Enhanced ROBO4 is mediated by up‐regulation of HIF‐1α/SP1 or reduction in miR‐125b‐5p/miR‐146a‐5p in diabetic retinopathy

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.