MicroRNA, miR144 Dysregulates TGf-b Signaling Cascade and Contributes to Development of BOS Following Human Lung Transplantation.

Abstract

Aim: Bronchiolitis Obliterans Syndrome (BOS) is characterized by a fibroproliferative cascade that is not amenable to current treatment regimens and remains the major impediment for the long term function of the transplanted organ. MicroRNAs have been shown to significantly impact several biological processes through their ability to regulate gene expression. The objective of the current study was to define the role of miR144 in the development of fibroproliferative cascade in lung transplant recipients. Methods: Lung biopsies were obtained from transplant recipients with and without BOS (n=10 each). Expression levels of miR144 and its gene target TGIF1 were analyzed by quantitative real-time PCR using specific primers. The functional role of the miR144 was evaluated by transfecting human fibroblasts and evaluating their response to TGF-b by analyzing the expression level of SMAD and fibrogenic growth factors by quantitative real time PCR. Results: Analysis of the miR144 in the lung biopsies demonstrated that there was 4.1±0.8 fold increases in the levels of miR144 in the BOS+ when compared to BOS-samples. Similarly, the level of TGIF1 was 3.6±1.2 fold lower in the BOS+ when compared to BOS- samples. These results strongly suggest that overexpression of miR144 in the lungs of BOS+ samples results in a significant reduction in the levels of TGIF1, a transcription factor that acts as a corepressor of SMAD and an inhibitor of the retinoic acid responsive element. These two pathways are implicated in the development of fibrosis and Th17 responses. To confirm we performed In vitro transfection analysis which demonstrated that overexpression of miR144 results in significant reduction in the expression of TGIF1 (4.6±0.7 fold) and significant increases in the expression of SMAD (5.3±1.4 folds), and FGF (4.1±0.9 fold) when compared to nontransfected fibroblasts. Conclusion: We conclude that miR144 is a critical regulator of the TGF-b signaling cascade and is overexpressed 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.