MicroRNA-144 dysregulates the transforming growth factor-β signaling cascade and contributes to the development of bronchiolitis obliterans syndrome after human lung transplantation

BACKGROUND: Bronchiolitis obliterans syndrome (BOS), chronic lung allograft rejection, remains an impediment for the function of the transplanted organ. In this study, we defined the role of the microRNA (miRNA) miR-144 in fibroproliferation leading to BOS. METHODS: Biopsy specimens were obtained from 20 lung transplant recipients with BOS((+)) and 19 without BOS((-)). Expression of miR-144 and its target, transforming growth factor-beta (TGF-beta)-induced factor homeobox 1(TGIF1), were analyzed by real-time polymerase chain reaction and Western blot. Overexpression of miR-144 and luciferase reporter genes were performed to elucidate miRNA-target interactions. The function of miR-144 was evaluated by transfecting fibroblasts and determining the response to TGF-beta by analyzing Sma- and Mad-related family (Smads), fibroblast growth factor, TGF-beta, and vascular endothelial growth factor. Smooth muscle actin-alpha-positive stress fibers and F-actin filaments in lung fibroblasts were analyzed by immunofluorescence. RESULTS: Analysis of miR-144 in the biopsy specimens demonstrated 4.1 +/- 0.8-fold increases in BOS+ compared with BOS- patients, with a significant reduction in TGIF1 (3.6 +/- 1.2-fold), a corepressor of Smads. In vitro transfection confirmed that over-expression of miR-144 results in a reduction in TGIF1 and an increase in SMAD2, SMAD4, fibroblast growth factor-6, TGF-beta, and vascular endothelial growth factor. Increasing miR-144 by transfecting, increased smooth muscle actin-alpha and fibronectin, and knockdown of miR-144 diminished fibrogenesis in MRC-5 fibroblasts. CONCLUSIONS: miR-144 is a critical regulator of the TGF-beta signaling cascade and is over-expressed in lungs with BOS. Therefore, miR-144 is a potential target toward preventing fibrosis leading to BOS after lung transplant. (C) 2015 International Society for Heart and Lung Transplantation. All rights reserved.