失调的 miR-125a 通过增强糖酵解促进血管生成。
Dysregulated miR-125a promotes angiogenesis through enhanced glycolysis.
机构信息
Molecular Rheumatology, School of Medicine, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland; EULAR Centre for Arthritis and Rheumatic Diseases, Vincent's University Hospital, Dublin Academic Health Care, University College Dublin, Dublin 4, Ireland.
EULAR Centre for Arthritis and Rheumatic Diseases, Vincent's University Hospital, Dublin Academic Health Care, University College Dublin, Dublin 4, Ireland.
出版信息
EBioMedicine. 2019 Sep;47:402-413. doi: 10.1016/j.ebiom.2019.08.043. Epub 2019 Aug 26.
BACKGROUND
Although neoangiogenesis is a hallmark of chronic inflammatory diseases such as inflammatory arthritis and many cancers, therapeutic agents targeting the vasculature remain elusive. Here we identified miR-125a as an important regulator of angiogenesis.
METHODS
MiRNA levels were quantified in Psoriatic Arthritis (PsA) synovial-tissue by RT-PCR and compared to macroscopic synovial vascularity. HMVEC were transfected with anti-miR-125a and angiogenic mechanisms quantified using tube formation assays, transwell invasion chambers, wound repair, RT-PCR and western blot. Real-time analysis of EC metabolism was assessed using the XF-24 Extracellular-Flux Analyzer. Synovial expression of metabolic markers was assessed by immunohistochemistry and immunofluorescent staining. MiR-125a CRISPR/Cas9-based knock-out zebrafish were generated and vascular development assessed. Finally, glycolytic blockade using 3PO, which inhibits Phosphofructokinase-fructose-2,6-bisphophatase 3 (PFKFB3), was assessed in miR-125a-/- ECs and zebrafish embryos.
FINDINGS
MiR-125a is significantly decreased in PsA synovium and inversely associated with macroscopic vascularity. In-vivo, CRISPR/cas9 miR-125a zebrafish displayed a hyper-branching phenotype. In-vitro, miR-125a inhibition promoted EC tube formation, branching, migration and invasion, effects paralleled by a shift in their metabolic profile towards glycolysis. This metabolic shift was also observed in the PsA synovial vasculature where increased expression of glucose transporter 1 (GLUT1), PFKFB3 and Pyruvate kinase muscle isozyme M2 (PKM2) were demonstrated. Finally, blockade of PFKFB3 significantly inhibited anti-miR-125a-induced angiogenic mechanisms in-vitro, paralleled by normalisation of vascular development of CRISPR/cas9 miR-125a zebrafish embryos.
INTEPRETATION
Our results provide evidence that miR-125a deficiency enhances angiogenic processes through metabolic reprogramming of endothelial cells. FUND: Irish Research Council, Arthritis Ireland, EU Seventh Framework Programme (612218/3D-NET).
背景
尽管新血管生成是慢性炎症性疾病（如炎症性关节炎和许多癌症）的一个标志，但针对血管的治疗药物仍然难以捉摸。在这里，我们发现 miR-125a 是血管生成的一个重要调节因子。
方法
通过 RT-PCR 定量测定 Psoriatic Arthritis (PsA) 滑膜组织中的 miRNA 水平，并将其与宏观滑膜血管生成进行比较。将 HMVEC 转染抗 miR-125a，使用管形成测定、Transwell 侵袭室、伤口修复、RT-PCR 和 Western blot 量化血管生成机制。使用 XF-24 细胞外通量分析仪实时分析 EC 代谢。通过免疫组织化学和免疫荧光染色评估滑膜代谢标志物的表达。生成基于 miR-125a CRISPR/Cas9 的敲除斑马鱼，并评估血管发育情况。最后，使用 3PO（抑制磷酸果糖激酶-果糖-2,6-二磷酸酶 3 (PFKFB3)）抑制糖酵解，在 miR-125a-/-EC 和斑马鱼胚胎中进行评估。
结果
miR-125a 在 PsA 滑膜中显著降低，与宏观血管生成呈负相关。在体内，CRISPR/cas9 miR-125a 斑马鱼表现出过度分支表型。在体外，miR-125a 抑制促进 EC 管形成、分支、迁移和侵袭，其代谢谱向糖酵解的转变与之平行。这种代谢转变也在 PsA 滑膜血管中观察到，其中葡萄糖转运蛋白 1 (GLUT1)、PFKFB3 和丙酮酸激酶肌肉同工酶 M2 (PKM2) 的表达增加。最后，PFKFB3 阻断显著抑制了体外抗 miR-125a 诱导的血管生成机制，同时使 CRISPR/cas9 miR-125a 斑马鱼胚胎的血管发育正常化。
结论
我们的研究结果提供了证据，表明 miR-125a 缺乏通过内皮细胞的代谢重编程增强血管生成过程。
资金
爱尔兰研究委员会、关节炎爱尔兰、欧盟第七框架计划（612218/3D-NET）。
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