Studies On The Function And Mechanism Of The Regulation Of VEGF And Robo4 By MiRNA In Diabetic Retinopathy

Diabetic retinopathy(DR)is one of severe complications in diabetes mellitus.It is the most important cause of blindness in working age.Depending on retinal neovascularization occurs,DR is divided into non-proliferative diabetic retinopathy(NPDR)and proliferative diabetic retinopathy(PDR).Vascular endothelial growth factor(VEGF)and Robo4 are the most important factors in the development of DR.VEGF plays an important role in the development of DR,which can increase vascular permeability and make changes in the interaction between retinal cells and capillary endothelial cells.The expression of VEGF and Robo4 increased simultaneously under high glucose,and promoted the development of DR.Therefore,it is very important to study the regulatory mechanisms of VEGF and Robo4 in the development of DR.In this study,we examined the expression correlation between VEGF and Robo4 in the FVM of PDR patients and in the retina of STZ-induced diabetic rats,and predicted the functions in the DR process.This part of the study was tested in human tissue samples,cells and animals:(1)In 13 cases of PDR,we analyzed the structure of the FVM by HE staining and the expression of VEGF and Robo4 in FVM,the co-localization of these two protein was further identified by confocal microscopy.It showed that Robo4 and VEGF played an important role in the development of DR.(2)To verify the expression of VEGF and Robo4 in the development of DR,HREC and HRPE were cultured under high glucose conditions,It is showed that both miRNA and protein levels of VEGF and Robo4 were improved by using RT-PCR and Western-Blot.(3)Diabetes rat was induced by STZ,and the expression of VEGF and Robo4 protein in retinal of diabetic rats was investigated by Western-Blot.After 4 to 8weeks of feeding,the expression of VEGF and Robo4 protein was improved in diabetic rats than that in normal groups.From the above studies,we predict that the interaction of VEGF and Robo4 hasthree stages:(1)In normal pHysiological state,Robo4 inhibits the expression of VEGF;(2)The expression of Robo4 was decreased,the expression of VEGF was increased in high glucose condition,which resulted in the occurrence of DR;(3)In the course of DR,the expression of Robo4 and VEGF was increased,and promoted the progression of DR.miRNA modulates gene expression in various manners:(1)by directly binding to DNA into the gene promoter region;(2)by miRNA binding to the 3?-untranslated region of target genes,leading to either posttranscriptional silencing and translational repression or RNA degradation;(3)The binding of miRNA to transcription factors and to DNA regions leads to epigenetic modification of the chromatin,which in turn participate to DR progression.In this study,we analyzed the expression of miRNA in retinal endothelial cells and retinal pigment epithelial cells under high glucose levels,and screened miRNA interacted with both VEGF and Robo4.The possibility of its application in DR therapy was discussed by diabetic rat models.(1)RT-PCR was used to study the expression of miRNA-15 family in retinal cells in high glucose conditions.The results showed that the expression of miRNA-15 a,miRNA-16,miRNA-424 in HREC and HRPE was decreased and the expression of miRNA-195 and miRNA-497 was increased.(2)In this study,we used software to predict that miRNA-15 a and miRNA-195 could act on both VEGF and Robo4,and study the effect of miRNA-15 a on both VEGF and Robo4 protein expression in diabetic rats.Based on the above studies,it indicated that miRNA-15 a targets VEGF and Robo4,and inhibit the expression of VEGF and Robo4 proteins.It can be used as a therapeutic target forPDR.In conclusion,we concluded that VEGF and Robo4 had synergistic effects on the development of PDR,and promoted the progression of the disease.The miRNA as a treatment target for DR was also evaluated.The study not only helps to understand the mechanism of DR progress,but also provides theoretical support for identifying the potential therapeutic target of DR patients,it greatly promotes the development and clinical application of miRNA.