Abstract 15507: Inhibition of MEF2A Using 3rd Generation Antisense Enhances Neovascularization via Posttranscriptional Regulation of 14q32 MicroRNAs miR-329 and miR-494
Introduction and Hypothesis: The 14q32 locus encodes 54 individual microRNAs. Inhibition of 14q32 microRNAs leads to improved blood flow recovery after ischemia. Snyder et al (Development 2013) showed that transcription factor Myocyte Enhancer Factor 2A (MEF2A) induces expression of 14q32 microRNAs. Therefore, we hypothesized that inhibition of MEF2A leads to decreased 14q32 microRNA expression and thus to increased blood flow recovery after ischemia.
Methods: We inhibited expression of MEF2A in a hindlimb ischemia model in mice using 3rd Generation Antisense (3GA) and studied effects on blood flow recovery, as well as on 14q32 microRNA expression.
Results: Treatment with 3GA-MEF2A improved blood flow recovery within 3 days (44% recovery versus 25% recovery in control) and persisted until 14 days after induction of ischemia (80% recovery versus 60% recovery in control). However, we found no evidence of transcriptional regulation of 14q32 microRNAs. Inhibition of MEF2A decreased expression of miR-329 (p=0.026) and miR-494 (p=0.06), but not of other 14q32 microRNAs. Furthermore, 14q32 microRNA precursor levels were unaffected by MEF2A inhibition. Using rt/qPCR primers specific for each stage of microRNA processing, i.e. pri-microRNA, pre-microRNA and mature microRNA, we found that expression of 14q32 microRNAs after hindlimb ischemia is not determined by transcription, but rather by posttranscriptional processing of 14q32 microRNA precursors. Therefore, we investigated whether MEF2A can act as ‘RNA Binding Protein’ and enhance posttranscriptional processing of miR-329 and miR-494. We performed immunoprecipitation on 3T3 cell lysates, using an anti-MEF2A antibody. Pri-miR-329 expression was too low in 3T3 cells to confirm or exclude MEF2A binding. However, we observed specific binding of MEF2A to pri-miR-494, but not pri-miR-487b, a 14q32 microRNA that was not regulated by MEF2A inhibition.
Conclusions: This study demonstrates a novel function for MEF2A in neovascularization via posttranscriptional regulation of 14q32 microRNAs miR-329 and miR-494. We show here for the first time that MEF2A can potentially function as RNA Binding Protein.
Author Disclosures: S. Welten: None. M. de Vries: None. E. Peters: None. S. Agrawal: Employment; Modest; Idera Pharmaceuticals. P. Quax: None. A. Nossent: None.
- © 2016 by American Heart Association, Inc.