| Background and objectiveTGF?1-induced tubular epithelial to mesenchymal transition(EMT)plays a key role in renal fibrosis as seen in diabetic kidney disease(DKD).High mobility group AT-hook 2(HMGA2)is reported to be involved in TGF?1-mediated EMT via multifactorial mechanisms.As significant regulatory factors post-transcription,specific microRNA(miRNA)is closely associated with the occurrence of EMT.Past studies regarding TGF?1/HMGA2 in EMT were always focused on the fields of tumor or other organs,however,the role of TGF?1/HMGA2 in EMT remains unclear in DKD especially in tubular EMT and renal fibrogenesis.This study aims to explore the effects of HMGA2 on tubular EMT and renal fibrogenesis induced by TGF?1,and further investigate the potential mechanism of miRNA targeting HMGA2 during renal fibrosis in DKD.Through the above study,we can find significant intervention and therapy targets in view of the effective signaling pathways,further optimizing the therapeutic avenues for DKD and renal fibrogenesis.Methods1.The cellular model in NRK-52E cells stimulated with TGF?1 was established,and the expression of HMGA2 and EMT-related genes before and after TGF?1 stimulation was examined by using the method of qRT-PCR and western blot.2.The function of HMGA2 in TGF?1-induced EMT and renal fibrogenesis was verified by using the method of siRNA transfection,and the expression of the downstream transcription factors of HMGA2 was examined by using the method of qRT-PCR and western blot.3.The upstream miRNA targeting HMGA2 was screened through bioinformatics method,and the expression of each screened miRNA before and after TGF?1 stimulation in NRK-52E cells was detected by using the method of qRT-PCR.And the expression of HMGA2 and its upstream miRNA was futher investigated in UUO mice model.4.Functional experiments were performed through transfection of miRNA mimics and inhibitors to further verify the role of the screened miRNA in TGF?1-induced EMT and renal fibrogenesis.The expression of HMGA2 and EMT-related factors was measured before and after transfection of miRNA mimics and inhibitors by using the method of qRT-PCR and western blot.Results1.The results from qRT-PCR and western blot indicated that the expression of HMGA2,?-SMA,vimentin,Col-I and FN was significantly increased in NRK52E cells 48 h after stimulation with 10 ng/ml TGF?1,and the expression of E-Cad was significantly decreased.2.The results from qRT-PCR and western blot indicated that transfection of siRNA-HMGA2 significantly prevented TGF?1-induced upregulation of HMGA2?a-SMA and vimentin and downregulation of E-Cad in NRK-52E cells.Gene expression analyses of transcription factors including Snail,Slug and Twist showed no significant changes before and after transfection of siRNA-HMGA2.3.The significant screened miRNAs were miRNA-let-7d,miRNA-98,and miRNA-33.The results from qRT-PCR indicated that the expression of miRNA-let-7d was significantly decreased both in TGF?1-induced NRK-52E cells and in the kidneys from UUO model,while the expression of HMGA2 was significantly increased.Gene expression analyses of miRNA-98 and miRNA-33 showed no significant changes before and after TGF?1 stimulation in NRK-52E cells.4.The results from qRT-PCR and western blot indicated that transfection of let-7d mimic significantly attenuated TGF?1-induced upregulation of HMGA2??-SMA,Col-I and FN and downregulation of E-Cad in NRK-52E cells,while transfection of let-7d inhibitor significantly enhanced TGF?1-induced upregulation of HMGA2?Col-I and FN and downregulation of E-Cad.Conclusions1.TGF?1 can upregulate the expression of HMGA2 and induce an EMT and fibrogenesis process characterized by phenotypic transition in NRK-52E cells.2.SiRNA targeting HMGA2 can inhibit TGF?1-induced EMT and fibrogenesis in NRK-52E cells,and HMGA2 is a meaningful target gene.3.miRNA-let-7d is a significant upstream miRNA targeting HMGA2,and miRNA-let-7d/HMGA2 may play a role in the process of TGF?1-induced EMT and renal fibrogenesis.4.Overexpression of miRNA-let-7d prevents TGF?1-induced EMT and renal fibrogenesis through downregulation of HMGA2 expression,while inhibition of miRNA,let-7d accelerates the progession of TGF?1-induced EMT and renal fibrogenesis through upregulation of HMGA2 expression.5.miRNA-let-7d regulates the progression of tubular EMT and renal fibrosis through regulation of HMGA2 expression,and intervention in this process may provide new approaches for the prevention and treatment of DKD and renal fibrogenesis. |
PDF Full Text Request