Transcriptional Profiling Of Peripheral Blood In Proliferative Diabetic Retinopathy Patients

Background: Diabetic retinopathy is one of the most serious complications in diabetic patients,and is also one of the major causes to blindness.When diabetic retinopathy comes to proliferative stage,accompanying with neovascularization,could lead to damage of structure in retina,such as vitreous hemorrhage and retinal detachment,which can cause irreversible blindness.For now,the best treatment of proliferative diabetic retinopathy is through surgery,to eliminate tractional effect caused by fibrovascular membrane,and save patients' sight.In order to seek for a new method to give patients with proliferative diabetic retinopathy intervenes in early stage to keep their sight,we chose RNA sequencing(RNA-seq)to select target genes.RNA-seq is considered as a major transcriptome profiling system.Elucidation of the human peripheral blood transcriptome by RNA-seq is essential for identifying candidate genes which might contribute to proliferative diabetic retinopathy.Method: There were six patients who were admitted in Tianjin Medical University Eye Hospital were selected.These six patients were divided into two groups according to the different clinical manifestation: three patient were diagnosed as proliferative diabetic retinopathy(PDR group),which was based on the diabetic retinopathy staging criteria(1987);the other three patients didn't have diabetes(control group).The patients were all drawn blood.The peripheral blood samples were frozen by liquid nitrogen and sent to the Novogene ? Company(Beijing,China)which specialized genomic methods.The Novogene? Company used standard extraction method to extract total RNA from human peripheral blood.After examination and qualification of total RNA samples,generation and inspection of libraries,and sequencing,we got raw data of the two groups.Finally,we obtained the results of differentially expressed genes by means of biological information analysis.Result: We divided six patients into two groups and generated human transcriptome.In total,we acquired 46111478-56703914 unique mapped reads which mapped in exon region 86.7%-93.2% across all the six peripheral blood samples.Gene expression level analysis and gene structure analysis showed that 82 differentially expressed genes(p<0.05,false discovery rate q<0.05)between the PDR group andcontrol group were revealed.Among them,there were 19 up-regulated genes and 63down-regulated genes.Clustering analysis dispalyed that these genes had similar translation patterns in same group.Gene ontology and pathway analysis demonstrated that the 82 differently expressed genes were enriched in specific biological process.According to gene functions related to proliferation,migration,differentiation and angiogenesis,we picked out 26 genes.Based on different biological signaling pathways,such as mitogen-activated protein kinases pathway and phosphatidylinositol 3-kinases/Akt pathway,we picked out 9 genes to verify by polymerase chain reaction.As a result,DUSP2 and DUSP5 were the most promising candidate genes affecting the formation of PDR.Conclusions: This study investigated the complexity of the human peripheral blood transcriptome in humans using RNA-seq.And we found out DUSP2 and DUSP5 might have connection with PDR,and RNA-seq could become a popular method to provide new targets to treat disease.