Published ahead of print on April 15, 2010, doi:10.1164/rccm.200911-1698OC
© 2010 American Thoracic Society doi: 10.1164/rccm.200911-1698OC
Inhibition and Role of let-7d in Idiopathic Pulmonary Fibrosis1 Division of Pulmonary, Allergy, and Critical Care Medicine, Dorothy P. and Richard P. Simmons Center for Interstitial Lung Disease, University of Pittsburgh School of Medicine, and 2 Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania; 3 Department of Pneumonology, Medical School, Democritus University of Thrace, and University Hospital of Alexandroupolis, Alexandroupolis, Greece; 4 Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania; 5 Instituto Nacional de Enfermedades Respiratorias, Mexico City, Mexico; 6 Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; 7 Facultad de Ciencias, Universidad Nacional Autónoma de México, Mexico City, Mexico; 8 Comprehensive Pneumology Center, Munich, Germany; and 9 Department of Computational Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania Correspondence and requests for reprints should be addressed to Naftali Kaminski, M.D., University of Pittsburgh Medical Center, NW 628 MUH, 3459 5th Avenue, Pittsburgh, PA 15261. E-mail: kaminskin{at}upmc.edu; or to Panayiotis V. Benos, Ph.D., Department of Computational Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261. E-mail: benos{at}pitt.edu Rationale: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and usually lethal fibrotic lung disease characterized by profound changes in epithelial cell phenotype and fibroblast proliferation. Objectives: To determine changes in expression and role of microRNAs in IPF. Methods: RNA from 10 control and 10 IPF tissues was hybridized on Agilent microRNA microarrays and results were confirmed by quantitative real-time polymerase chain reaction and in situ hybridization. SMAD3 binding to the let-7d promoter was confirmed by chromatin immunoprecipitation, electrophoretic mobility shift assay, luciferase assays, and reduced expression of let-7d in response to transforming growth factor-β. HMGA2, a let-7d target, was localized by immunohistochemistry. In mice, let-7d was inhibited by intratracheal administration of a let-7d antagomir and its effects were determined by immunohistochemistry, immunofluorescence, quantitative real-time polymerase chain reaction, and morphometry.
Measurements and Main Results: Eighteen microRNAs including let-7d were significantly decreased in IPF. Transforming growth factor-β down-regulated let-7d expression, and SMAD3 binding to the let-7d promoter was demonstrated. Inhibition of let-7d caused increases in mesenchymal markers N-cadherin-2, vimentin, and Conclusions: Our results indicate a role for microRNAs in IPF. The down-regulation of let-7d in IPF and the profibrotic effects of this down-regulation in vitro and in vivo suggest a key regulatory role for this microRNA in preventing lung fibrosis. Clinical trial registered with www.clinicaltrials.gov (NCT 00258544).
Key Words: epithelial–mesenchymal transition • HMGA2 (high-mobility group AT-hook 2) • microRNA • transforming growth factor-β
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-smooth muscle actin (ACTA2) as well as HMGA2 in multiple epithelial cell lines. let-7d was significantly reduced in IPF lungs and the number of epithelial cells expressing let-7d correlated with pulmonary functions. HMGA2 was increased in alveolar epithelial cells of IPF lungs. let-7d inhibition in vivo caused alveolar septal thickening and increases in collagen, ACTA2, and S100A4 expression in SFTPC (pulmonary-associated surfactant protein C) expressing alveolar epithelial cells. 
