Chen Yen-Chou, Chen Bing-Chang, Yu Chung-Chi, Lin Shin-Hua, Lin Chien-Huang
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.
J Cell Physiol. 2016 Oct;231(10):2236-48. doi: 10.1002/jcp.25341. Epub 2016 Mar 14.
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.
尽管在肺纤维化的病理生理学中已证实微小RNA（miRNA）失调与细胞内信号级联破坏有关，但miRNA与肺纤维化中细胞内信号级联之间的关系仍不清楚。使用人胚肺成纤维细胞系WI-38，我们观察到内皮素-1（ET-1）和凝血酶诱导分化标志物α-平滑肌肌动蛋白（α-SMA）和波形蛋白的表达，同时结缔组织生长因子（CTGF）蛋白表达增加。CTGF小干扰RNA（siRNA）降低CTGF蛋白表达可显著阻断WI-38细胞中ET-1和凝血酶诱导的α-SMA和波形蛋白表达。丝裂原活化蛋白激酶（MAPK）细胞外信号调节激酶ERK、c-Jun氨基末端激酶（JNK）和p38的激活促成了WI-38细胞中ET-1和凝血酶诱导的CTGF、α-SMA和波形蛋白表达。使用TargetScan Human、miRanda和PicTar预测算法来预测在CTGF mRNA的3'非翻译区（UTR）具有结合位点的miRNA。miR-19a、-19b和-26b是CTGF的候选miRNA。通过荧光素酶测定法，使用含有CTGF mRNA 3'-UTR的SV40启动子-IRES驱动的荧光素酶作为报告质粒，验证了候选miRNA与CTGF mRNA的3'-UTR的直接结合。ET-1和凝血酶降低了候选miRNA水平。候选miRNA过表达显著抑制WI-38细胞中ET-1和凝血酶诱导的CTGF表达，并降低α-SMA和波形蛋白表达。此外，在博来霉素诱导的肺纤维化小鼠的肺组织中，候选miRNA水平降低，气管内应用miR-19a、-19b和26b可减轻博来霉素诱导的肺纤维化严重程度。本研究首次证明MAPK激活与miR-19a、-19b和-26b表达降低之间的相互作用导致肺成纤维细胞分化。《细胞生理学杂志》231: 2236 - 2248，2016年。© 2016威利期刊公司
