Let-7d: A regulator of epithelial-mesenchymal transition (EMT) and idiopathic pulmonary fibrosis (IPF). K. Pandit1, D. Corcoran1, H. Yousef1, D. Handley1, A. Ben-Yehudah1, A. Pardo2, M. Selman2, O. Eickelberg3, M. Yarlagadda1, P. Ray1, P. Benos1, N. Kaminski1 1) University of Pittsburgh, USA; 2) Instituto Nacional de Enfermedades Respiratorias, Mexico; 3) University of Giessen Lung Center, Germany.
Introduction: IPF is a chronic, lethal lung disease resulting in death within 3-5 years of diagnosis. TGF- plays a central role in the pathogenesis of IPF, with SMAD3 being its effector molecule. In this study, we investigated the effect of TGF- on the microRNA, let-7d and the role of this microRNA in the pathogenesis of IPF. Methods and Results: Discriminative motif analysis of upstream regions of intergenic microRNAs recognized a SMAD3 binding site in the promoter of let-7d. Electrophoretic mobility shift assay and SMAD3 chromatin immunoprecipitation were performed to confirm the let-7d/SMAD3 binding. Stimulation of A549 cells with recombinant TGF-, led to a decrease in let-7d (p<0.05) 6h later and a corresponding increase in HMGA2 (p<0.05) as quantified by qRT-PCR. HMGA2 is a key regulator of the TGF--induced EMT known to be expressed only during embrogenesis and carcinogenesis. Transfection of A549 cells with a let-7d inhibitor resulted in an increase in HMGA2 and markers of EMT including N-cadherin, vimentin and -smooth muscle actin, by qRT-PCR and immunofluorescence. Let-7d is downregulated in IPF lungs (p<0.05) compared to control lungs by qRT-PCR. In control lungs, let-7d was localized in the bronchial and alveolar epithelial cells by in situ hybridization. HMGA2, a target of let-7d, is increased 12-fold in IPF lungs by qRT-PCR and localizes to alveolar epithelial cells by immunohistochemistry. Ongoing experiments include inhibition of let-7d in the lungs of mice by intratracheal administration of a chemically-modified oligonucleotide to study fibrotic changes in the lung tissue. Conclusion: Our results suggest that let-7d is under direct transcriptional regulation of TGF- and is a key mediator of EMT. Thus, let-7d occupies a strategic place in the TGF- signaling pathway; its downregulation in IPF suggests that it plays a major role in the pathogenesis of this devastating disease.