Research On The Regulation And Mechanism Of Mir-203A-3P On OBRB Function In Diabetic Retinopathy

Purpose: The destruction of blood retinal barrier(BRB)and diabetic macular edema(DME)are the main characteristics of DR,but there is no safe and effective treatment strategies.By simulating the environment of diabetes in vivo and in vitro,the regulatory effect and potential mechanism of miR-203a-3p on the blood retinal barrier(o BRB)were studied.Methods:(1)Impact of miR-203a-3p on o BRB function: firstly,Intraperitoneal injection of 1% STZ solution to establish a diabetic rat model,in situ hybridization was used to determine the location of miR-203a-3p in the retina of diabetic rats;the expression of miR-203a-3p and SOCS3 in ARPE-19 cells cultured in high glucose and hypoxia were detected by Western blot and RT-PCR respectively;then ARPE-19 was transfected with inhibitor-miR-203a-3p,mimic-miR-203a-3p and their negative control oligonucleotides,permeability measurement and transepithelial resistance(TER)were used to detect the diffusion rate of FITC-dextran in ARPE-19 cell layer from the top of ARPE-19 to the base and the transepithelial resistance of paracellular pathway,western blot was used to detect the expression of claudin-1,ZO-1,and occludin in ARPE-19;then,we use lentivirus plasmid to overexpress SOCS3 in ARPE-19 cell layer or use STAT inhibitor WP1066 to observe FITC-dextran diffusion rate and transepithelial resistance of paracellular pathway,the expression of p-STAT,occludin and other tight junction proteins,to verify that whether SOCS3 affects the o BRB function by regulating the STAT pathway.(2)The effect of miR-203a-3p on apoptosis of ARPE-19 induced by hypoxia: ARPE-19 was transfected with mimic-miR-203a-3p and its negative control oligonucleotide,Flow cytometry,TUNEL staining and Western blot were used to detect the effect of mimic-miR-203a-3p on the apoptosis rate,morphology,exoression of cleaved caspase 3,Bax,Bcl2 of ARPE-19 induced by hypoxia;Luciferase experiment was used to verify the specific targeting relationship between miR-203a-3p and SOCS3;then,we used lentivirus plasmid to overexpress SOCS3 in ARPE-19 or SP600125,a JNK specific inhibitor,to verify whether SOCS3 can affect hypoxia induced RPE apoptosis by regulating JNK pathway.Results: The effect of miR-203a-3p on the function of o BRB: compared with normal rats,the expression of miR-203a-3p in RPE layer and INL layer of diabetic rats increased significantly;the expression of miR-203a-3p in ARPE-19 cells was upregulated by high glucose and hypoxia culture in vitro,but the trend of SOCS3 expression was opposite;the transfection of mimic-miR-203a-3p increased the FITCdextran diffusion,decreased the trans epithelial resistance of the paracellular pathway,and decreased the expression of claudin-1 and other tight junction proteins in ARPE-19 single cell layer cultured in high glucose and hypoxia,while the transfection of inhibitormiR-203a-3p had the opposite effect;overexpression of SOCS3 or application of WP1066 ameliorated the damage of o BRB caused by high glucose and hypoxia.(2)The effect of miR-203a-3p on apoptosis of ARPE-19 cells induced by hypoxia: the transfection of mimic-miR-203a-3p increased the apoptosis rate of ARPE-19 cells induced by hypoxia,upregulated the expression of cleaved caspase 3 and Bax,downregulated the expression of Bcl-2;Luciferase assay confirmed that miR-203a-3p had specific targeting effect on SOCS3;overexpression of SOCS3 or application of SP600125 could reverse the promoting effect of miR-203a-3p on apoptosis of ARPE-19 induced by hypoxia.Conclusion: miR-203a-3p has an specific inhibitory effect on SOCS3,not only through the regulation of STAT signaling pathway,but also through the specific inhibition of SOCS3,it enhances JNK / c-Jun signaling pathway,promotes the apoptosis of ARPE-19 cells induced by hypoxia,which may be an important mechanism leading to the damage of o BRB.