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Inhibitor of growth 4 (ING4), a potential tumor suppressor, is implicated in cell migration and angiogenesis. However, its effects on diabetic retinopathy (DR) have not been elucidated. In this study, we aimed to evaluate ING4 expression in normal and diabetic rats and clarify its effects on hypoxia‐induced dysfunction in human retinal pigment epithelial (ARPE‐19) cells. A Type 1 diabetic model was generated by injecting rats intraperitoneally with streptozotocin and then killed them 4, 8, or 12 weeks later. ING4 expression in retinal tissue was detected using western blot analysis, reverse transcription quantitative real‐time polymerase chain reaction (RT‐qPCR), and immunohistochemistry assays. After transfection with an ING4 overexpression lentiviral vector or small interfering RNA (siRNA), ARPE‐19 migration under hypoxia was tested using wound healing and transwell assays. The angiogenic effect of conditioned medium (CM) from ARPE‐19 cells was examined by assessing human retinal endothelial cell (HREC) capillary tube formation. Additionally, western blot analysis and RT‐qPCR were performed to investigate the signaling pathways in which ING4, specificity protein 1 (Sp1), matrix metalloproteinase 2 (MMP‐2), MMP‐9, and vascular endothelial growth factor A (VEGF‐A) were involved. Here, we found that ING4 expression was significantly reduced in the diabetic rats' retinal tissue. Silencing ING4 aggravated hypoxia‐induced ARPE‐19 cell migration. CM collected from ING4 siRNA‐transfected ARPE‐19 cells under hypoxia promoted HREC angiogenesis. These effects were reversed by ING4 overexpression. Furthermore, ING4 suppressed MMP‐2, MMP‐9, and VEGF‐A expression in an Sp1‐dependent manner in hypoxia‐conditioned ARPE‐19 cells. Overall, our results provide valuable mechanistic insights into the protective effects of ING4 on hypoxia‐induced migration and angiogenesis regulation in ARPE‐19 cells. Restoring ING4 may be a novel strategy for treating DR. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The catabolic process of autophagy is arousing the attention of researchers studying diabetic retinopathy (DR), but the role and molecular mechanism of autophagy in DR are still unclear. An in vivo diabetic rat model and in vitro hyperglycemic-exposed retinal pigment epithelium (RPE) cell cultures were established to mimic early DR. Transmission electron microscopy and mRFP-GFP-LC3 adenovirus transfection were applied for autophagic flux analysis. MicroRNA (miR)-19a-3p, members of the phosphate and tensin homolog (PTEN)/Akt/mammalian target of rapamycin (mTOR) pathway, and the autophagy-related proteins light chain (LC)3II/I and p62 were detected. Annexin V, transwell, Cell Counting Kit-8, fluorescein isothiocyanate-dextran monolayer permeability assay, and transepithelial electrical resistance were performed to evaluate the effects of regulating autophagy on RPE cells under the DR condition. Autophagy was aberrantly activated in DR as evidenced by autophagosome accumulation. Further mechanistic experiments revealed that DR induced PTEN expression, thus inhibiting Akt/mTOR phosphorylation and stimulating aberrant autophagy and apoptosis. Notably, these events could be reversed by miR-19a-3p directly targeting PTEN. Downregulation of autophagy by miR-19a-3p overexpression, PTEN knockdown, or 3-methyladenine (3-MA) treatment inhibited autophagosome formation and thus effectively ameliorated hyperglycemia-induced RPE cell apoptosis, increased migration, inhibited viability, and enhanced monolayer permeability under the DR condition. Our findings suggest that upregulation of miR-19a-3p inhibits aberrant autophagy by directly targeting PTEN, thus protecting RPE cells against DR damage. miR-19a-3p may represent a novel therapeutic target for inducing protective autophagy in early DR. [ABSTRACT FROM AUTHOR]

Retinal pigment epithelium (RPE) cell-based gene expression studies performed under hypoxia and/or hyperglycemia show huge potential for modeling cell responses in diabetic retinopathy, retinopathy of prematurity and other retinal diseases. However, normalization of gene expression on RPE cells under those conditions has commonly been done using either GAPDH or β-actin as reference genes without any validation of their expression stability. Therefore, we aimed to establish a suitable set of reference genes for studies on RPE cells cultured under both normal culturing glucose and atmospheric oxygen tension (normoxia, 21%), under a low oxygen tension (hypoxia, 1%), under a high glucose growth medium (25 mmol/l) and under the combination of the two changed conditions above for distinct time points taking together from 24 h to 7 days. Quantitative real-time PCR (qRT-PCR) was applied on RNA obtained from a cell line, ARPE-19. Stability of 14 commonly used reference genes was assessed and ranked according to their stability values using the geNorm and NormFinder softwares with the aim to find the most stable expressed gene under all conditions. Our findings confirm that HPRT1, GUSB and PPIA are the most suitable reference genes for RPE cell gene expression experiments subjected to hypoxia and/or hyperglycemia. To emphasize the importance of selecting the most stably expressed reference genes for obtaining reliable results, mRNA expression levels of hypoxia induced factor-1α were analyzed vs the best reference genes, the worst ones and the most commonly used ones. These reference genes gave the most reliable normalization for comparative analyses of gene transcription under those conditions. [ABSTRACT FROM AUTHOR]