Adenosine-to-Inosine Editing of MicroRNA-487b Alters Target Gene Selection After Ischemia and Promotes Neovascularization

Rationale: Adenosine-to-inosine (A-to-I) editing of microRNAs has the potential to cause a shift in target-site selection. 29-O-ribose-methylation (29OMe) of adenosine residues however, has been shown to inhibit A-to-I-editing. Objective: To investigate whether angiomiR miR487b is subject to A-to-I-editing and/or 29OMe during neovascularization. Methods and Results: cDNA was prepared from C57BL/6-mice subjected to hindlimb ischemia. Using Sanger-sequencing and endonuclease digestion, we identified and validated A-to-I-editing of the miR487b seed-sequence. In the gastrocnemius muscle, pri-miR487b editing increased from 6.7±0.4% before to 11.7±1.6% (P=0.02) 1 day after ischemia. Edited pri-miR487b is processed into a novel microRNA, miR487b-ED, which is also upregulated following ischemia. We confirmed editing of miR487b in multiple human primary vascular cell-types. siRNA-mediated knockdown demonstrated that editing is ADAR1&2-dependent. Using 9Reverse-Transcription at Low dNTP concentrations followed by Quantitative-PCR9 (RTL-Q), we found that the same adenosine-residue is methylated in mice and human primary cells. In the murine gastrocnemius, the estimated methylation fraction increased from 32.8±14% before to 53.6±12% 1 day after ischemia. siRNA knockdown confirmed that methylation is Fibrillarin-dependent. Although we could not confirm that methylation directly inhibits editing, we do show that ADAR1&2 and Fibrillarin negatively influence each other9s expression. Using multiple luciferase reporter gene assays, we could demonstrate that editing results in a complete switch of target-site selection. In human primary cells, we confirmed the shift in miR487b targeting after editing, resulting in a miR487b-ED targetome that is enriched for multiple pro-angiogenic pathways. Furthermore, overexpression of miR-487b-ED, but not miR-487b-WT, stimulates angiogenesis in both in vitro and ex vivo assays. Conclusions: MiR487b is edited in the seed-sequence in mice and humans, resulting in a novel, pro-angiogenic microRNA with a unique targetome. The rate of miR487b editing, as well as 29OMe, is increased in murine muscle tissue during post-ischemic neovascularization. Our findings suggest miR487b editing plays an intricate role in post-ischemic neovascularization.