miR-146b disrupts the epidermal growth factor receptor/transforming growth factor β receptor profibrotic feedforward loop to inhibit lung fibroblast proliferation and differentiation.

Idiopathic pulmonary fibrosis (IPF) is a progressive and often fatal lung disease. Despite recent treatment advancements, survival rates for IPF patients remain low, reflecting the urgent need for a deeper understanding of disease mechanisms. A key feature of IPF progression is the excessive growth of fibroblasts and their transformation into myofibroblasts, driven by a profibrotic feedforward loop involving mediators like transforming growth factor (TGF) α and β1. These factors activate the epidermal growth factor receptor (EGFR) and TGFβ1 receptor (TGFβR1), perpetuating fibrosis. MicroRNAs play a vital role in regulating gene expression, and many are disrupted in IPF. Through RNA sequencing and quantitative reverse-transcription polymerase chain reaction, we identified miR-146b as one of the most significantly reduced miRNAs in human lung fibroblasts (HLF) following TGFβ1 treatment, with a confirmed 70% decrease in expression. Although miR-146b is normally abundant in lung fibroblasts, its levels are significantly lower in lung fibroblasts from IPF patients (HLF-F) and in mice with experimental pulmonary fibrosis. HLF-F exhibited greater responses to profibrotic signals compared with nonfibrotic fibroblasts. Elevating miR-146b levels in HLF-F reduced TGFα-induced proliferation and inhibited TGFβ1-induced myofibroblast differentiation by 60%-90%. Conversely, deletion of the miR-146b gene enhanced fibrotic responses in mouse lung fibroblasts. Additionally, miR-146b directly targets critical signaling pathways, including EGFR, Jun, TGFβR1, and SMAD3, thereby suppressing fibroblast proliferative and fibrogenic activities. Thus, targeting miR-146b presents a promising therapeutic strategy to inhibit lung fibroblast proliferation and differentiation by modulating the profibrotic feedforward loop of EGFR and TGFβR signaling pathways. SIGNIFICANCE STATEMENT: Excess fibroblast proliferation and differentiation into myofibroblasts in response to profibrotic mediators are considered key steps in idiopathic pulmonary fibrosis progression. This study identified miR-146b as an important endogenous antifibrotic factor via inhibition of both lung fibroblast proliferation and differentiation. The repression of miR-146b by transforming growth factor β1 also uncovers a new mechanism linking the factors that drive fibrosis and excessive cell growth in idiopathic pulmonary fibrosis, opening up new avenues for slowing disease progression and improving patient outcomes via restoration of miR-146b expression.