Expression and Regulation of let-7d in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and usually lethal fibrotic lung disease with as yet unknown etiology and treatment. IPF lungs have a definitive gene expression signature, but the role of microRNAs in IPF has not been studied. MicroRNA genes are short, non-coding RNAs that function as post-transcriptional gene regulators. We hypothesized that microRNAs are differentially expressed in IPF and play a role in the pathogenesis of the disease. The microRNA expression profile of IPF lungs was characterized using microRNA microarrays. We identified 18 significantly decreased and 28 significantly increased microRNAs in samples obtained from patients with IPF compared to controls. Promoter analysis of the decreased microRNAs recognized a SMAD3 binding site in the promoter of let-7d. SMAD3 binding and responsiveness to TGF-β of the let-7d promoter were confirmed by SMAD3 ChIP, EMSA, luciferase assays and reduced expression of let-7d in response to TGF-β. In situ hybridization confirmed a significant reduction in let-7d expression in IPF lungs and localized it to alveolar epithelial cells in the control lungs. To study the role of let-7d, we determined the consequences of loss of function of let-7d by inhibiting its expression in lung epithelial cells. In-vitro inhibition of let-7d caused increases in epithelial mesenchymal transition (EMT) markers N-cadherin (CDH2), vimentin (VIM), alpha-smooth muscle actin (ACTA2) and high mobility group AT-hook 2 (HMGA2). HMGA2 is a proven let-7d target that was increased in IPF lungs and localized to alveolar epithelial cells. We reproduced our in vitro results in vivo. In mice, intratracheal administration of a let-7d inhibitor caused alveolar septal thickening, increases in collagen, ACTA2 and CDH2 and decreases in the epithelial markers, CDH1 and ZO1. We colocalized the mesenchymal markers FSP1 and ACTA2 with the epithelial marker SPC, indicative of EMT.Our results indicate a definitive role for microRNAs in IPF. The downregulation of let-7d in IPF and the pro-fibrotic effects of this downregulation in vitro and in vivo suggest a key regulatory role for this microRNA in preventing lung fibrosis. Deciphering their mechanism of action of microRNAs has great public health significance. It would add new insight into the pathophysiology of the disease and aid in reducing the mortality by development of therapeutic interventions