| BackgroundDiabetic nephropathy(DN)is a common and serious complication of diabetes,20% to 40% of diabetic patients will progress to DN.DN is the primary cause of end-stage renal disease(ESRD)and also the fatal cause of diabetes in western countries.In China,the morbidity of DN patients is also increasing year by year which has become the main cause of ESRD.Results of the epidemiological survey of nearly 100,000 adults in China in 2010: the morbidity of diabetes among Chinese adults was 11.6%,with about 114 million patients.Prediabetes morbidity was estimated at 50.1%,with approximately 493 million patients.According to the calculation of 50% diabetic patients will progress to ESRD in 10 ~ 20 years,there will be 15 million new patients with uremia caused by diabetic nephropathy in China in the next 10 ~ 20 years,which will bring heavy burden to our national physical and mental health and the development of social economic.However,the occurrence of diabetic nephropathy is caused by multiple factors,the pathogenesis is still not clear and the treatment is relatively limited,current clinical treatment can only alleviate the progress of the disease but cannot reverse or cure the disease.Therefore,it is of great significance for our clinical and scientific researchers to study the pathogenesis of diabetic nephropathy and find new therapeutic targets for the early prevention and clinical treatment of the disease.Human renal glomerular endothelial cells,one of the glomerular innate cells which has a special fenstration and polysaccharide protein complex covering on the surface.It is located at the innermost part of the glomerular capillaries and constitute the glomerular selective filtration barrier together with the basement membrane and podocyte,therefore it plays an important role in the selective filtration of macromolecular substances such as proteins.In addition,endothelial cells,podocytes and mesangial cells can interact through autocrine,paracrine and many other pathways.Due to its special location,it is highly susceptible to glucose,lipids and inflammatory factors in the blood.Therefore,we believed that the special structure and function of endothelial cells play an important role in the development of diabetic nephropathy.MicroRNAs(miRNAs)are small endogenous non-coding RNAs with a length of about 20 bp that have been discovered in recent years.They mainly bind to the 3' untranslated region of the target mRNA to induce silencing complexes to degrade the target mRNA or inhibit its translation,thereby regulating the expression of downstream factors to affect various physiological and pathological processes of human body.However,one miRNA can regulate multiple target genes to induce different effects,and the mechanism of its involvement in the occurrence and development of DN is still not clear,so more studies are needed to explore the relationship between miRNA and the pathogenesis of DN.Previous studies have shown that miR-155 is involved in the process of cell differentiation,proliferation,apoptosis and the regulation of various biological processes.The early research of our group carried out the serum microRNAs detection for patients with diabetes in the early stage,and found that compared with the healthy control group,the levels of serum miR-150,miR-155-5p,miR-30 e and miR-3196 were significantly decreased in the early stage of diabetic nephropathy group,and the expression of miR-155 was significantly different between the microalbuminuria group and the massive proteinuria group(P<0.05),which was positively correlated with eGFR in DN patients,and negatively correlated with the excretion rate of urinary protein suggesting that miR-155 may be correlated with the pathogenesis of DN.ObjectiveThe regulatory effect of miR-155 on target gene ETS-1 and downstream effector factors was detected by transfection of miR-155 mimic and inhibitor into high glucose cultured glomerular endothelial cells,and the effect of miR-155 on cell apoptosis and inflammatory response was also studied to investigate the role of miR-155 in glomerular endothelial cell dysfunction under high glucose environment.Research object and methodsPrimary human glomerular endothelial cells were adopted to investigate the changes of cell morphology,apoptosis,the expression of ETS-1 and its downstream factors VCAM-1 and MCP-1 in each group under high glucose with miR-155 mimic and inhibitor transfection conditions.1.The effect of high glucose on endothelial cell inflammation and apoptosis.Human glomerular endothelial cells at logarithmic growth stage were randomly divided into three groups: normal glucose group(NG: 5.6 mmol/L D-glucose),mannitol control group(HM: 5.6 mmol/L D-glucose +24.4 mmol/L mannitol)and high glucose group(HG: 30 mmol/L D-glucose).The protein expression of ETS-1,VCAM-1,MCP-1 and Cleaved caspase-3 were detected by western blot.The localization and fluorescence intensity of ETS-1,VCAM-1 and MCP-1 were observed by immunofluorescence.2.The effect of high glucose with miR-155 transfection on ETS-1 expression and endothelial cell inflammation and apoptosis.Human glomerular endothelial cells were transfected with miR-155 mimic and miR-155 inhibitor to alter the expression of miR-155,mimic control and inhibitor control were control groups.Western blot was used to detect the protein expressions of ETS-1,VCAM-1,MCP-1,and Cleaved caspase-3 respectively when miR-155 was overexpressed and inhibited.The localization and fluorescence intensity of ETS-1,VCAM-1 and MCP-1 were observed by immunofluorescence.3.The regulation of VCAM-1 and MCP-1 by transfection of ETS-1 siRNA under high glucose.ETS-1 siRNA was transfected into human glomerular endothelial cells under high glucose environment to down-regulate the expression of ETS-1 mRNA.Scramble RNA was control group.Western blot was used to detect the protein expressions of VCAM-1 and MCP-1 when ETS-1 is interfered.The localization and fluorescence intensity of ETS-1,VCAM-1 and MCP-1 were observed by immunofluorescence.Results1.The results of western blot showed that the glomerular endothelial cells were significantly damaged,the expressions of inflammatory proteins ETS-1,VCAM-1,and MCP-1 were up-regulated,the expression of apoptosis-related protein Cleaved caspase-3 was increased when stimulated with high glucose(P < 0.05).Immunofluorescence results showed that ETS-1,VCAM-1 and MCP-1 fluorescence were enhanced by high glucose stimulation,which was consistent with western blot.2.After transfected with miR-155 mimic,miR-155 inhibitor into high-glucose cultured glomerular endothelial cells,the western blot results showed that the expressions of ETS-1,VCAM-1,MCP-1 and Cleaved caspase-3 proteins were decreased when mir-155 was overexpressed,the expressions of ETS-1,VCAM-1,MCP-1 and Cleaved caspase-3 proteins were increased when miR-155 was inhibited(P<0.05).There was no significant difference between the transfection control groups and the high glucose groups.Immunofluorescence results were consistent with western blot.3.After transfection of ETS-1 siRNA into human glomerular endothelial cells cultured under high glucose,Western blot results showed that the expressions of ETS-1,VCAM-1 and MCP-1 proteins were all decreased significantly(P<0.05).No significant difference was observed between the Scramble RNA group and the high glucose control group.Immunofluorescence results were consistent with western blot.Conclusions1.The expression of inflammatory cytokines and the apoptosis of endothelial cells were both increased under the stimulation of high glucose.2.The expression of ETS-1 was decreased when miR-155 was overexpressed and increased when miR-155 was inhibited in high glucose cultured glomerular endothelial cells,miR-155 may be involved in endothelial cell injury by negatively regulate the expression of ETS-1.3.Compared miR-155 mimic group with ETS-1 siRNA group,the expressions of ETS-1 and its downstream factors VCAM-1 and MCP-1 were consistent,indicating that ETS-1 may mediate the inflammatory response of endothelial cells by regulating the expression of protein VCAM-1 and MCP-1. |
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