Alterations And Validation Of Micro RNA Expression Profile Induced By Oxidative Stress In HUVECs

Objective: With the development of economy and people lifestyle changes, the trend of aging increase rapidly in modern society. The morbidity of Diabetic Retinopathy(DR) is rising year after year and influences on quality of life seriously. DR is one of diabetic microvascular complications,chronic hyperglycemia is the pathogenic basis, and metabolism, endocrine and blood factors promote its occurrence,and development.The pathogenesis of diabetic retinopathy are mainly the polyol pathway, protein kinase C activation, advanced glycation end products formation and receptor activation, hexosamine pathway and the levels of oxidative stress rise higher.Among them,elevated levels of oxidative stress which is an early event and initial stage in the pathogenesis of DR. For early prevention and treatment of DR,to reseach the effects and molecular mechanisms of oxidative damage in the pathogenesis of DR has important significance.Micro RNAs(mi RNAs) are a class of endogenous, approximately 22 nucleotide(nt), non-coding small RNA. It is well known that mi RNAs play key roles in regulating target gene expression at the posttranscriptional level. A number of studies have witnessed that under pathological conditions, mi RNAs play an important role via its targets.But there is no report about mi RNAs alterations and the regulation mechanism of target gene in the early onset of DR induced by oxidative stress. Hydrogen peroxide(H2O2) is a stable, uncharged and freely diffusible reactive oxygen species,and vascular endothelial cell injury is an important feature in the pathogenesis of DR.We stimulated the primary human umbilical vein endothelial cells(HUVECs) with H2O2 to develop a cell model of oxidative stress and studied the expression profiles of oxidative stress-induced mi RNAs. the different expressed mi RNAs were validated using real-time PCR assay and the bioinformatic analysis were performed in present study.To clarify the roles of mi RNAs in the process of oxidative stress leading to DR and its molecular mechanism.At the same time,we can provide s new perspective for the treatment of DR.Methods: Primary cultures of HUVECs: HUVECs were isolated by collagenase digestion method,The HUVECs were plated onto poly-L-lysine-coated 25cm2 dish incubation at 37℃in a humidified atmosphere of 95 % air and 5 % CO2. The medium was changed after 24 h and after this, according to the cell growth, medium was changed.When the primary cell fusion was more than 90%, the cells were subcultured with 0.05% trypsin digestion of-0.02%EDTA solution.The fourth generation of endothelial cells were for determination of cell viability and apoptosis, necrosis. RNA was extracted from the collected cells,using for gene chip screening and Real-time PCR verification.Identifications of primary cultured HUVECs: The first generation of HUVECs were incubated with Anti-VWF antibody to identify endothelial cells.viability assay: The fourth generation of primary cultured HUVECs were treated with 0, 100, 200, 400, 800μM H2O2(Sigma) diluted with supplemented medium for 8h、16h、24h at 37 ℃in a humidified atmosphere of 95 % air and 5 % CO2. The cell viability was determined using the Cell Counting Kit-8(CCK-8).Measurement of cell apoptosis and death: According to CCK-8 assay the suitable H2O2 concentration, 200μM and 24 h were selected to treat the HUVECs detecting the apoptotic and dead cells by Annexin V-FITC/PI fluorescent staining.Microarray:RNAs from primary cultured HUVECs stimulated with 0 or 200μM H2O2 for 24 h were isolated. The mi RNAs profiles from primary HUVECs were determined via Lian Chuan biological technology company. Bioinformatic analysis of mi RNAs: According to the result of microarray, we selected mi RNAs to perform bioinformatic analysis using the DAVID(Database for Annotation,Visualization and Interrogated Discovery) bioinformatic resources. Real-time PCR: We performed Real-time PCR to validate the result of microarray.Statistical analysis: All data were presented as mean±SD. Statistical analysis was determined by the independent Student t test and one-way ANOVA using SPSS 13.3 software. A P value cut-off 0.05 was considered significant.Results:1 The first generation of HUVECs were spindle shaped or polygonal cells, whose cell body is full,surface is smooth and boundary is clear.2 CCK-8 assay revealed loss of cell viability significantly after stimulating with H2O2 for 24 h. Cell viability assay also showed the dose-dependent and time-dependent excitotoxicity of H2O2 on primary cultured HUVECs.3 Primary cultured HUVECs were subjected to Annexin V-FITC/PI fluorescent staining. We found that the apoptosis and death rate of HUVECs exposured to 200 μM H2O2 for 24 h was increased significantly compared to that of normal cultured HUVECs.4 The result of microarray showed that there were 40 deregulated mi RNAs in H2O2- or control- treated HUVECs.Based on the bioinformatics analysis results, pathology of DR and previous reports, we chose 7 mi RNAs, mi R-638、mi R-1246、mi R-1275、mi R-4267、mi R-4324、mi R-4734 and mi R-15b-5p,to perform real-time RT-PCR confirmation.5 We used DAVID platform to analyse functional annotation of deregulated mi RNA- target genes. Bioinformatics and statistical analysis showed that these mi RNAs may be involved in the regulation ofcell apoptosis, MAPK signaling pathway,type II diabetes mellitus,calcium signaling pathway,vascular smooth muscle contraction,insulin signaling pathway,cancer development,p53 signaling pathway,and so on. 33 target genes predicted by 21 mi RNAs including mi R-4267 took part in cell apoptosis.6 mi R-638、mi R-1246、mi R-4267 displayed a consistent upregulation in HUVECs with H2O2 stimulation for 24 h.7 Enrichment for Gene Ontology(GO) terms for biological processes and molecular functions of mi R4267 targets were tested. Analysis of mi R-4267 targrts revealed that mi R-4267 related to the cell apoptosis.Conclusion:1 The results of CCK-8 assay suggested that the cell viability was significantly reduced after H2O2 stimulation. The detection of Annexin V-FITC/PI fluorescent staining showed that the rate of cell apoptosis and death obviously increased after H2O2 stimulation. Our study demonstrated that the apoptosis of HUVECs induced by oxidative stress may be one of the most important pathgenesis of DR.2 Profiles of oxidative stress-related mi RNAs showed that the deregulated mi RNAs may be represented in oxidative stress leading to DR in the process of the molecular mechanism.3 The results of bioinformatic analysis provided more evidence that mi RNAs induced by oxidative stress may play important roles in the pathogenesis of DR by influencing cell apoptosis.