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Antifibrotic Effect of microRNA-16 in Human Lung Fibroblasts
Description
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A2295 - Antifibrotic Effect of microRNA-16 in Human Lung Fibroblasts
Author Block: M. Inomata, K. Kamio, A. Azuma, K. Matsuda, T. Kashiwada, N. Kokuho, K. Atsumi, H. Hayashi, K. Fujita, Y. Saito, S. Abe, M. Seike, A. Gemma; Department of Internal Medicine, Division of Pulmonary Medicine, Infectious Diseases and Oncology, Nippon Medical School, Tokyo, Japan.
RATIONALE
Exosomes are 40-100-nm nano-sized vesicles that are released from many cell types, and several components including microRNAs (miRNAs) have been identified within exosomes. Idiopathic pulmonary fibrosis (IPF) is a progressive disease with unknown etiology, and its pathological hallmark is the abnormal deposition of extracellular matrix in the lung parenchyma as a result of pathological proliferation and activation of lung fibroblasts which contribute to the initiation and progression of IPF. Several miRNAs have been reported to be involved in the pathogenesis of pulmonary fibrosis. Transforming growth factor (TGF)-β1 also plays an important role in the pathogenesis of pulmonary fibrosis. It regulates the expression of secreted protein acidic and rich in cysteine (SPARC), which is a matricellular protein, through the activation of mammalian target of rapamycin complex 2 (mTORC2). Membrane-associated guanylate kinase, WW and PDZ domain-containing protein 2 (MAGI2) is a scaffold protein required for PTEN, and it is diminished after TGF-β1 stimulation resulting in myofibroblast differentiation. Therefore, we investigated the role of microRNAs in the pathogenesis of pulmonary fibrosis through the regulation of TGF pathway in human lung fibroblasts.
METHODS
Bleomycin (100 mg/kg) was infused with osmotic pumps into C57BL/6 mice on day 0. After the bleomycin challenge, exosomes were isolated from plasma using ExoQuick™ (System Biosciences) on day 7, 14, 21, and 28. MicroRNAs in exosomes were compared between the groups with or without bleomycin on day 7, 14, 21, 28 using microRNA array (3D-Gene®, TORAY). Next, differentially up-regulated microRNAs were evaluated using a mimic microRNA in human fetal lung fibroblasts (HFL-1 cells) treated with TGF-β1 (1 ng/ml) for 96 h. Fibrosis markers were quantified by quantitative real-time PCR and western blotting.
RESULTS
In bleomycin-treated groups, microRNA-16 in exosomes increased by 10.32-fold on day 14 when compared with that in saline-treated groups. Pretreatment of HFL-1 cells with a mimic microRNA-16 (50 nM) decreased mRNA expression of smooth muscle actin, collagen type I, and SPARC in HFL-1 cells treated with TGF-β1. The amount of TGF-β1-induced phospho-Smad2 and Smad3 as assessed by western blotting was suppressed by mimic microRNA-16 pretreatment. Pretreatment of HFL-1 cells with mimic microRNA-16 reversed MAGI2 protein expression, which was down-regulated by
TGF-β1.
CONCLUSIONS
This is the first study demonstrating the antifibrotic effect of microRNA-16 through TGF-β1 and mTORC2 pathway, together with inhibition of myofibroblast differentiation through the up-regulation of MAGI2.
Author Block: M. Inomata, K. Kamio, A. Azuma, K. Matsuda, T. Kashiwada, N. Kokuho, K. Atsumi, H. Hayashi, K. Fujita, Y. Saito, S. Abe, M. Seike, A. Gemma; Department of Internal Medicine, Division of Pulmonary Medicine, Infectious Diseases and Oncology, Nippon Medical School, Tokyo, Japan.
RATIONALE
Exosomes are 40-100-nm nano-sized vesicles that are released from many cell types, and several components including microRNAs (miRNAs) have been identified within exosomes. Idiopathic pulmonary fibrosis (IPF) is a progressive disease with unknown etiology, and its pathological hallmark is the abnormal deposition of extracellular matrix in the lung parenchyma as a result of pathological proliferation and activation of lung fibroblasts which contribute to the initiation and progression of IPF. Several miRNAs have been reported to be involved in the pathogenesis of pulmonary fibrosis. Transforming growth factor (TGF)-β1 also plays an important role in the pathogenesis of pulmonary fibrosis. It regulates the expression of secreted protein acidic and rich in cysteine (SPARC), which is a matricellular protein, through the activation of mammalian target of rapamycin complex 2 (mTORC2). Membrane-associated guanylate kinase, WW and PDZ domain-containing protein 2 (MAGI2) is a scaffold protein required for PTEN, and it is diminished after TGF-β1 stimulation resulting in myofibroblast differentiation. Therefore, we investigated the role of microRNAs in the pathogenesis of pulmonary fibrosis through the regulation of TGF pathway in human lung fibroblasts.
METHODS
Bleomycin (100 mg/kg) was infused with osmotic pumps into C57BL/6 mice on day 0. After the bleomycin challenge, exosomes were isolated from plasma using ExoQuick™ (System Biosciences) on day 7, 14, 21, and 28. MicroRNAs in exosomes were compared between the groups with or without bleomycin on day 7, 14, 21, 28 using microRNA array (3D-Gene®, TORAY). Next, differentially up-regulated microRNAs were evaluated using a mimic microRNA in human fetal lung fibroblasts (HFL-1 cells) treated with TGF-β1 (1 ng/ml) for 96 h. Fibrosis markers were quantified by quantitative real-time PCR and western blotting.
RESULTS
In bleomycin-treated groups, microRNA-16 in exosomes increased by 10.32-fold on day 14 when compared with that in saline-treated groups. Pretreatment of HFL-1 cells with a mimic microRNA-16 (50 nM) decreased mRNA expression of smooth muscle actin, collagen type I, and SPARC in HFL-1 cells treated with TGF-β1. The amount of TGF-β1-induced phospho-Smad2 and Smad3 as assessed by western blotting was suppressed by mimic microRNA-16 pretreatment. Pretreatment of HFL-1 cells with mimic microRNA-16 reversed MAGI2 protein expression, which was down-regulated by
TGF-β1.
CONCLUSIONS
This is the first study demonstrating the antifibrotic effect of microRNA-16 through TGF-β1 and mTORC2 pathway, together with inhibition of myofibroblast differentiation through the up-regulation of MAGI2.
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