Background: Bronchiolitis Obliterans Syndrome (BOS) is characterized by a fibroproliferative cascade that is not amenable to current treatment regimens and remains the major impediment for long term function of the transplanted organ. The objective of the study was to define the role of miR-144 in the development of fibroproliferative cascade in lung transplant recipients (LTxR).

Materials and Methods: Lung biopsies were obtained from LTxR with (n=20) and without BOS (n=19). Expression levels of miR-144 and its gene target, TGFβ-induced factor homeobox 1 (TGIF1), were analyzed by quantitative real-time PCR (qRT-PCR). Luciferase reporter genes were used to elucidate direct miRNA-target interactions. The functional role was evaluated by transfecting human fibroblasts with miR-144. Immunofluorescence of SMA-α-positive stress fiber and F-actin filaments in lung fibroblasts by transfecting miR-144 mimics.

Results: Analysis of the miR-144 in the lung biopsies demonstrated that there was 4.1±0.8 fold increases in the levels of miR-144 in BOS+ patients compared to BOS-. Similarly, the level of TGIF1 was 3.6±1.2 fold lower in BOS+ when compared to BOS- samples. Direct miR-144 targeting of TGIF1 by miR-144 demonstrated that over expression of miR-144 in lungs of BOS+ patient's results in a significant reduction in the levels of TGIF1, a transcription factor that acts as a corepressor of Smads. In vitro transfection analysis also demonstrated that over expression of miR-144 results in significant reduction in expression of TGIF1 (4.6±0.7 fold) and significant increases in the expression of Smad2 (p<0.01, 4.3±0.8 folds), Smad4 (p<0.01, 4.1±0.4 folds), FGF (p<0.01, 4.1±0.9), TGFβ (p<0.0,1, 3.5±0.6), and VEGF (p=0.035, 1.7±0.4) when compared to control-transfected fibroblasts. Furthermore, increasing miR-144 levels in fibroblasts by transfecting miR-144 mimics increased protein expression of SMA-α and Fibronectin compared to those in control miR-transfected MRC-5 cells, while knockdown of miR-144 diminishes fibrogenesis in lung fibroblasts.

Conclusions: We conclude that miR-144 is a critical regulator of TGFβ signaling cascade and is over expressed in lungs of patients diagnosed with BOS and therefore can be a potential target for therapeutic intervention towards preventing the development of fibrosis and BOS development following human lung transplantation.

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