Transforming Growth Factor (TGF)-β1-induced miR-133a Inhibits Myofibroblast Differentiation and Pulmonary Fibrosis

Peng Wei  ^{1   2   3} , Yan Xie  ² , Peter W Abel  ² , Yapei Huang  ² , Qin Ma  ⁴ , Linghai Li  ⁵ , Junfeng Hao  ¹ , Dennis W Wolff  ⁶ , Taotao Wei  ^{7   8} , Yaping Tu  ⁹

• ¹ National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
• ² Department of Pharmacology and Neuroscience, Creighton University School of Medicine, Omaha, NE, 68178, USA.
• ³ University of Chinese Academy of Sciences, Beijing, 100049, China.
• ⁴ Key Laboratory of Genomics and Precision Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 100101, China.
• ⁵ Department of Anesthesiology, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing, 101149, China.
• ⁶ Kansas City University of Medicine and Biosciences-Joplin, Joplin, MO, 64804, USA.
• ⁷ National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China. weitt@moon.ibp.ac.cn.
• ⁸ University of Chinese Academy of Sciences, Beijing, 100049, China. weitt@moon.ibp.ac.cn.
• ⁹ Department of Pharmacology and Neuroscience, Creighton University School of Medicine, Omaha, NE, 68178, USA. Yat60399@creighton.edu.

Abstract

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 strategies to systematically study TGF-β1-induced alternations of miRNAs in human lung fibroblasts (HFL). Our data show that miR-133a was significantly upregulated by TGF-β1 in a time- and concentration-dependent manner. Surprisingly, miR-133a inhibits TGF-β1-induced myofibroblast differentiation whereas miR-133a inhibitor enhances TGF-β1-induced myofibroblast differentiation. Interestingly, quantitative proteomics analysis indicates that miR-133a attenuates myofibroblast differentiation via targeting multiple components of TGF-β1 profibrogenic pathways. Western blot analysis confirmed that miR-133a down-regulates TGF-β1-induced expression of classic myofibroblast differentiation markers such as ɑ-smooth muscle actin (ɑ-SMA), connective tissue growth factor (CTGF) and collagens. miRNA Target Searcher analysis and luciferase reporter assays indicate that TGF-β 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-β1-induced miR-133a as an anti-fibrotic factor. It functions as a feed-back negative regulator of TGF-β1 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.