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Transforming growth factor (TGF)-?1-induced miR-133a inhibits myofibroblast differentiation and pulmonary fibrosis.

ABSTRACT: Transforming growth factor (TGF)-?1, a main profibrogenic cytokine in the progression of idiopathic pulmonary fibrosis (IPF), induces differentiation of pulmonary fibroblasts to myofibroblasts that produce high levels of collagen, leading to concomitantly loss of lung elasticity and function. Recent studies implicate the importance of microRNAs (miRNAs) in IPF but their regulation and individual pathological roles remain largely unknown. We used both RNA sequencing and quantitative RT-PCR strategies to systematically study TGF-?1-induced alternations of miRNAs in human lung fibroblasts (HFL). Our data show that miR-133a was significantly upregulated by TGF-?1 in a time- and concentration-dependent manner. Surprisingly, miR-133a inhibits TGF-?1-induced myofibroblast differentiation whereas miR-133a inhibitor enhances TGF-?1-induced myofibroblast differentiation. Interestingly, quantitative proteomics analysis indicates that miR-133a attenuates myofibroblast differentiation via targeting multiple components of TGF-?1 profibrogenic pathways. Western blot analysis confirmed that miR-133a down-regulates TGF-?1-induced expression of classic myofibroblast differentiation markers such as ?-smooth muscle actin (?-SMA), connective tissue growth factor (CTGF) and collagens. miRNA Target Searcher analysis and luciferase reporter assays indicate that TGF-? receptor 1, CTGF and collagen type 1-alpha1 (Col1a1) are direct targets of miR-133a. More importantly, miR-133a gene transferred into lung tissues ameliorated bleomycin-induced pulmonary fibrosis in mice. Together, our study identified TGF-?1-induced miR-133a as an anti-fibrotic factor. It functions as a feed-back negative regulator of TGF-?1 profibrogenic pathways. Thus, manipulations of miR-133a expression may provide a new therapeutic strategy to halt and perhaps even partially reverse the progression of IPF.

SUBMITTER: Wei P

PROVIDER: S-EPMC6739313 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Transforming growth factor (TGF)-β1-induced miR-133a inhibits myofibroblast differentiation and pulmonary fibrosis.

Wei Peng P Xie Yan Y Abel Peter W PW Huang Yapei Y Ma Qin Q Li Linghai L Hao Junfeng J Wolff Dennis W DW Wei Taotao T Tu Yaping Y

Cell death & disease 20190911 9

Transforming growth factor (TGF)-β1, a main profibrogenic cytokine in the progression of idiopathic pulmonary fibrosis (IPF), induces differentiation of pulmonary fibroblasts to myofibroblasts that produce high levels of collagen, leading to concomitantly loss of lung elasticity and function. Recent studies implicate the importance of microRNAs (miRNAs) in IPF but their regulation and individual pathological roles remain largely unknown. We used both RNA sequencing and quantitative RT-PCR strate ...[more]

PMID: 31511493

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Project description:Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disease with irreversible loss of lung tissue and function. Myofibroblasts in the lung are key cellular mediators of IPF progression. Transforming growth factor (TGF)-β1, a major profibrogenic cytokine, induces pulmonary myofibroblast differentiation, and emerging evidence has established the importance of microRNAs (miRs) in the development of IPF. The objective of this study was to define the pro-fibrotic roles and mechanisms of miRs in TGF-β1-induced pulmonary myofibroblast differentiation. Using RNA sequencing, we identified miR-424 as an important TGF-β1-induced miR in human lung fibroblasts (HLFs). Quantitative RT-PCR confirmed that miR-424 expression was increased by 2.6-fold in HLFs in response to TGF-β1 and was 1.7-fold higher in human fibrotic lung tissues as compared to non-fibrotic lung tissues. TGF-β1-induced upregulation of miR-424 was blocked by the Smad3 inhibitor SIS3, suggesting the involvement of this canonical TGF-β1 signaling pathway. Transfection of a miR-424 hairpin inhibitor into HLFs reduced TGF-β1-induced expression of classic myofibroblast 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. In addition, TGF-β1 induced Smad3 phosphorylation in HLFs, and this response was reduced by the miR-424 inhibitor. In silico analysis identified Slit2, a protein that inhibits TGF-β1 profibrogenic signaling, as a putative target of regulation by miR-424. Slit2 is less highly expressed in human fibrotic lung tissues than in non-fibrotic lung tissues, and knockdown of Slit2 by its siRNA enhanced TGF-β1-induced HLF differentiation. Overexpression of a miR-424 mimic down-regulated expression of Slit2 but not the Slit2 major receptor ROBO1 in HLFs. Luciferase reporter assays showed that the miR-424 mimic represses Slit2 3' untranslated region (3'-UTR) reporter activity, and mutations at the seeding regions in the 3'-UTR of Slit2 abolish this inhibition. Together, these data demonstrate a pro-fibrotic role of miR-424 in TGF-β1-induced HLF differentiation. It functions as a positive feed-back regulator of the TGF-β1 signaling pathway by reducing expression of the negative regulator Slit2. Thus, targeting miR-424 may provide a new therapeutic strategy to prevent myofibroblast differentiation and IPF progression.

Dataset Information

Transforming growth factor (TGF)-?1-induced miR-133a inhibits myofibroblast differentiation and pulmonary fibrosis.

Publications

Transforming growth factor (TGF)-β1-induced miR-133a inhibits myofibroblast differentiation and pulmonary fibrosis.

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