Eye Center, The Second Hospital of Jilin University, Changchun, Jilin, China.
Department of Ophthalmology, Shanghai General Hospital (Shanghai first people hospital), Shanghai Jiaotong University Medical School, Shanghai, China.
J Cell Mol Med. 2019 Jul;23(7):4723-4737. doi: 10.1111/jcmm.14369. Epub 2019 May 15.
Retinal cell damage caused by diabetes leads to retinal microvascular injury. Roundabout 4 (ROBO4) is involved in angiogenesis, which varies with the development of diabetic retinopathy (DR). Here, we explored the transcriptional regulation and microRNA-mediated modulation of ROBO4 expression and related retinal cell function in DR. A streptozotocin-induced type I diabetic animal model was established to detect the expression of hypoxia inducible factor-1α (HIF-1α), specificity protein 1 (SP1) and ROBO4. Retinal pigment epithelium (RPE) cells were cultured under hyperglycaemia or hypoxia and used for mechanistic analysis. Furthermore, roles of miR-125b-5p and miR-146a-5p were evaluated, and their targets were identified using luciferase assays. The cell functions were evaluated by MTS assays, permeability analysis and migration assays. The development of DR increased the levels of HIF-1α, SP1 and ROBO4 both in the DR model and in hyperglycaemic/hypoxic RPE cells. They were co-expressed and up-regulated in diabetic retinas and in RPE cells under hyperglycaemia/hypoxia. Knockdown of HIF-1α significantly inhibited SP1 and ROBO4, whereas SP1 down-regulation abolished ROBO4 expression in RPE cells under hyperglycaemia/hypoxia. miR-125b-5p and miR-146a-5p were down-regulated by hyperglycaemia and/or hypoxia. Up-regulation of miRNAs reversed these changes and resulted in recovery of target gene expression. Moreover, luciferase assays confirmed miR-125b-5p targeted SP1 and ROBO4, and miR-146a-5p targeted HIF-1α and ROBO4 directly. The decreased cell viability, enhanced permeability, and increased cell migration under DR conditions were mitigated by knockdown of HIF-1α/SP1/ROBO4 or up-regulation of miR-125b-5p/miR-146a-5p. In general, our results identified a novel mechanism that miR-125b-5p/miR-146a-5p targeting HIF-1α/SP1-dependent ROBO4 expression could retard DR progression.
糖尿病引起的视网膜细胞损伤导致视网膜微血管损伤。Roundabout 4（ROBO4）参与血管生成，其变化与糖尿病视网膜病变（DR）的发展有关。在这里，我们探讨了 ROBO4 表达的转录调控和 microRNA 介导的调节以及 DR 中相关视网膜细胞功能。建立链脲佐菌素诱导的 1 型糖尿病动物模型，以检测缺氧诱导因子-1α（HIF-1α）、特异性蛋白 1（SP1）和 ROBO4 的表达。在高糖或缺氧条件下培养视网膜色素上皮（RPE）细胞，进行机制分析。此外，评估了 miR-125b-5p 和 miR-146a-5p 的作用，并通过荧光素酶测定鉴定其靶标。通过 MTS 测定、通透性分析和迁移测定评估细胞功能。DR 的发展增加了 DR 模型中和高糖/缺氧 RPE 细胞中 HIF-1α、SP1 和 ROBO4 的水平。它们在糖尿病视网膜和高糖/缺氧下的 RPE 细胞中共同表达和上调。HIF-1α 的敲低显著抑制了 SP1 和 ROBO4，而 SP1 的下调消除了高糖/缺氧下 RPE 细胞中 ROBO4 的表达。miR-125b-5p 和 miR-146a-5p 被高糖和/或缺氧下调。miRNA 的上调逆转了这些变化，并导致靶基因表达的恢复。此外，荧光素酶测定证实 miR-125b-5p 靶向 SP1 和 ROBO4，miR-146a-5p 直接靶向 HIF-1α 和 ROBO4。在 DR 条件下，细胞活力下降、通透性增强和迁移增加通过 HIF-1α/SP1/ROBO4 的敲低或 miR-125b-5p/miR-146a-5p 的上调得到缓解。总之，我们的结果确定了一种新的机制，即 miR-125b-5p/miR-146a-5p 通过靶向 HIF-1α/SP1 依赖性 ROBO4 表达来减缓 DR 的进展。
