MiR-146b inhibits lung fibroblast proliferation and differentiation by targeting EGFR and TGFβR signaling pathways

Abstract

<b>Abstract ID 29836</b> <b>Poster Board 163</b> Idiopathic pulmonary fibrosis (IPF) is the most common type of pulmonary fibrosis. It is progressive and usually fatal. Current treatments have not significantly improved survival, leaving an unmet need for novel anti-fibrotic therapies. Excess fibroblast proliferation and differentiation into myofibroblast in response to profibrotic mediators such as transforming growth factor (TGF) α and β1 are considered key steps in IPF progression. TGFα and TGFβ1 bind and stimulate the epidermal growth factor receptor (EGFR) and TGFβ receptors (TGFβR1/2), respectively. Upregulation of EGFR and TGFβRs and their signaling in lung fibroblasts play an important role in the pathogenesis of IPF. Thus, suppressing EGFR/TGFβR-dependent fibroblast proliferation and differentiation are attractive targets for preventing IPF progression and death. MicroRNAs (miRs) are important posttranscriptional gene expression regulators and several miRs are known to be dysregulated in IPF, but the mechanisms for their dysregulation and pathological effects remain largely unknown. We used both RNA sequencing and quantitative RT-PCR strategies to systematically study TGF-β1-indued alternations of miRNAs in human lung fibroblasts (HLF). While miR-21, miR-145, miR-424 and miR-133a were among the TGFβ1-induced miRNAs that were previously shown to promote or inhibit pulmonary fibrosis, miR-146b is one of the most reduced miRNAs in TGFβ1-treated cells. Quantitative RT-PCR confirmed that miR-146b expression was significantly reduced by 70% in HLFs in response to TGF-β1 treatment. Interestingly, normal expression of miR-146b is highest in lung fibroblasts but was markedly downregulated by about 50% in IPF patients or mice with experimental pulmonary fibrosis. Using mouse precision-cut lung slices as an <i>ex vivo</i> 3D model of fibrosis, we found that miR-146b deficiency exacerbated fibrotic pathohistological changes in response to profibrotic mediators TGFα and TGFβ1. Human lung fibroblasts from IPF patients (HLF-F) also show stronger fibrotic mediator responses than non-fibrotic HLF-N. Increasing expression of miR-146b in HLF-F suppresses TGFα-stimulated proliferation and blocks TGFβ1-induced myofibroblast differentiation by 60-90%, whereas deletion of miR-146b in lung fibroblasts significantly increases fibrotic responses. RNA-sequencing and KEGG pathway analysis revealed that miR-146b downregulates the cell cycle pathway and TGFβ signaling. Western blot analysis further confirmed miR-146b targets EGFR/TGFβR-mediated proliferative and fibrogenic signaling pathways. Together we identified miR-146b as an important endogenous anti-fibrotic factor in lung fibroblasts. It inhibits lung fibroblast proliferation and differentiation by targeting EGFR and TGFβR signaling pathways. Importantly, TGFβ1 repression of miR-146b provides a novel mechanism that integrates the profibrotic and hyper-proliferative aspects of human IPF. (Supported by grants from NIH R01HL116849 to YT, R35HL144481 to BBM and SKH; Nebraska State LB595 to PWA).