RESUMO
PURPOSE: The apoptosis of human retinal pigment epithelial cells (RPEs) plays a critical role in the pathogenesis of diabetic retinopathy (DR), but the molecular mechanisms remain unclear. In this study, we explored the function of miR-203a-3p in CoCl2-induced RPEs apoptosis. METHODS: The cellular localization of miR-203a-3p was assessed by in situ hybridization. Luciferase reporter assays were performed to validate that suppressor of cytokine signaling 3(SOCS3) as a direct target of miR-203a-3p. Effects of miR-203a-3p manipulation on RPEs apoptosis were evaluated using TdT-mediated dUTP Nick-End Labeling (TUNEL) and Flow Cytometry. Expression levels of miR-203a-3p was analyzed by RT-PCR, the expression of target proteins was detected by western blot. RESULTS: miR-203a-3p was found to be located in the RPE layer of the retinas from normal and diabetic rats and SOCS3 was a direct target of miR-203a-3p. miR-203a-3p mimics resulted in improved CoCl2-induced apoptosis of RPEs, overexpression of SOCS3 or c-Jun N-terminal kinase (JNK) inhibitor SP600125 reversed the pro-apoptotic effect of miR-203a-3p, to a certain extent. CONCLUSIONS: Our data implied a crucial role of miR-203a-3p as a novel regulator of CoCl2-induced RPEs apoptosis through SOCS3. Deregulation of miR-203a-3p/SOCS3/JNK/c-Jun cascade thus may serve as an important contributor to RPEs apoptosis in DR.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Retinopatia Diabética/metabolismo , Células Epiteliais/patologia , MicroRNAs/metabolismo , Epitélio Pigmentado da Retina/patologia , Animais , Antimutagênicos , Apoptose , Técnicas de Cultura de Células , Cobalto , Diabetes Mellitus Experimental/etiologia , Diabetes Mellitus Experimental/patologia , Retinopatia Diabética/etiologia , Retinopatia Diabética/patologia , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Humanos , Ratos , Epitélio Pigmentado da Retina/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/metabolismoRESUMO
OBJECTIVE: To evaluate the role of CCAAT/enhancer-binding protein ß (C/EBP ß (C/EBP. METHODS: Rats with OIR were exposed to alternating hypoxic and hyperopic conditions for 14 days. Then, the rats with OIR were assigned randomly to groups that received intravitreal injections of either shRNA lentiviral particles targeting C/EBP ß (C/EBP ß (C/EBP ß (C/EBP ß (C/EBP ß (C/EBP ß (C/EBP. RESULTS: In OIR rats, the expression levels of C/EBP ß (C/EBP P < 0.01). The p-C/EBP ß (C/EBP ß (C/EBP ß (C/EBP ß (C/EBP ß (C/EBP P < 0.01). The p-C/EBP ß (C/EBP ß (C/EBP ß (C/EBP P < 0.01). The p-C/EBP. CONCLUSIONS: C/EBP ß shRNA inhibits RNV in OIR. A potential mechanism may be that the activity of C/EBP ß increases with its overexpression, which in turn aggravates the amount of the retinal vascular damage and promotes transcription of VEGF. C/EBP ß might be a new therapeutic target for preventing RNV.ß (C/EBP ß (C/EBP ß (C/EBP.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Doenças Retinianas/metabolismo , Neovascularização Retiniana/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Proteína beta Intensificadora de Ligação a CCAAT/genética , Modelos Animais de Doenças , Inativação Gênica , Oxigênio , RNA Interferente Pequeno , Ratos , Ratos Sprague-Dawley , Doenças Retinianas/induzido quimicamente , Doenças Retinianas/genética , Neovascularização Retiniana/genéticaRESUMO
PURPOSE: To evaluate the changes of oxidative DNA damage (in the form of 8-OHdG) and three key DNA base-excision repair (BER) proteins, human 8-oxoguanine DNA glycosylase 1 (hOGG1), apurinic/apyrimidinic endonuclease 1 (APE1) and DNA polymerase ß (Pol ß), in lens epithelium cells (LECs), cortex and nucleus of lenses with age-related cataract (ARC) and age-matched controls. METHODS: A total of 90 patients with ARC and 21 control subjects were enrolled. The samples included the anterior lens capsules (mainly composed of LECs) and various portions of lens. An ELISA assay was used to assess the 8-OHdG levels of genomic DNA extracted. Immunofluorescence and Western blot were used to analyze the localization and quantification of three BER proteins, respectively. RESULTS: The 8-OHdG levels in lenses with ARC were higher than those of controls, and were not different among ARC subtypes. The 8-OHdG levels were the highest in the nucleus, followed by the LECs and cortex. The repair proteins were predominantly detected in the cellular nuclei of the LECs and superficial cortical cells. In the LECs, the protein levels of the three BER enzymes were higher in ARC than in controls. In the cortex, a downward trend of the levels of three BER enzymes was found with the increasing opaque degrees. In the nucleus, no enzymes were detected. CONCLUSIONS: Our findings indicate that the oxidative DNA damage increases in lenses with ARC, and the three BER enzymes compensatively increase in the LECs, while decreasing in the opaque cortex. The results suggest that the oxidative DNA damage may be related ARC and the alteration of DNA repair enzyme levels in ARC is associated with the location and opaque degrees of lens.
