| BackgroudTubulointerstitial fibrosis(TIF)plays an important role in the progression of renal fibrosis in diabeticnephropathy(DN).Accumulating evidence supports a crucial effect of early growth response factor 1(Egr1)on renal fibrosis in DN,but the underlying mechanisms are not entirely clear.MicroRNAs(miRNAs),as a post-transcriptional regulator that leads to fast degradation of target genes or blocks translation by binging to specific sequences,especially the 3’ untranslated region(3’UTR)of the target gene mRNAs.MiRNAs have been gradually regarded as an important contributing factor of renal fibrosis in DN.It is not clear that which miRNA can regulate the expression of Egrl by binding to Egrl 3’UTR in TIF.ObjectiveWe performed this study including in vivo and in vitro experiments to answer these questions:1.whether Egrl plays a crucial role in TIF of DN;2.whether miR-181a-5p can act as an upstream regulator of Egr1;3.whether miR-181a-5p can prevent TIF through suppressing Egr1 expression in DN.We hope that this study will improve the better understanding of DN pathogenesis and the development of novel effective therapies for DN.Methods1.Male Otsuka-Long-Evans-Tokushima-Fatty(OLETF)rats,a model of spontaneous type 2 diabetes,and male Long-Evans-Tokushima-Otsuka(LETO)rats used as nondiabetic controls were used in this experiment.We sacrificed 3 OLETF rats and 3 LETO rats to analysis at the time point of 40 weeks.PAS staining and Masson staining were conducted to determine the pathological feature of the kidney of 40-weeks-age OLETF rats.Immunohistochemical analysis was performed to investigate the expression of Egr1 and TGF-β1 in kidney of 40-weeks-age OLETF rats.QRT-PCR and Western blot analysis were conducted to determine the mRNA and protein levels of Egr1,TGF-β1,FN and COL Ⅰ in renal tissues.2.HK-2 cells were transfected with pENTER-Egr1 plasmid or siEgr1 respectively.QRT-PCR and Western blot analysis were conducted to determine the mRNA and protein levels of Egr1,TGF-β1,FN and COL Ⅰ in cells.3.HK-2 cells incubated in serum-free culture were treated with high glucose(30 mM)or mannitol(30 mM)as a control for osmolality respectively.QRT-PCR and Western blot analysis were conducted to determine the time-dependent mRNA and protein expression of Egr1.4.Analysis of public available algorithms was performed to identify which miRNAs could target Egr1 3’UTR.MiR-181a-5p was determined through qRT-PCR analysis of HK-2 cells treated with high glucose(30 mM).Renal tissues were detected by qRT-PCR to investigate the expression of miR-181a-5p mRNA.Dual-luciferase activity assay was conducted to determine whether miR-181a-5p directly targets the 3’UTR of Egr1 mRNA.5.HK-2 cells were transfected with miR-181a-5p mimics or inhibitor respectively.QRT-PCR and Western blot analysis were conducted to determine the mRNA and protein levels of Egrl,TGF-β1,FN and COL I in cells.6.HK-2 cells were transfected with siEgr1,miR-181a-5p inhibitor or siEgrl with miR-181a-5p inhibitor respectively.QRT-PCR and Western blot analysis were conducted to determine the mRNA and protein levels of Egr1,TGF-β1,FN and COL I in cells.Results1.PAS staining and Masson staining demonstrated that the kidney of 40-weeks-age OLETF rats revealed the pathological feature of DN.Immunohistochemistry analysis of renal tissues revealed that expression of Egr1 and TGF-β1 was significantly elevated in the DM group.It was shown that Egrl,TGF-β1 FN and COL I mRNA and protein levels were dramatically increased in renal tissues of DM group.2.It was shown that overexpression of Egr1 significantly increased levels of Egr1,TGF-β1,FN and COL I by qRT-PCR and western blot analysis in HK-2 cells transfected with pENTER-Egrl plasmid for 48h.By contrast,silenced Egrl expression by siEgrl resulted in a decreased expression of Egr1,TGF-β1,FN and COL I in HK-2 cells transfected with siEgrl for 48h.3.It was shown a time-dependent increased expression of Egr1 by qRT-PCR and western blot analysis in HK-2 cells treated with high glucose.The expression of Egr1 mRNA and protein rose from 0.5 h and peaked at 1 h,and rapidly decreased to baseline at 2 h or so.It was illustrated that mannitol had no effect on the expression of Egr1 mRNA and protein.4.MiR-181a-5p and other 8 miRNAs were predicted to have potential target sites in 3’UTR of Egrl gene according to analysis of public available algorithms.We treated HK-2 cells with mannitol(30 mM),HG(30 mM)and NG(5 mM)for 48 h,respectively.As illustrated by qRT-PCR analysis,the level of miR-181a-5p decreased in a time dependent manner in HK-2 cells cultured with HG.The expression of miR-181a-5p mRNA declined from 0.5 h and minimized at 1 h,then rapidly returned to baseline at 2 h or so.Meanwhile,cells cultured with mannitol had no significant changes of miR-181 a-5p expression.Furthermore,statistical analysis showed that miR-181a-5p mRNA level was negatively correlated with Egrl mRNA level in HK-2cells treated with HG(r =0.6879,P<0.001).It was shown significant down-regulated expression of miR-181a-5p in DM group by qRT-PCR analysis.Dual-luciferase activity assay demonstrated that miR-181a-5p can directly target the 3’UTR of Egrl mRNA.5.It was shown that the mRNA and protein levels of Egrl,TGF-β1,FN,and COL I significantly down-regulated in HK-2 cells transfected with mimics.In contrast,HK-2 cells transfected with inhibitor revealed remarkably increased expressions of Egr1,TGF-β1 FN,and COL I mRNA and protein levels.6.It was illustrated by qRT-PCR and western blot analysis that the decreased expression of Egrl,TGF-β1,FN,and COL I were rescued when Egrl-silenced cells were treated with miR-181a-5p inhibitor.Conclusions1.The renal tissue of 40-weeks-age OLETF rat had revealed the pathological feature of DN and fibrosis.2.Egrl aggravated fibrosis by promoting the synthesis of ECM through upregulaing TGF-β1 signaling pathway in HK-2 cells.3.MiR-181a-5p regulated the expression of Egrl by directly targeting the 3’UTR of Egrl mRNA.4.MiR-181 a-5p prevented renal tubulointerstitial fibrosis through suppressing Egr1 expression in HK-2 cells. |
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