Functional analysis of miR-21-3p, miR-30b-5p and miR-150-5p shuttled by extracellular vesicles from diabetic subjects reveals their association with diabetic retinopathy
Functional analysis of miR-21-3p, miR-30b-5p and miR-150-5p shuttled by extracellular vesicles from diabetic subjects reveals their association with diabetic retinopathy
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DOI：
10.1016/j.exer.2019.04.015
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发表时间：
2019-07-01
影响因子：
3.4
通讯作者：
Beltramo, Elena
中科院分区：
文献类型：
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作者：
Mazzeo, Aurora;Lopatina, Tatiana;Beltramo, Elena
内皮细胞和周细胞之间相互作用的改变引起的微血管功能障碍是糖尿病视网膜病变发病机制中的关键事件。在类糖尿病条件下培养的间充质干细胞产生的细胞外小泡进入周细胞，引起周细胞脱离和迁移，刺激血管生成。我们最近发现，糖尿病视网膜病变患者的EV具有不同于健康对照组的miRNA图谱，并在视网膜微血管的体外模型中确定了视网膜病变的特征。特别是囊泡内miR-150-5p、miR-21-3p和miR-30b-5p的作用被低估。在这项工作中，我们进一步鉴定了糖尿病视网膜病变患者的EV，并研究了miR-150-5p、miR-21-3p和miR-30b-5p在微血管细胞内的功能。将miR-21-3p、miR-30b-5p和miR-150-5p的模拟物或抑制剂适当地导入人视网膜周细胞和内皮细胞，以评价它们促进细胞迁移和管状形成的能力。对糖尿病视网膜病变患者(DR组)、无视网膜病变患者(NODR组)和健康对照组(CTR组)的EV进行了mRNA和蛋白质谱分析。微血管细胞内miR-150-5p、miR-21-3p和miR-30b-5p的调控证实了它们参与了异常血管生成。MRNA分析显示7个参与血管生成的基因有不同的表达，随后的蛋白质分析证实DR组HIF-1α表达增加。由于所有这些分子都与糖尿病的缺氧性视网膜损伤有关，我们的数据支持了从循环EV中提取的miR-150-5p、miR-21-3p和miR-30b-5p作为预测糖尿病视网膜病变的生物标志物的可能性的假设。
Microvascular dysfunctions due to altered interactions between endothelial cells (ECs) and pericytes are key-events in the pathogenesis of diabetic retinopathy. Extracellular vesicles (EVs) derived from mesenchymal stem cells cultured in diabetic-like conditions enter pericytes, cause their detachment and migration, and stimulate angiogenesis. We recently showed that EVs from diabetic patients with retinopathy have different miRNA profiling patterns from healthy controls, and determine features of retinopathy in in vitro models of retinal microvasculature. In particular, a role for intra-vesicle miR-150-5p, miR-21-3p and miR-30b-5p was hypothe-sized. In this work, we further characterized EVs from subjects with diabetic retinopathy and investigated miR-150-5p, miR-21-3p and miR-30b-5p functions inside microvascular cells. Human retinal pericytes and ECs were transfected with mimics or inhibitors, as appropriate, of miR-21-3p, miR-30b-5p and miR-150-5p, to evaluate their ability in promoting cell migration and tube formation. mRNA and protein profiling of EVs extracted from diabetic subjects with (DR group) or without retinopathy (noDR group), and healthy controls (CTR group) were also performed. Modulation of miR-150-5p, miR-21-3p and miR-30b-5p inside microvascular cells confirmed their involvement in abnormal angiogenesis. mRNA analysis revealed differing expression of 7 genes involved in angiogenesis, while subsequent protein analysis confirmed increased expression of HIF-1 alpha in DR group. Since all these molecules are involved in the hypoxia-induced retinal damage characteristic of the disease, our data reinforce the hypothesis of a potential use of miR-150-5p, miR-21-3p and miR-30b-5p extracted from circulating EVs as prognostic biomarkers for diabetic retinopathy.