Date of Award
Master of Biomedical Science
College of Graduate Studies
Jeffrey A. Jones
Paul J. McDermott
Michael R. Zile
Background: A significant increase in the myocardial abundance of Membrane-Type 1 Matrix Metalioproteinase (MT1-MMP) has been identified in patients suffering Dilated Cardiomyopathy (DCM). These increased levels likely contribute to DCM; however, the molecular basis for up-regulation of MT1-MMP in the context of DCM remains unknown. Of significant relevance, microRNAs fine-tune protein translation by interrupting ribonucleoprotein:mRNA complexes, inducing degradation, or more commonly, translational repression of the message. Therefore, the aim of this study was to identify an endogenous microRNA responsible for the specific alterations correlating with disease state, and demonstrate a method of translational regulation of MT1-MMP. Methods and results: To identify potential regulatory microRNAs, a bioinformatics approach was taken to examine the MT1-MMP transcript for putative interaction sites. As a result, miR-133a was selected for further investigation. Using a commercially available reporter vector, constructed by cloning the 3' untranslated region (UTR) of the human MT1-MMP transcript downstream of a luciferase open reading frame, miR-133a was confirmed to directly target MT1-MMP's 3'UTR and selectively reduce translation with a significantly high level of efficiency in HT1080 cells. Investigations continued in a myocardial fibroblast model of DCM, developed and validated by this laboratory, and through reverse-transcription quantitative PCR (RT-qPCR), have, most interestingly, identified miR-133a microRNA levels to be significantly reduced in DCM myocardial fibroblasts (n=5) when compared to normal (n=4). Utilizing tools for permanent over expression and inhibition of miR-133a, the present study has successfully demonstrated, by protein immunoblot, a significant reduction in MT1-MMP protein abundance with miR-133a over expression, as well as, a significant increase in MT1-MMP protein abundance with miR-133a knockdown, in both normal and DCM myocardial fibroblasts. These findings were not accompanied by any significant change in MT1-MMP mRNA levels, and did not elicit induction of an interferon response, determined by RT-qPCR. Conclusions: The present study identifies aberrant miR-133a microRNA expression in DCM, confirms MT1-MMP as a target of miR-133a and demonstrates a method of translational regulation of MT1-MMP by direct modulation of miR-133a in normal and DCM myocardial fibroblasts. These findings hold diagnostic and therapeutic relevance for one of the most urgent public health problems in the United States.
Akerman, Adam William, "Regulation of Membrane-Type 1 Matrix Metalloproteinase Abundance by Selective Modulation of MicroRNA-133a" (2011). MUSC Theses and Dissertations. 14.
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