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miR-200a Prevents renal fibrogenesis through repression of TGF-β2 expression.

Wang B, Koh P, Winbanks C, Coughlan MT, McClelland A, Watson A, Jandeleit-Dahm K, Burns WC, Thomas MC, Cooper ME, Kantharidis P - Diabetes (2010)

Bottom Line: TGF-β1 and TGF-β2 also downregulated expression of miR-200a.The renal expression of miR-141 and miR-200a was also reduced in mouse models representing early and advanced kidney disease. miR-200a and miR-141 significantly impact on the development and progression of TGF-β-dependent EMT and fibrosis in vitro and in vivo.These miRNAs appear to be intricately involved in fibrogenesis, both as downstream mediators of TGF-β signaling and as components of feedback regulation, and as such represent important new targets for the prevention of progressive kidney disease in the context of diabetes.

View Article: PubMed Central - PubMed

Affiliation: JDRF Danielle Alberti Memorial Centre for Diabetes Complications, Melbourne, Australia.

ABSTRACT

Objective: Progressive fibrosis in the diabetic kidney is driven and sustained by a diverse range of profibrotic factors. This study examines the critical role of microRNAs (miRNAs) in the regulation of the key fibrotic mediators, TGF-β1 and TGF-β2.

Research design and methods: Rat proximal-tubular epithelial cells (NRK52E) were treated with TGF-β1 and TGF-β2 for 3 days, and expression of markers of epithelial-to-mesenchymal transition (EMT) and fibrogenesis were assessed by RT-PCR and Western blotting. The expression of miR-141 and miR-200a was also assessed, as was their role as translational repressors of TGF-β signaling. Finally, these pathways were explored in two different mouse models, representing early and advanced diabetic nephropathy.

Results: Both TGF-β1 and TGF-β2 induced EMT and fibrogenesis in NRK52E cells. TGF-β1 and TGF-β2 also downregulated expression of miR-200a. The importance of these changes was demonstrated by the finding that ectopic expression miR-200a downregulated smad-3 activity and the expression of matrix proteins and prevented TGF-β-dependent EMT. miR-200a also downregulated the expression of TGF-β2, via direct interaction with the 3' untranslated region of TGF-β2. The renal expression of miR-141 and miR-200a was also reduced in mouse models representing early and advanced kidney disease.

Conclusions: miR-200a and miR-141 significantly impact on the development and progression of TGF-β-dependent EMT and fibrosis in vitro and in vivo. These miRNAs appear to be intricately involved in fibrogenesis, both as downstream mediators of TGF-β signaling and as components of feedback regulation, and as such represent important new targets for the prevention of progressive kidney disease in the context of diabetes.

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miR-200a represses the expression of ECM proteins. A: NRK52E cells were transfected with miR-200a (100 nmol/l), and RNA was harvested after 3 days for real-time QPCR analysis. miR-200a resulted in significantly decreased expression of several ECM proteins including αSMA, collagen (Col) I and IV, and fibronectin (Fibr) (*P < 0.05 compared with control transfected cells). Expression of TGF-β2 was also significantly decreased as was PAI-1, which is downstream of the TGF-β signaling pathway. The expression of E-cadherin (E-cad) was significantly elevated. B: Western analysis demonstrated a significant decrease in αSMA and collagen I by miR-200a, consistent with the RNA expression analysis (*P < 0.05 compared with control transfected cells). The Westerns were quantified and also shown in graph format below the Western blots (*P < 0.05 compared with control). C: NRK52E cells were cotransfected with the p(CAGA)12 SMAD3 activity reporter construct, a β-galactosidase construct, and miR-200a. Four h later, the cells were treated with TGF-β1, and cells were harvested after 3 days. TGF-β1 resulted in increased SMAD3 activity with miR-C, which was strongly inhibited by miR-200a (*P < 0.00005 compared with miR-C control; #P < 0.0005 compared with miR-C with TGF-β1; $P < 0.002 compared with miR-200a control). D: Western analysis of phospho-SMAD3 and total SMAD3 levels in miR-200a-transfected NRK52E demonstrating reduced SMAD3 phosphorylation relative to total SMAD3 protein (*P < 0.05 compared with miR-C control). The Westerns were quantified and shown in graph format (*P < 0.05 compared with miR-C).
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Figure 4: miR-200a represses the expression of ECM proteins. A: NRK52E cells were transfected with miR-200a (100 nmol/l), and RNA was harvested after 3 days for real-time QPCR analysis. miR-200a resulted in significantly decreased expression of several ECM proteins including αSMA, collagen (Col) I and IV, and fibronectin (Fibr) (*P < 0.05 compared with control transfected cells). Expression of TGF-β2 was also significantly decreased as was PAI-1, which is downstream of the TGF-β signaling pathway. The expression of E-cadherin (E-cad) was significantly elevated. B: Western analysis demonstrated a significant decrease in αSMA and collagen I by miR-200a, consistent with the RNA expression analysis (*P < 0.05 compared with control transfected cells). The Westerns were quantified and also shown in graph format below the Western blots (*P < 0.05 compared with control). C: NRK52E cells were cotransfected with the p(CAGA)12 SMAD3 activity reporter construct, a β-galactosidase construct, and miR-200a. Four h later, the cells were treated with TGF-β1, and cells were harvested after 3 days. TGF-β1 resulted in increased SMAD3 activity with miR-C, which was strongly inhibited by miR-200a (*P < 0.00005 compared with miR-C control; #P < 0.0005 compared with miR-C with TGF-β1; $P < 0.002 compared with miR-200a control). D: Western analysis of phospho-SMAD3 and total SMAD3 levels in miR-200a-transfected NRK52E demonstrating reduced SMAD3 phosphorylation relative to total SMAD3 protein (*P < 0.05 compared with miR-C control). The Westerns were quantified and shown in graph format (*P < 0.05 compared with miR-C).

Mentions: Ectopic expression of miR-200a after transfection with premiR-200a resulted in decreased expression of several ECM genes, including collagen I and IV and fibronectin, compared with premiRNA-control transfected NRK52E cells (Fig. 4A). Some of these changes in ECM protein expression were also observed at the protein level (Fig. 4B). miR-200a also caused reduced expression of the mesenchymal marker, αSMA, and increased expression of the epithelial marker, E-cadherin mRNA levels (Fig. 4A), consistent with previous reports that the miR-200 family targets the transcriptional repressors of E-cadherin, ZEB1, and ZEB2 (12,18–21). Finally the expression of PAI-1, a downstream target of TGF-β signaling, was also decreased (Fig. 4A).


miR-200a Prevents renal fibrogenesis through repression of TGF-β2 expression.

Wang B, Koh P, Winbanks C, Coughlan MT, McClelland A, Watson A, Jandeleit-Dahm K, Burns WC, Thomas MC, Cooper ME, Kantharidis P - Diabetes (2010)

miR-200a represses the expression of ECM proteins. A: NRK52E cells were transfected with miR-200a (100 nmol/l), and RNA was harvested after 3 days for real-time QPCR analysis. miR-200a resulted in significantly decreased expression of several ECM proteins including αSMA, collagen (Col) I and IV, and fibronectin (Fibr) (*P < 0.05 compared with control transfected cells). Expression of TGF-β2 was also significantly decreased as was PAI-1, which is downstream of the TGF-β signaling pathway. The expression of E-cadherin (E-cad) was significantly elevated. B: Western analysis demonstrated a significant decrease in αSMA and collagen I by miR-200a, consistent with the RNA expression analysis (*P < 0.05 compared with control transfected cells). The Westerns were quantified and also shown in graph format below the Western blots (*P < 0.05 compared with control). C: NRK52E cells were cotransfected with the p(CAGA)12 SMAD3 activity reporter construct, a β-galactosidase construct, and miR-200a. Four h later, the cells were treated with TGF-β1, and cells were harvested after 3 days. TGF-β1 resulted in increased SMAD3 activity with miR-C, which was strongly inhibited by miR-200a (*P < 0.00005 compared with miR-C control; #P < 0.0005 compared with miR-C with TGF-β1; $P < 0.002 compared with miR-200a control). D: Western analysis of phospho-SMAD3 and total SMAD3 levels in miR-200a-transfected NRK52E demonstrating reduced SMAD3 phosphorylation relative to total SMAD3 protein (*P < 0.05 compared with miR-C control). The Westerns were quantified and shown in graph format (*P < 0.05 compared with miR-C).
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Figure 4: miR-200a represses the expression of ECM proteins. A: NRK52E cells were transfected with miR-200a (100 nmol/l), and RNA was harvested after 3 days for real-time QPCR analysis. miR-200a resulted in significantly decreased expression of several ECM proteins including αSMA, collagen (Col) I and IV, and fibronectin (Fibr) (*P < 0.05 compared with control transfected cells). Expression of TGF-β2 was also significantly decreased as was PAI-1, which is downstream of the TGF-β signaling pathway. The expression of E-cadherin (E-cad) was significantly elevated. B: Western analysis demonstrated a significant decrease in αSMA and collagen I by miR-200a, consistent with the RNA expression analysis (*P < 0.05 compared with control transfected cells). The Westerns were quantified and also shown in graph format below the Western blots (*P < 0.05 compared with control). C: NRK52E cells were cotransfected with the p(CAGA)12 SMAD3 activity reporter construct, a β-galactosidase construct, and miR-200a. Four h later, the cells were treated with TGF-β1, and cells were harvested after 3 days. TGF-β1 resulted in increased SMAD3 activity with miR-C, which was strongly inhibited by miR-200a (*P < 0.00005 compared with miR-C control; #P < 0.0005 compared with miR-C with TGF-β1; $P < 0.002 compared with miR-200a control). D: Western analysis of phospho-SMAD3 and total SMAD3 levels in miR-200a-transfected NRK52E demonstrating reduced SMAD3 phosphorylation relative to total SMAD3 protein (*P < 0.05 compared with miR-C control). The Westerns were quantified and shown in graph format (*P < 0.05 compared with miR-C).
Mentions: Ectopic expression of miR-200a after transfection with premiR-200a resulted in decreased expression of several ECM genes, including collagen I and IV and fibronectin, compared with premiRNA-control transfected NRK52E cells (Fig. 4A). Some of these changes in ECM protein expression were also observed at the protein level (Fig. 4B). miR-200a also caused reduced expression of the mesenchymal marker, αSMA, and increased expression of the epithelial marker, E-cadherin mRNA levels (Fig. 4A), consistent with previous reports that the miR-200 family targets the transcriptional repressors of E-cadherin, ZEB1, and ZEB2 (12,18–21). Finally the expression of PAI-1, a downstream target of TGF-β signaling, was also decreased (Fig. 4A).

Bottom Line: TGF-β1 and TGF-β2 also downregulated expression of miR-200a.The renal expression of miR-141 and miR-200a was also reduced in mouse models representing early and advanced kidney disease. miR-200a and miR-141 significantly impact on the development and progression of TGF-β-dependent EMT and fibrosis in vitro and in vivo.These miRNAs appear to be intricately involved in fibrogenesis, both as downstream mediators of TGF-β signaling and as components of feedback regulation, and as such represent important new targets for the prevention of progressive kidney disease in the context of diabetes.

View Article: PubMed Central - PubMed

Affiliation: JDRF Danielle Alberti Memorial Centre for Diabetes Complications, Melbourne, Australia.

ABSTRACT

Objective: Progressive fibrosis in the diabetic kidney is driven and sustained by a diverse range of profibrotic factors. This study examines the critical role of microRNAs (miRNAs) in the regulation of the key fibrotic mediators, TGF-β1 and TGF-β2.

Research design and methods: Rat proximal-tubular epithelial cells (NRK52E) were treated with TGF-β1 and TGF-β2 for 3 days, and expression of markers of epithelial-to-mesenchymal transition (EMT) and fibrogenesis were assessed by RT-PCR and Western blotting. The expression of miR-141 and miR-200a was also assessed, as was their role as translational repressors of TGF-β signaling. Finally, these pathways were explored in two different mouse models, representing early and advanced diabetic nephropathy.

Results: Both TGF-β1 and TGF-β2 induced EMT and fibrogenesis in NRK52E cells. TGF-β1 and TGF-β2 also downregulated expression of miR-200a. The importance of these changes was demonstrated by the finding that ectopic expression miR-200a downregulated smad-3 activity and the expression of matrix proteins and prevented TGF-β-dependent EMT. miR-200a also downregulated the expression of TGF-β2, via direct interaction with the 3' untranslated region of TGF-β2. The renal expression of miR-141 and miR-200a was also reduced in mouse models representing early and advanced kidney disease.

Conclusions: miR-200a and miR-141 significantly impact on the development and progression of TGF-β-dependent EMT and fibrosis in vitro and in vivo. These miRNAs appear to be intricately involved in fibrogenesis, both as downstream mediators of TGF-β signaling and as components of feedback regulation, and as such represent important new targets for the prevention of progressive kidney disease in the context of diabetes.

Show MeSH
Related in: MedlinePlus