Rationale: Adenosine-to-inosine editing of microRNAs has the potential to cause a shift in target site selection. 2′-O-ribose-methylation of adenosine residues, however, has been shown to inhibit adenosine-to-inosine editing.

Objective: To investigate whether angiomiR miR487b is subject to adenosine-to-inosine editing or 2′-O-ribose-methylation during neovascularization.

Methods and Results: Complementary DNA was prepared from C57BL/6-mice subjected to hindlimb ischemia. Using Sanger sequencing and endonuclease digestion, we identified and validated adenosine-to-inosine 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, edited miR487b, which is also upregulated after ischemia. We confirmed editing of miR487b in multiple human primary vascular cell types. Short interfering RNA–mediated knockdown demonstrated that editing is adenosine deaminase acting on RNA 1 and 2 dependent. Using reverse-transcription at low dNTP concentrations followed by quantitative-PCR, 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. Short interfering RNA knockdown confirmed that methylation is fibrillarin dependent. Although we could not confirm that methylation directly inhibits editing, we do show that adenosine deaminase acting on RNA 1 and 2 and fibrillarin negatively influence each other’s 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 edited miR487b targetome that is enriched for multiple proangiogenic pathways. Furthermore, overexpression of edited miR487b, but not wild-type miR487b, 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, proangiogenic microRNA with a unique targetome. The rate of miR487b editing, as well as 2′-O-ribose-methylation, is increased in murine muscle tissue during postischemic neovascularization. Our findings suggest miR487b editing plays an intricate role in postischemic neovascularization.

基本原理： microRNA的腺苷到肌苷编辑可能会导致靶位点选择发生变化。然而，腺苷残基的2'-O-核糖甲基化已显示抑制腺苷到肌苷的编辑。

目的：研究在新生血管形成过程中angiomiR miR487b是否进行腺苷到肌苷编辑或2'-O-核糖甲基化。

方法与结果：从C57BL / 6-小鼠后肢缺血中制备互补DNA。使用Sanger测序和核酸内切酶消化，我们鉴定并验证了miR487b种子序列的腺苷到肌苷的编辑。在腓肠肌中，pri-miR487b编辑从以前的6.7±0.4％增加到11.7±1.6％（ P= 0.02）缺血后1天。编辑后的pri-miR487b被加工成新的microRNA，编辑后的miR487b，在缺血后也被上调。我们证实了在多种人类原代血管细胞类型中对miR487b的编辑。短干扰RNA介导的敲低表明，编辑是腺苷脱氨酶作用于RNA 1和2的依赖。使用低dNTP浓度的逆转录，然后进行定量PCR，我们发现相同的腺苷残基在小鼠和人类原代细胞中被甲基化。在鼠腓肠肌中，估计的甲基化分数从缺血前1天的32.8±14％增加到缺血后1天的53.6±12％。短暂的干扰RNA敲低证实甲基化是纤维蛋白依赖性的。尽管我们无法确认甲基化会直接抑制编辑，我们确实表明，作用于RNA 1和2的腺苷脱氨酶和原纤维蛋白对彼此的表达产生负面影响。使用多种萤光素酶报告基因检测，我们可以证明编辑结果可以完全切换目标位点。在人类原代细胞中，我们确认了编辑后miR487b靶向的转变，从而导致编辑后的miR487b靶向组富集了多种促血管生成途径。此外，在体外和离体试验中，编辑过的miR487b（而不是野生型miR487b）的过表达刺激血管生成。导致编辑的miR487b靶基因组富集多种促血管生成途径。此外，在体外和离体试验中，编辑过的miR487b（而不是野生型miR487b）的过表达刺激血管生成。导致编辑的miR487b靶基因组富集多种促血管生成途径。此外，在体外和离体试验中，编辑过的miR487b（而不是野生型miR487b）的过表达刺激血管生成。

结论： MiR487b在小鼠和人类的种子序列中进行了编辑，从而产生了具有独特靶标的新型促血管生成microRNA。在缺血后新血管形成过程中，鼠肌肉组织中miR487b编辑的速率以及2'-O-核糖甲基化的速率增加。我们的发现表明，miR487b编辑在缺血后新血管形成中起着复杂的作用。