Mechanisms Of Type 2 Diabetic Retinopathy-associated MicroRNAs And Their Downstream Target Genes

Background and purpose:Type 2 diabetes mellitus(T2DM)is a common chronic metabolic disease with blindness risk.Diabetic retinopathy(DR)is the major cause of blindness in patients with T2 DM.The current understanding of the molecular pathology of DR lies in the role of oxidative stress,inflammation,and angiogenesis in the development and progression of DR.In recent years,there have been significant advances in the understanding of the molecular mechanisms of DR,with biological pathways such as response to glucose,cell division,protein kinase activity,and mesenchymal cell development also involved in the development and progression of DR.However,its pathogenesis is unclear and there is no effective treatment.Therefore,Bioinformatics algorithms will be used to quantify unknown microRNAs and known microRNAs in peripheral blood of patients with DR,to identify their enrichment,and to indicate biological functions.At the same time,our previous findings suggest a reduced enrichment of mi R-204-3p in peripheral blood of patients with DR.Therefore,this study amis to use a cell model to verify whether its expression is down-regulated in human retinal endothelial cells.Methods:1)Prediction of unknown microRNAs as well as known microRNAs in peripheral blood using bioinformatics algorithms,analysis of microRNA expression differences between diabetic patients with and without retinopathy using the DESeq2 package,and prediction of potential target genes through mi RDB and multi Mi R databases.To further explore the function of these target genes,enrichment analysis was performed using metascape.2)In vitro validation experiments: human retinal endothelial cells(h RMECs)were used to construct a high glucose injury model using 25 m M glucose,and the success of the model was verified using CCK8 cell viability assay and migration assay(Transwell assay).q RT-PCR was used to detect changes in the expression of mi R-204-3p,VEGFA and VEGFR.Results :1)Peripheral blood microRNA sequencing data sets from 108 type 2 diabetic patients(42 DR,66 T2DM)were analyzed using the DESeq2 package.Differential analysis of known microRNAs identified 225 statistically significant microRNAs,of which 17 were significantly different in expression(padj≤0.05,|log2Fold Change|>2).Pathway enrichment analysis of the target genes showed that these microRNAs were involved in multiple biological pathways,including enzyme-linked receptor protein signaling pathways,cancer pathways and MAPK cascade regulation.The enriched genes for these pathways were derived from the target genes of 15 significantly enriched microRNAs.2)We identified 422 unknown microRNAs in the peripheral blood of T2 DM patients,of which 9 unknown microRNAs were significantly enriched in the peripheral blood of patients with DR.Target gene prediction and pathway analysis revealed that the target genes of these 9 microRNAs were associated with key biological processes and pathways such as brain development,neuronal differentiation,signaling and metabolism.3)In our cell experiment,we detected the effects of high glucose on cell viability,VEGFA,VEGFR,and mi R-204-3p.No significant changes in cell viability were observed between 2 and 7 days.VEGF and VEGFR expression levels did not change significantly between the high glucose group and the control group at 3 days;the relative expression of VEGF and VEGFR was significantly higher compared to the control group at 5 and 7 days of continuous high glucose culture.The relative expression of mi R-204-3p was significantly higher in the high glucose group compared to the control group at 7 days of continuous incubation,in contrast to the significantly lower expression trend in the plasma of DR patients.At 9 and 14 days,the relative expression of mi R-204-3p was significantly lower compared to the control group,in contrast to the significantly lower expression trend in the plasma of DR patients.HRMECs were significantly more capable of migrating in the high glucose group than in the control group after 14 days of high glucose induction at 25 m M.Conclusions:1)The study validated the role of known microRNAs in DR by regulating MAPK cascade,angiogenesis and other signaling pathways.2)Our study identified 9 novel microRNAs and found that these unknown microRNAs may be involved in neuronal differentiation and morphogenesis,affecting the occurrence and development of DR.3)Human retinal microvascular endothelial cells can be effectively simulated under a pathological state of continuous high glucose culture for 9 days.