MicroRNA 144 Involved in the Modulation of TGF-b Signaling and Fibrosis Is Upregulated in Lung Biopsies from Transplant Recipients Undergoing Chronic Rejection - Bronchiolitis Obliterans Syndrome (BOS)

Abstract

Introduction: Development of BOS following human lung transplantation (LTx) remains a major impediment for long term function of the transplanted lungs. BOS is a fibroproliferative process resulting in progressive decline in pulmonary function and eventual allograft failure. The immune mechanisms leading to the development of BOS remains poorly defined. MicroRNAs (miR), through the regulation of gene expression, control various biological processes. Specifically, miR 144 has been reported to upregulate TGF-b signaling leading to fibrosis and therefore may play a significant role in the pathogenesis of BOS. Objective: The objective of this study was to determine whether miR 144 is upregulated in the lung biopsies from LTx recipients diagnosed with BOS and to define the role of miR 144 in the development of BOS following human LTx. Methods: Serum concentrations of TGF-β1 and IL-6 were determined by ELISA. Levels of miR 144 in the lung biopsies of BOS+ and BOS-LTx recipients (n=15 each) were analyzed by quantitative real time PCR (qPCR). The levels of downstream targets of miR 144 TGFB-induced factor homeobox 1 (TGIF-1), SMAD, TGF-b1, IL-6, and connective tissue growth factor (CTGF) were determined by qPCR using gene specific primers. Results: Analysis of the serum levels of TGF-β1 and IL-6 demonstrated a significant increase in the levels of TGF-β1 (430 vs 110 ng/ml, p< 0.05) and IL-6 (278 vs 63 ng/ml, p< 0.05) levels in BOS+ patients. Based on this we analyzed miR 144 levels in the lung biopsies from a different cohort of LTx which showed significant increase (3.4±1.2 fold) in the levels of miR 144 in the lung biopsies of BOS+ LTx when compared to the BOS- LTx (p< 0.05). qPCR analysis for the down stream targets also demonstrated significant decrease in the expression of TGIF (3.8±0.9 fold) and marked increases in SMAD (4.3±1.2 fold), TGFβ1 (5.3 fold increase), IL-6 (2.9±0.9 fold increase) and CTGF (3.9±1.1 fold increase) in comparison to BOS- LTx recipients. Analysis of TGF-β and IL-6 in serum samples from part of this cohort of BOS+ LTx also demonstrated significant increases in the levels of TGF-β (262ng/ml) and IL-6 (62.6ng/ml). Conclusion: Our results demonstrated increased levels of miR 144 in the lung biopsies from BOS+ LTx recipients. We propose that miR144 can down regulate the expression of TGIF-1, a negative regulator of SMAD which is critical for the TGF-β signaling cascade. This will result in increased expression of TGF-β1, IL-6 and CTGF which can promote the development of fibrosis, a hallmark of the lesion for the development of BOS in LTx recipients. Therefore, agents that can target miR 144 may aid in the prevention of fibrosis development and therefore BOS following LTx.