ABSTRACT
The dysregulated microRNAs (miRNAs) are involved in diabetic retinopathy progression. Epithelial mesenchymal transition (EMT) and cell permeability are important events in diabetic retinopathy. However, the function and mechanism of miR-195 in EMT and cell permeability in diabetic retinopathy remain largely unclear. Diabetic retinopathy models were established using streptozotocin (STZ)-induced diabetic mice and high glucose (HG)-stimulated ARPE-19 cells. Retina injury was investigated by hematoxylin-eosin (HE) staining. EMT and cell permeability were analyzed by western blotting, immunofluorescence, wound healing, and FITC-dextran assays. MiR-195 expression was detected via qRT-PCR. YY1, VEGFA, Snail1, and Smurf2 levels were detected via western blotting. The interaction relationship was analyzed via ChIP, Co-IP, or dual-luciferase reporter assay. The retina injury, EMT, and cell permeability were induced in STZ-induced diabetic mice. HG induced EMT and cell permeability in ARPE-19 cells. MiR-195, YY1, VEGFA, and Snail1 levels were enhanced, but Smurf2 abundance was reduced in STZ-induced diabetic mice and HG-stimulated ARPE-19 cells. VEGFA knockdown decreased Snail1 expression and attenuated HG-induced EMT and cell permeability. YY1 silence reduced VEGFA and Snail1 expression, and mitigated HG-induced EMT and cell permeability. YY1 could bind with VEGFA and Snail1, and it was degraded via Smurf2-mediated ubiquitination. MiR-195 knockdown upregulated Smurf2 to decrease YY1 expression and inhibited HG-induced EMT and cell permeability. MiR-195 targeted Smurf2, increased expression of YY1, VEGFA, and Snail1, and promoted HG-induced EMT and cell permeability. MiR-195 promotes EMT and cell permeability of HG-stimulated ARPE-19 cells by increasing VEGFA/Snail1 via inhibiting the Smurf2-mediated ubiquitination of YY1.
Subject(s)
Diabetic Retinopathy/metabolism , Epithelial Cells/metabolism , Epithelial-Mesenchymal Transition , MicroRNAs/metabolism , Retinal Pigment Epithelium/metabolism , Ubiquitin-Protein Ligases/metabolism , YY1 Transcription Factor/metabolism , Animals , Cell Line , Diabetic Retinopathy/genetics , Diabetic Retinopathy/pathology , Disease Models, Animal , Epithelial Cells/pathology , Glucose/metabolism , Humans , Male , Mice, Inbred C57BL , MicroRNAs/genetics , Permeability , Proteasome Endopeptidase Complex/metabolism , Proteolysis , Retinal Pigment Epithelium/pathology , Signal Transduction , Snail Family Transcription Factors/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitination , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism , YY1 Transcription Factor/geneticsABSTRACT
Long noncoding RNAs (lncRNAs) are important regulators in various diseases including diabetic retinopathy (DR). In this study, DR patients exhibited significantly increased expression of serum LncRNA-OGRU compared with normal individuals. Streptozotocin (STZ)-challenged rats with DR also had higher OGRU expression in retinas than that of the control group, which was confirmed in Müller cells upon high glucose (HG) stimulation. OGRU knockdown remarkably decreased vascular endothelial growth factor (VEGF) and transforming growth factor-ß1 (TGF-ß1) expression in HG-incubated Müller cells. HG-induced inflammatory response and oxidative stress in vitro were markedly mitigated by OGRU knockdown through restraining IκBÉ/nuclear factor kappa beta (NF-κB) and improving nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathways, respectively. Further studies indicated that OGRU suppression greatly restored miR-320 expression, and a negative correlation between them was detected in DR patients. We also found that miR-320 over-expression considerably restrained TGF-ß1 signaling, and hindered inflammation and reactive oxygen species (ROS) production in HG-stimulated Müller cells. Additionally, OGRU knockdown or miR-320 over-expression could dramatically down-regulate ubiquitin-specific peptidase 14 (USP14) expression levels in HG-incubated Müller cells, and miR-320 could directly target USP14. Notably, OGRU/miR-320 axis-mediated TGF-ß1 signaling, inflammation and ROS were largely dependent on USP14. Intriguingly, our results showed that USP14 directly interacted with transforming growth factor-beta type 1 receptor (TßR1), and impeded TßR1 ubiquitination and degradation. Furthermore, USP14 could also facilitate IκBÉ deubiquitination and degradation, exacerbating IκBÉ phosphorylation and NF-κB activation. Finally, our in vivo studies confirmed that OGRU knockdown considerably ameliorated DR progression in STZ-challenged rats through mediating the mechanisms observed in vitro. Collectively, these findings implicated that LncRNA-OGRU mediated DR progression through competing for miR-320 to regulate USP14 expression, and thus LncRNA-OGRU/miR-320/USP14 axis may be considered as a therapeutic target for DR treatment.
