Supplementary Material for: Reciprocal Antagonism between MicroRNA-138 and SIRT1 and Its Implications for the Angiogenesis of Endothelial Cells

MicroRNAs and sirtuins are important epigenetic regulators of gene expression and both contribute significantly to postnatal vascular development. However, the crosstalk between miRNAs and sirtuins in the modulation of angiogenesis has rarely been discussed. Here, we investigated the interactions between miR-138 and sirtuins in the process of angiogenesis. We found that overexpression of miR-138 markedly suppressed the proliferation, migration, and tube-forming capacities of the endothelial cells. And, miR-138 inhibitor-treated endothelial cells showed a reversed phenotype. Furthermore, miR-138 plays a negative role in vascular development in vivo. Western blot and qPCR assays demonstrated that SIRT1 was silenced by miR-138, and a luciferase reporter assay showed that miR-138 bound to the 3′-UTR of SIRT1. The re-expression of SIRT1 alleviated miR-138-mediated suppression of angiogenesis. Furthermore, silencing SIRT1 could boost the level of miR-138. And, upon miR-138 inhibitor treatment, SIRT1 silencing no longer reduced the angiogenic ability of endothelial cells significantly. These results demonstrated that the circuitry involving miR-138 and SIRT1 may participate in vascular homeostasis and also offered the possibility of identifying a new approach in the treatment of angiogenic diseases.

微RNA（MicroRNAs）与去乙酰化酶（sirtuins）均为重要的基因表达表观遗传调控因子，二者均可显著参与产后血管发育的调控过程。然而，微小RNA与去乙酰化酶在血管生成调控中的交叉对话却鲜有报道。本研究探究了miR-138与去乙酰化酶在血管生成过程中的相互作用。研究发现，miR-138过表达可显著抑制内皮细胞的增殖、迁移及管腔形成能力；经miR-138抑制剂处理的内皮细胞则呈现出表型逆转的现象。此外，miR-138在体内可对血管发育发挥负调控作用。蛋白质免疫印迹（Western blot）与实时荧光定量聚合酶链反应（qPCR）实验结果显示，miR-138可沉默SIRT1的基因表达；荧光素酶报告基因实验证实，miR-138可直接结合至SIRT1的3′非翻译区（3′-UTR）。恢复SIRT1的表达可缓解miR-138介导的血管生成抑制效应。进一步研究显示，沉默SIRT1可上调miR-138的表达水平；且当使用miR-138抑制剂处理时，沉默SIRT1便不再能显著降低内皮细胞的血管生成能力。上述结果表明，miR-138与SIRT1构成的调控环路可能参与血管稳态的维持，同时也为血管生成相关性疾病的新型治疗策略开发提供了潜在方向。