Abstract 15481: The Vasoactive MicroRNA miR-487b-3p Undergoes A-to-I Editing of the Seed-Sequence During Post-Ischemic Neovascularization

• Published in: Circulation (New York, N.Y.) Vol. 134; no. Suppl_1 Suppl 1; p. A15481
• Main Authors: van der Kwast, Reginald, Quax, Paul, Nossent, Anne Yael
• Format: Journal Article
• Language: English
• Published: by the American College of Cardiology Foundation and the American Heart Association, Inc 11.11.2016
• ISSN: 0009-7322

IntroductionWe have previously shown that miR-487b-3p plays an important role in cardiovascular pathology. MiR-487b-3p regulates arteriogenesis and angiogenesis in a murine hindlimb ischemia model. In contrast to most microRNAs, that can have up to several hundred potential target genes, miR-487b-3p only has 21 potential conserved target genes. It has been shown for several microRNAs that, under pathological conditions, Adenosine residues in the mature microRNA sequence are edited to Inosine. In Watson-Crick base-pairing Inosine behaves as a Guanine residue and thus, ‘A-to-I editing’ in microRNAs can have a major impact on target gene selection.HypothesisGiven the narrow range of potential miR-487b-3p target genes, we hypothesized that miR-487b-3p undergoes A-to-I editing under pathological conditions.Methods and ResultscDNA was made from total RNA isolated from muscle tissue of mice subjected to hindlimb ischemia. We used Sanger sequencing to identify A-to-I editing sites in the miR-487b precursor sequence (pri-miR-487b) and located a potential site in the sequence of miR-487b-3p, changing the seed sequence from 5’-ATCGTAC-3’ to 5’-ITCGTAC-3’. We used digestion by restriction endonuclease PfeI to confirm heterozygosity of the site. As PfeI cuts the unedited pri-miR-487b, but not the edited pri-miR-487b, synthetic cDNA sequences containing either the edited of unedited residue were used as control to evaluate the efficiency and specificity of the digestion. We quantified the amount of edited pri-mir-487b relative to the amount of unedited pri-miR-487b and found that, within 24 hours after hindlimb ischemia, the rate of A-to-I editing is increased 2- to 3-fold. We found a similar increase in A-to-I editing in the mature miR-487b-3p using custom TaqMan small RNA rt/qPCR assays. Finally, using human primary arterial fibroblasts, we could confirm A-to-I editing of the exact same site in the human miR-487b-3p.ConclusionsMiR-487b-3p is subject to A-to-I editing in the seed sequence of the microRNA, both in mice and in humans. Under conditions of ischemia, the rate of A-to-I editing of miR-487b-3p is increased, likely increasing the range of potential target genes.