cardiac hypertrophy and failure. Our data reveals that miR-199a is also upregulated in b1 and b2 adrenergic receptor overexpressing mice hearts and by isoproterenol, thus suggesting an increase in miR-199a expression to the activation of b-adrenergic pathway. Pretreatment of cells with Isoproterenol before subjecting them to HPC or caAKT abrogated Hif-1a and Sirt1 expression, making the cells more susceptible to hypoxic damage. This suggests an antagonistic effect induced by the AKT survival pathway
Thus, we conclude that miR-199a is a “master regulator” of a hypoxia-triggered pathway and its knockdown can be exploited for preconditioning the cells. Furthermore, activation of the beta-adrenergic pathway can counteract the survival effects of preconditioning by upregulation of miR-199a.


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