Inhibition and role of let-7d in idiopathic pulmonary fibrosis

KV Pandit, D Corcoran, H Yousef… - American journal of …, 2010 - atsjournals.org
KV Pandit, D Corcoran, H Yousef, M Yarlagadda, A Tzouvelekis, KF Gibson, K Konishi…
American journal of respiratory and critical care medicine, 2010atsjournals.org
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 …
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 α-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.
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).
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