| [Objective] Long term hyperglycemia caused by type II diabetes mellitus(T2DM)can lead to microvascular complications,such as Diabetic Nephropathy(DN).DN is a long-term complication of poor glycemic control,which can lead to progressive renal damage and cardiovascular risk.Current studies have shown that the pathogenesis of diabetic nephropathy is caused by multiple factors such as hyperglycemia,hypertension,renal hemodynamic changes,genetic and metabolic interactions.The aim of this study was to investigate the expression of miR-25-5p and AFF1 in DN,and analyze the correlation between miR-25-5p-AFF1 pathway which regulated the apoptosis related proteins in human glomerular endothelial cells and DN.[Method] Bioinformatics methods were used to study the target genes of miR-25-5p,and three methods were used to predict the downstream target genes regulated by miR-25-5p.Total RNA was extracted from 30 DN patients and 30 healthy adults.The expression levels of miR-25-5p,aff1 and NFIX were detected by RT-q PCR;the targeted binding of miR-25-5p and aff1 was verified by double luciferase assay;the expression of miR-25-5p and aff1 was detected by RT-q PCR after high glucose treatment;after high glucose treatment,the expression of miR-25-5p and AFF1 was detected by RT q PCR;after high glucose treatment,the expression of miR-25-5p was detected by Western blot After transfection with miR-25-5p,the effects of miR-25-5p on AFF1 and apoptosis related proteins were observed.[Result]:1.The fasting blood glucose,serum creatinine,blood urea nitrogen,urinary protein,glycosylated hemoglobin and urinary albumin excretion rate of diabetic nephropathy patients were significantly higher than those of healthy people(P <0.05),while glomerular filtration rate was significantly lower than that of healthy people(P < 0.05).2.Three softwares,Tar Base,mirdb and targetscan,were used to predict the target genes regulated by miR-25-5p.The results showed that there were 28 target genes predicted by Tar Base,50 target genes predicted by mirdb and 1878 target genes predicted by targetscan.Through the intersection analysis of the three softwares,only two genes were predicted by the three softwares: aff1 and NFIX.3.Analysis of the relationship between miR-25-5p and aff1 and NFIX: there was a negative correlation between the expression of miR-25-5p and AFF1,AFF1 may be the downstream target gene of miR-25-5p.4.Double luciferase assay confirmed that AFF1 was the target gene of miR-25-5p.miR-25-5p interacted with the corresponding 3’-UTR sequence of AFF1,and finally down regulated the expression of AFF1 gene.5.Morphological changes of human glomerular endothelial cells cultured in high glucose in vitro: compared with untreated group,the volume of cells cultured with high glucose began to round,and the number of suspension apoptotic cells increased6.The expression of miR-25-5p was down regulated and the expression of AFF1 was up-regulated in high glucose culture.It is suggested that high glucose can cause down regulation of miR-25-5p and up regulation of AFF1 expression.7.The expression of Bcl-2 was down regulated and the expression of Bax was increased in high glucose culture.It is suggested that high glucose can promote apoptosis.8.Effect of miR-25-5p on aff1 and apoptosis related proteins: miR-25-5p up regulate can target AFF1,up regulate Bcl-2,and down regulate Bax protein,inhibit apoptosis of human glomerular endothelial cells.[Conclusion]:1.The expression of miR-25-5p in serum of DN patients was higher than that of healthy people,while the expression of AFF1 was decreased.miR-25-5p and AFF1 were related to the occurrence of DN.2.Up regulation of miR-25-5p can specifically inhibit AFF1,and inhibit apoptosis of human glomerular endothelial cells by up regulating Bcl-2 and down regulating Bax protein,which may be related to the occurrence and development of DN. |
