| Background and objective:Renal interstitial fibrosis is the ultimate outcome of various chronic kidney diseases progress.The epithelial mesenchymal transdifferentiation(EMT)of renal tubule is an important pathogenesis of renal interstitial fibrosis.Wnt/β-catenin signaling pathway is a complex,conservative cell-cell biological pathway,which regulates cell function and phenotypes in biological development and disease.β-catenin is the key molecular of this signaling pathway and plays an important role in various fibrosis diseases.MicroRNAs(miRNAs)are a group of small non-coding RNA in vivo,which participate in regulating cell proliferation,differentiation and apoptosis.With the intensive study of miR-200 a in cancer,it brings our attention to its role in fibrosis diseases.Researches have reported miR-200 a plays an important role in the liver fibrosis and pulmonary fibrosis.However the role of miR-200 a in renal fibrosis remains unclear.In this study,we used TGF-β1 to induce EMT of human tubular epithelial cells(HK-2)in vitro,and observed the miR-200 a and β-catenin expression.Next dual luciferase reporter system was used to confirm that miR-200 a is target for β-catenin gene(CTNNB1).Then we demonstrated the influence of up-and down-regulated miR-200 a expression on β-catenin and EMT.We used β-catenin siRNA to disprove the importance of β-catenin in progress of miR-200 a regulates EMT.Finally,we determine that miR-200 a inhibits EMT by directly targeting β-catenin.These provide a new experimental basis for the intervention of renal interstitial fibrosis and a new target for the treatment of the disease.Methods:1.To evaluate the influence of TGF-β1 on miR-200 a and β-catenin in HK-2 cells,cells were cultured in vitro and divided into four groups: 0h group,12 h group,24 h group,48 h group,namely induced with TGF-β1(10ng/ml)for 12 h,24h,48 h.QPCR was used to detect the miR-200 a expression.QPCR and Western Blot analysis were used to detect the β-catenin,E-cadherin,α-SMA and FN expression.2.In order to evaluate the influence of agomir(miR-200 a agonist)and antagomir(miR-200 a inhibitors)on miR-200 a expression in HK-2 cells,cells were divided into four groups: control group,cultured with DMEM/F12 containing 10% FBS;NC-miRNA group,transfected with NC-miRNA(negative control miRNA);agomir group,transfected with agomir(50n M);antagomir group,transfected with antagomir(100n M).48 hours later,q PCR was used to detect the miR-200 a expression.3.Using dual luciferase report plasmid(the wide-type/mutant-type of β-catenin gene,CTNNB1 UTR WT/MT)verify the target relationship between miR-200 a and β-catenin.HK-2 cells were divided into four groups: control group,transfected with CTNNB1 UTR WT/MT plasmid;NC-miRNA group,transfected with CTNNB1 UTR WT/MT plasmid and NC-miRNA;antagomir group,transfected with CTNNB1 UTR WT/MT plasmid and antagomir;agomir group,transfected with CTNNB1 UTR WT/MT plasmid and agomir.48 hours later,the Dual-Luciferase? Reporter Assay system was used to measure the luciferase activity.4.To explore the influence of agomir on β-catenin and EMT induced by TGF-β1,HK-2 cells were divided into four groups: control group,cultured with DMEM/F12 containing 10% FBS;TGF-β1 group,treated with TGF-β1;NC-miRNA+TGF-β1 group,transfected with NC-miRNA and treated with TGF-β1;agomir+TGF-β1 group,transfected with agomir and treated with TGF-β1.48 hours later,q PCR and Western Blot analysis were used to test the expression of β-catenin,E-cadherin,α-SMA and FN.Cell immunofluorescence was used to test the E-cadherin and α-SMA expression.5.To explore the influence of antagomir on β-catenin and EMT,HK-2 cells divided into three groups: control group,cultured with DMEM/F12 containing 10% FBS;NC-miRNA group,transfected with NC-miRNA;antagomir group,transfected with antagomir.48 hours later,q PCR and Western Blot analysis were used to test the expression of β-catenin,E-cadherin,α-SMA and FN.Cell immunofluorescence was used to test the E-cadherin and α-SMA expression.6.To evaluate the influence of β-catenin siRNA on β-catenin expression,HK-2 cells were divided into three groups: control group,cultured with DMEM/F12 containing 10% FBS;NC-siRNA group,transfected with NC-siRNA;si R-β-catenin group,transfected with β-catenin siRNA(50n M).48 hours later,q PCR and Western Blot analysis were used to test the β-catenin expression.7.To explore the influence of β-catenin siRNA on EMT induced by antagomir,HK-2 cells were divided into four groups: control group,cultured with DMEM/F12 containing 10% FBS;antagomir group,transfected with antagomir;antagomir+NC-siRNA group,transfected with antagomir and NC-siRNA;antagomir+si R-β-catenin group,transfected with antagomir and β-catenin siRNA.48 hours later,q PCR and Western Blot analysis were used to test the E-cadherin,α-SMA and FN expression.Results:1.miR-200 a expression was high level at 0h,dramatically decreased after TGF-β1 stimulation for 12 h and dropped to a minimum at 48h(comparison with 0h,P < 0.05).The mRNA and protein expression of β-catenin were low level at oh,but significantly increased at 24 h then up to the most at 48h(comparison with 0h,P < 0.05).The E-cadherin expressionin were high level at 0h,but significantly decreased at 24 h then dropped to a minimum at 48h(comparison with 0h,P < 0.05).The expressions of α-SMA and FN were opposite to the E-cadherin expression at the mRNA and protein levels,significantly increased at 12 h then up to the most at 48h(comparison with 0h,P < 0.05).It suggests that miR-200 a is down-regulated and β-catenin is up-regulated during TGF-β1-induced EMT.2.miR-200 a expression had no significant difference between control group and NC-miRNA group(P > 0.05).Compared with NC-miRNA group,miR-200 a expression was significantly increased in agomir group(P < 0.05),and obviously decreased in antagomir group(P < 0.05).It shows that agomir can increase miR-200 a expression and antagomir can decrease its expression.3.Dual luciferase assays demonstrated that the delivery of the CTNNB1 UTR MT construct exerted no influence on the luciferase activity(P > 0.05).Compared with the NC-miRNA group,co-transfection of antagomir and the CTNNB1 UTR WT construct induced a significant increase in luciferase activity(P < 0.05),whereas the co-transfection with agomir cause an obvious reduction in luciferase activity(P < 0.05).It demonstrates that miR-200 a directly targets for the gene of β-catenin,CTNNB1.4.The mRNA and protein levels of β-catenin,E-cadherin,α-SMA and FN had no significant difference between the NC-miRNA+TGF-β1 group and the TGF-β1 group(P > 0.05).Compared with control group,the levels of β-catenin,α-SMA and FN were significantly increased in TGF-β1 group(P < 0.05),but the levels of E-cadherin obviously decreased in TGF-β1 group(P < 0.05).Compared with NC-miRNA+TGF-β1 group,the levels of β-catenin,α-SMA and FN were significantly decreased in agomir+TGF-β1 group(P < 0.05),and the levels of E-cadherin significantly increased in agomir+TGF-β1 group(P < 0.05).The result of cell immunofluorescence was consistent with the Western Blot.It suggests that the up-regulation of miR-200 a inhibits β-catenin and attenuates TGF-β1-induced EMT5.The levels of β-catenin,E-cadherin,α-SMA and FN had no significant difference between the NC-miRNA group and the control group(P > 0.05).Compared with NC-miRNA group,the levels of β-catenin,α-SMA and FN were significantly increased in antagomir group(P < 0.05),and the levels of E-cadherin were significantly decreased in antagomir group(P < 0.05).The result of cell immunofluorescence was consistent with the Western Blot.It shows that down-regulation of miR-200 a increase β-catenin and induces EMT.6.The mRNA and protein levels of β-catenin were no significant difference between the control group and the NC-siRNA group(P > 0.05).Compared with NC-siRNA group,β-catenin levels were significantly decreased in si R-β-catenin group(P < 0.05).It suggests that β-catenin siRNA can inhibites β-catenin expression.7.The levels of E-cadherin,α-SMA and FN had no significant difference between the antagomir group and the antagomir+NC-siRNA group(P > 0.05).Compared with control group,the levels of E-cadherin were significantly decreased in antagomir group(P < 0.05),and the levels of α-SMA and FN were obviously increased in antagomir group(P < 0.05).Compared with antagomir+NC-siRNA group,the levels of E-cadherin were significantly increased in antagomir+si R-β-catenin group(P < 0.05),and the levels of α-SMA and FN were obviously decreased in antagomir+si R-β-catenin group(P < 0.05).It suggests that β-catenin siRNA inhibites the EMT induced by miR-200 a down-regulation.Disproving miR-200 a directly regulates β-catenin to affect tubular EMT.Conclusion:1.miR-200 a is down-regulated and β-catenin is up-regulated during TGF-β1-induced EMT.2.miR-200 a directly targets for the gene of β-catenin,CTNNB1.3.The up-regulation of miR-200 a inhibits β-catenin and attenuates TGF-β1-induced EMT,and its down-regulation increase β-catenin and induce EMT.4.β-catenin siRNA inhibites the EMT induced by miR-200 a down-regulation.Disproving miR-200 a directly regulates β-catenin to affect tubular EMT.In a word,miR-200 a inhibits TGF-β1-induced EMT by directly targeting β-catenin in human tubular epithelial cell. |
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