Abstract
Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disease with irreversible loss of lung tissue and function. The myofibroblast is the key cellular mediator of IPF progression. Transforming growth factor (TGF)-β1, a major profibrogenic cytokine, induces differentiation of pulmonary fibroblasts to myofibroblasts. Emerging evidence has implicated the importance of microRNAs (miRs) in the development of IPF. The objective of our study was to identify novel and specific miRs that are dysregulated in TGF-β1-induced pulmonary myofibroblast differentiation and development of pulmonary fibrosis and their molecular mechanisms. Using the RNA sequencing, we identified miR-424 as an important miRNA that is up-regulated by TGF-β1 in human lung fibroblasts (HLFs) while miR-146b is down-regulated in TGF-β1 treated cells.
Quantitative RT-PCR (qRT-PCR) confirmed that miR-424 expression was increased in HLF in response to TGF-β1 and was higher in lung tissues of IPF patients as compared to normal subjects. We found that the canonical TGF-β1 signaling pathway was involved in TGF-β1-induced miR-424 up-regulation. Transfection of miR-424 inhibitor in HLF reduced TGF-β1-induced expression of differentiation markers including ɑ-smooth muscle actin (ɑ-SMA) and connective tissue growth factor (CTGF), whereas a miR-424 mimic significantly enhanced TGF-β1-induced myofibroblast differentiation. Moreover, TGF-β1-induced Smad3 phosphorylation in HLF was reduced by a miR-424 inhibitor. In silico analysis identified Slit2, an inhibitory protein of TGF-β1 profibrogenic signaling, as a putative target of miR-424. Together, this study demonstrates a pro-fibrotic role of miR-424 in TGF-β1-induced HLF differentiation. It functions as a positive feedback regulator of the TGF-β1 signaling pathway via targeting Slit2.
MiR-146b was confirmed by qRT-PCR as a micro-RNA that is significantly decreased both in TGF-β1-treated HLFs and in HLFs derived from IPF patients. Overexpression of miR-146b inhibited HLFs differentiation and proliferation while the deletion of miR-146b enhanced these processes in mouse lung fibroblasts. MiR-146b plays anti-fibrotic and anti-proliferation roles by regulating the expression of Smad3 and cyclin D1, respectively, indicating the potential pathologic importance of miR-146b in the development of pulmonary fibrosis.
Taken together, these studies identified two novel miRs that are likely involved in pulmonary fibrosis, and targeting these miRNAs may provide a new therapeutic strategy for IPF.