New Exploration Of Treatment Target For Diabetic Retinopathy Based On RNA-seq Technology

Objective:Diabetic retinopathy is a common and serious complication of diabetes.It has been found that high glucose can affect the function of vascular endothelial cells and play a crucial role in diabetic retinopathy.Based on RNA transcriptome analysis technology,we evaluated the effect of high glucose status on retinal vascular endothelial cells,aiming to fully explore and analyze the biological information of the effect of high glucose status on retinal endothelial cells,so as to provide a more accurate entry point for the treatment of diabetic retinopathy.Methods:Retinal microvascular endothelial cells(RF/6A)were divided into control group and the high glucose group(25 mmol/L glucose continued stimulus for 24hours),retinal vascular endothelial cell proliferation,migration ability and tube formation ability under the stimulation of high glucose were detected by MTT cell proliferation test,scratch test,lumen formation experiment and transwell detection.On this basis of high glucose cell model successfully established,RNA transcriptome analysis technology was applied to complete transcriptome sequencing,differentially expressed genes and functional enrichment analysis of the two groups of cells.Based on the transcriptome data,we screened out first eight differentially expressed genes between the two groups,and verified the expression of these genes in vivo(diabetic and non-diabetic patients blood,STZ induced diabetic rat model)and in vitro(RF/6A,HRCEC,RPE and Muller cells stimulated by 4HNE to simulate microenvironment in diabetic retinopathy)using RT-PCR,immunofluorescence and western.Results:Cell proliferation and migration experiments showed that the proliferation rate and migration rate of the high glucose group increased significantly compared with the normal group,indicating the successful establishment of the high glucose model.Differential gene expression profiles of vascular endothelial cells induced by high glucose were obtained by RNA-seq,and a total of 449 differentially expressed genes were found by comparing the sequencing results of the two groups.GO and Pathway analysis showed that differentially expressed genes were mainly concentrated in TGF-βsignaling pathways,which further affected energy metabolism and protein synthesis of cells.Six genes with significant expression were screened out from the differential genes,named SMAD9,BMP4,DAB1,ATP1A3,CHST5 and UNC5C.These genes have been linked to cancer,cardiovascular disease and other diseases,but their association with DR has not been proven.BMP4 has been shown to be involved in regulating VEGF.BMP4,SMAD9,DAB1,and CHST5 are associated with extracellular matrix metabolism.ATP1A3 regulates Na~+/K~+ ATPase system,causing ganglion cell ischemia and apoptosis.In addition,UNC5C can also induce the apoptosis of ganglion cells.RT-PCR,immunofluorescence and western results showed that the expressions of SMAD9 and BMP4 were up-regulated in the blood of diabetic patients,retina of STZ induced diabetic rats and several cell models,and the expression trends of genes were consistent with the transcriptomic data.In addition,after upregulating the expression of BMP4 in RF/6A,HRCEC,RPE and Muller cells,we found that BMP4 could significantly promote the proliferation and migration of these retinal cells,activate the expression of its downstream factor SMAD9,and significantly up-regulate the expression of related fibrosis factors and VEGF.Conclusion:The effects of high glucose on retinal vascular endothelial cells are various,such as destroying the transmembrane conduction and extracellular matrix metabolism of retinal vascular endothelial cells,thus affecting their functions.By analyzing transcriptome data and validating the results with multiple models,BMP4and SMAD9 may be new targets for the early treatment of diabetic retinopathy in the future.