title: miR-19a, -19b, and -26b Mediate CTGF Expression and Pulmonary Fibroblast Differentiation.
abstract: Although microRNA (miRNA) dysregulation with intracellular signaling cascade disruption has been demonstrated in the pathophysiology of pulmonary fibrosis, the relationship between miRNAs and intracellular signaling cascades in pulmonary fibrosis remains unclear. Using the human embryonic lung fibroblast cell line WI-38, we observed endothelin-1 (ET-1)- and thrombin-induced expression of the differentiation markers α-smooth muscle actin (α-SMA) and vimentin along with increased connective tissue growth factor (CTGF) protein expression. Decreased CTGF protein expression by CTGF siRNA significantly blocked ET-1- and thrombin-induced α-SMA and vimentin expression in WI-38 cells. Activation of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase ERK, c-Jun N-terminal kinase (JNK), and p38 contributed to ET-1- and thrombin-induced CTGF, α-SMA, and vimentin expression in WI-38 cells. TargetScan Human, miRanda, and PicTar prediction algorithms were used to predict miRNAs with binding sites in the 3' untranslated region (UTR) of CTGF mRNA. miR-19a, -19b, and -26b were candidate miRNAs of CTGF. Direct binding of the candidate miRNAs to the 3'-UTR of CTGF mRNA was verified through luciferase assay by using SV40-promoter-IRES-driven luciferase containing the 3'-UTR of CTGF mRNA as a reporter plasmid. ET-1 and thrombin reduced candidate miRNA levels. Candidate miRNA overexpression significantly suppressed ET-1- and thrombin-induced CTGF expression and reduced α-SMA and vimentin expression in the WI-38 cells. Furthermore, candidate miRNA levels were decreased in the lung tissues of mice with bleomycin-induced pulmonary fibrosis, and intratracheal application of miR-19a, -19b, and 26b reduced the pulmonary fibrotic severity induced by bleomycin. This study is the first to demonstrate crosstalk between MAPK activation and reduction in miR-19a, -19b, and -26b expression leading to lung fibroblast differentiation. J. Cell. Physiol. 231: 2236-2248, 2016. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan.|Cancer Research Center and Orthopedics Research Center, Taipei Medical University Hospital, Taipei, Taiwan.
authors.forename: Yen-Chou
authors.initials: YC
authors.lastname: Chen
authors.affiliation: School of Respiratory Therapy, College of Medicine, Taipei Medical University, Taipei, Taiwan.
authors.forename: Bing-Chang
authors.initials: BC
authors.lastname: Chen
authors.affiliation: Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan.
authors.forename: Chung-Chi
authors.initials: CC
authors.lastname: Yu
authors.affiliation: Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan.
authors.forename: Shin-Hua
authors.initials: SH
authors.lastname: Lin
authors.affiliation: Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan.
authors.forename: Chien-Huang
authors.initials: CH
authors.lastname: Lin
mesh_terms: D000199:Actins / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D055513:Connective Tissue Growth Factor / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics*; D010766:Phosphorylation; D011658:Pulmonary Fibrosis / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics
chemical_list: D000199:Actins; D055513:Connective Tissue Growth Factor; D048031:JNK Mitogen-Activated Protein Kinases; D035683:MicroRNAs; C549214:MIRN19A microRNA, human; C528163:MIRN26 microRNA, human