Research On Effects And Mechanism Of MiR-21 In The Pathogenesis Of Diabetic Nephropathy

Objective:To observe the expressions of microRNA-21(miR-21)and Ski-related novel protein(SnoN)in the renal fibrosis diabetic process and in the HG(high-glucose)-induced renal tubule epithelial cells.Morever,we also aim to investigate the possible mechanism of miR-21 and SnoN in the diabetic nephropathy process preliminarily.Methods:1.The diabetic rats were established by tail-vein injection of Streptozotocin,and the other group was normal control(NC)group.After8?16?24 weeks,the rats were sacrificed to detect relative biochemical parameters and renal index,and to observe the changes of pathomorphology by HE staining as well.Meanwhile,immunohistochemistry and Western blot were employed to detect the protein expression of SnoN ? transforming growth factor-?1(TGF-?1)? Smad3 ?p-Smad3(Ser423 /425)?E-cadherin??-SMA and collagen? in the renal tissue.In addition,the expression of SonN m RNA and miR-21were detected by Real-time PCR(qRT-PCR).2.NRK-52 E cells were randomly divided into normal glucose(NG)group and high glucose(HG)group,each group was cultured for 2h,12 h,24h and 48 h.Immunocytochemistry was used to identify the expressions of E-cadherinin and?-SMA in NRK-52 E cells.Western blot was used to detect the protein expression of SnoN?TGF-?1?Smad3?p-Smad3(Ser423 /425)?E-cadherin??-SMA and collagen?under different conditions.The expressions of miR-21 and SnoN mRNA were assessed by qRT-PCR.3.Immunoflurescence cytochemistry was used to identify expressions of E-cadherinin and ?-SMA in NRK-52 E cells;Interference technology was used to overexpress and inhibit miR-21 expression in renal tubular epithelial cells.The renal tubular epithelial cells treated with NG group and HG group for 48 h.Western blot was used to detect the proteins expression of SnoN,E-cadherin and Col-?;qRT-PCR was employed to detect SnoN mRNA and Smad7 mRNA.4.Interference technology was used to knock-down and increase SnoN expression in renal tubular epithelial cells.The renal tubular epithelial cells treated with NG group and HG group for 48 h.Western blot was used to detect the proteins expression of SnoN,E-cadherin and Col-?;qRT-PCR was employed to detect SnoN mRNA and miR-21.Results:1.Rat model of DM was established and compared with normal control the diabetic kidneys developed renal interstitium lesions.HG-induced tubular EMT,characterized by increased ?-SMA expression and deseased E-cadherin expression in renal tubular epithelial cells both in vitro and in vivo.Compared with NC group and NG group,the expressions of TGF-?1?p-Smad3(Ser423 /425)and?-SMA protein increased in DM group and HG group(p<0.05),while the protein level of SnoN decreased but the level of SnoN m RNA increased(p<0.05),the level of miR-21 markedly increased in DM group and HG group(p<0.05).Moreover,miR-21 and SnoN protein were negatively correlated by correlation analysis.2.Overexpression and suppression of miR-21 in RTECs by transgenic technology could obviously promote HG-induced EMT.Meanwhile,overexpression of miR-21 could down-regulate both m RNA and protein of SnoN.3.Under high glucose condition,BMP-7 could up-regulate the expression of SnoN in RTECs.Knockdown of SnoN in RTECs by transgenic technoloty could obviously up-regulate SnoN expression and promote HG-induced EMT.On the contrary,increasing of SnoN could decrease the expression of miR-21.Conclusion:1.TGF-?1 may up-regulate the expression of miR-21but restrain the translational expression of SnoN,aggravating fibrosis.2.Overexpression miR-21 in RTECs could down-regulate SnoN protein expression,promoting HG-induced EMT,the reason can be caused by some indirect effect.However,there is no relevant research reports.3.Increasing of SnoN expression in HG-induced RTECs could suppress the expression of miR-21,promoting HG-induced EMT.Conversely,knockdown of SnoN could up-regulate miR-21 expression.However,the regulatory mechanism still need further elucidation.