Volume 22, Issue 6, Pages 1122-1133 (June 2014) The Antifibrotic Effects and Mechanisms of MicroRNA-26a Action in Idiopathic Pulmonary Fibrosis  Haihai Liang, Chaoqian Xu, Zhenwei Pan, Ying Zhang, Zhidan Xu, Yingzhun Chen, Tianyu Li, Xuelian Li, Ying Liu, Longtao Huangfu, Ying Lu, Zhihua Zhang, Baofeng Yang, Samuel Gitau, Yanjie Lu, Hongli Shan, Zhimin Du  Molecular Therapy  Volume 22, Issue 6, Pages 1122-1133 (June 2014) DOI: 10.1038/mt.2014.42 Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Downregulation of miR-26a in the lungs of mice with experimental pulmonary fibrosis and in IPF patients. (a) Hematoxylin–eosin (HE) and Masson Trichrome staining of mouse lung sections showing interstitial fibrosis with collagen deposition 28 days after injection of bleomycin (BLM) (10 × 20; original magnification ×200). (b) Increased hydroxyproline content in the lungs of mice treated with BLM compared with the content in lungs of control group mice. (c) Increased collagen (Col) I, Col III, matrix metalloproteinase (MMP)-2, and MMP-9 mRNA expression in the BLM-treated mice compared with expression in control animals, measured by real-time PCR. (d) Western blot analysis of proteins in the TGF-β signaling pathway in BLM-treated mice as compared with the same in saline animals. (e) Real-time PCR analysis of expression deregulation of miRs in the lungs of mice with experimental pulmonary fibrosis. (f) Downregulation of miR-26a in IPF patients. Both the PCR and protein assessments are shown after normalization compared with internal controls. Mean ± SEM; n = 5 mice in each group; *P < 0.05; **P < 0.01 versus saline or normal group. CTGF, connective tissue growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; miR, microRNA; PCR, polymerase chain reaction; TGF, transforming growth factor. Molecular Therapy 2014 22, 1122-1133DOI: (10.1038/mt.2014.42) Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Inhibition of miR-26a caused pulmonary fibrosis in mice. (a) Real-time PCR analysis of miR-26a expression in mice treated with antagomiR-26a as compared with saline group. (b) Hematoxylin–eosin (HE) and Masson Trichrome staining of mouse lung sections showing interstitial fibrosis with collagen deposition 21 days after intratracheal injection of antagomiR-26a (original magnification ×200). (c) Increased hydroxyproline content in mice treated with antagomiR-26a. (d) Real-time PCR quantification of relative levels of collagen I (Col I) and Col III mRNAs in antagomiR-26a-treated and saline-treated mice. (e) Western blot analysis of TGF-β1 and CTGF proteins in antagomiR-26a-treated mice as compared with control animals. (f) Real-time PCR analysis of expression of Col I and Col III mRNAs in MRC-5 cells. Both the PCR and protein assessments are shown after normalization compared with internal controls. Mean ± SEM; n = 4; *P < 0.05, **P < 0.01 versus saline or control group. CTGF, connective tissue growth factor; miR, microRNA; PCR, polymerase chain reaction; TGF, transforming growth factor. Molecular Therapy 2014 22, 1122-1133DOI: (10.1038/mt.2014.42) Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 miR-26a attenuates TGF-β1-induced fibrogenesis in cultured MRC-5 cells. miR-26a suppresses TGF-β1-induced increase in (a) collagen content, mRNA levels of (b) collagenase (Col) I and (c) Col III in cultured MRC-5 cells, whereas the CTGF mask alleviates this antifibrotic effect of miR-26a. n = 5, *P < 0.05, **P < 0.01 versus control, respectively; #P < 0.05 versus TGF-β1; &P < 0.05 versus TGF-β1+miR-26a. (d) 5-ethynyl-2′-deoxyuridine (EdU) fluorescence staining detects the newly synthesized DNA. (e) Western blot shows that miR-26a inhibited the TGF-β1-induced differentiation in MRC-5 cells. (f) Fluorescence-labeled smooth muscle α-actin (α-SMA) protein was visualized by fluorescence microscopy. miR-26a suppressed TGF-β1-induced expression of α-SMA in MRC-5 cells. α-SMA was stained in red and nuclei in blue (original magnification ×200). Both the PCR and protein assessments are shown after normalization compared with internal controls. n = 3, **P < 0.01 versus control, #P < 0.05 versus TGF-β1. SMA, smooth muscle α-actin. CTGF, connective tissue growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; miR, microRNA; PCR, polymerase chain reaction; TGF, transforming growth factor. Molecular Therapy 2014 22, 1122-1133DOI: (10.1038/mt.2014.42) Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 miR-26a posttranscriptionally regulates CTGF. (a) miR-26a reduced TGF-β1-induced upregulation of CTGF protein in MRC-5 cells. **P < 0.01 versus control, #P < 0.05 versus TGF-β1. (b) Sequence alignment showing miR-26a:CTGF complementarity for mouse, rat, and human genes. The matched base pairs are outlined by dashed red rectangles. The Genbank accession numbers of the genes are indicated in the brackets, and the positions of the target sites are numbered. (c) Compared with control, transfection of miR-26a with the luciferase reporter gene vector containing the wild-type 3′-untranslated region (UTR) of CTGF resulted in a significant decrease of luciferase activity. Coapplication of miR-26a with AMO alleviated the reduction of luciferase activity, whereas NC showed no effects. miR-26a has no effect on the mut-CTGF construct. (d) Compared with control, transfection of miR-26a resulted in a significant decrease of CTGF. Coapplication of miR-26a with AMO alleviated the reduction of CTGF, whereas NC showed no effects. (e) Real-time PCR shows that miR-26a had no effects on CTGF mRNA level. AMO: miR-26a inhibitor; NC: negative control. Both the PCR and protein assessments are shown after normalization compared with internal controls. Mean ± SEM; n = 4, represents four independent experiments under each condition; *P < 0.05, **P < 0.01 versus control; #P < 0.05 versus miR-26a; &P < 0.05 versus mut-CTGF. CTGF, connective tissue growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; miR, microRNA; PCR, polymerase chain reaction; TGF, transforming growth factor. Molecular Therapy 2014 22, 1122-1133DOI: (10.1038/mt.2014.42) Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 miR-26a prevents experimental pulmonary fibrosis in mice. (a) Schematic representation of protocol used to study the ability of miR-26a to prevent (subpart (A)) and to attenuate (subparts (B,C)) experimental pulmonary fibrosis. Mice were pretreated with agomiR-26a for 3 days followed by injection of bleomycin (BLM) for 28 days. miR-26a significantly alleviated (b) collagen deposition and (c) area of fibrosis induced by BLM, whereas agomiR-NC had no effects. miR-26a markedly decreased (d) hydroxyproline content and (e) mRNAs of collagenase (Col) I, Col III, matrix metalloproteinase (MMP)-2, and MMP-9 in the lungs of mice treated with BLM. (f) miR-26a inhibited BLM-induced upregulation of Col1a2, Smad4, and CTGF protein levels. Both the PCR and protein assessments are shown after normalization compared with internal controls. n = 3, *P < 0.05, **P < 0.01 versus saline; #P < 0.05 versus BLM. NC, negative control. CTGF, connective tissue growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; miR, microRNA; PCR, polymerase chain reaction; TGF, transforming growth factor. Molecular Therapy 2014 22, 1122-1133DOI: (10.1038/mt.2014.42) Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Enhancement of miR-26a expression attenuates experimental pulmonary fibrosis. Administration of agomiR-miR-26a after 7 or 14 days infusion of bleomycin (BLM) attenuated (a) collagen deposition and (b) area of fibrosis, whereas agomiR-NC had no effects. miR-26a markedly decreased (c) hydroxyproline content and (d) mRNAs of collagenase (Col) I, Col III, matrix metalloproteinase (MMP)-2, and MMP-9 mRNAs in the lungs of mice treated with BLM. (e) miR-26a inhibited BLM-induced upregulation of Col1a2, Smad4, and CTGF protein levels. Both the PCR and protein assessments are shown after normalization compared with internal controls. n = 3, *P < 0.05, **P < 0.01 versus saline; #P < 0.05, ##P < 0.01 versus BLM. NC, negative control. CTGF, connective tissue growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; miR, microRNA; PCR, polymerase chain reaction; TGF, transforming growth factor. Molecular Therapy 2014 22, 1122-1133DOI: (10.1038/mt.2014.42) Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 A positive feedback loop between miR-26a and Smad3 is involved in the lung fibrosis. (a) Real-time PCR confirmed the effect of Smad3 on expression of miR-26a. (b) Inhibition of Smad3 with sh-Smad3 induced downregulation of CTGF in MRC-5 cells. (c) Sequence alignment showing the miR-26a:Smad4 complementarity for mouse, rat, and human genes. The matched base pairs are marked with dashed red rectangles. The Genbank accession numbers of the genes are indicated in the brackets, and the positions of the target sites are numbered. (d) Compared with control, transfection of miR-26a with the luciferase reporter gene vector containing the wild-type 3′-UTR of Smad4 resulted in a significant decrease of luciferase activity. Coapplication of miR-26a with AMO alleviated the reduction of luciferase activity, whereas NC showed no effects. miR-26a has no effect on the mut-Smad4 construct. (e) Compared with control, transfection of miR-26a resulted in a significant decrease of Smad4. Coapplication of miR-26a with AMO alleviated the reduction of Smad4, whereas NC showed no effects. (f) Real-time PCR shows that miR-26a had no effects on the expression of Smad4 mRNA. n = 4, represents three independent experiments under each condition. (g) Western blot and (h) immunofluorescence cell staining (original magnification ×400) analysis of nuclear translocation of p-Smad3 in the MRC-5 cells transfected with miR-26a or AMO. Both the PCR and protein assessments are shown after normalization compared with internal controls. n = 3, *P < 0.05, **P < 0.01 versus control; #P < 0.05, ##P < 0.01 versus miR-26a; &P < 0.05 versus mut-Smad4. NC, negative control. AMO: miR-26a inhibitor; CTGF, connective tissue growth factor; DAPI, 4',6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; miR, microRNA; PCR, polymerase chain reaction; TGF, transforming growth factor. Molecular Therapy 2014 22, 1122-1133DOI: (10.1038/mt.2014.42) Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 8 Proposed model for the role of miR-26a in pulmonary fibrosis. In response to stimuli, the TGF-β1 signaling pathway is activated, which in turn downregulates expression of miR-26a by stimulating phosphorylation and nuclear translocation of Smad3 in lung fibroblasts. Reduction of miR-26a results in increase in CTGF, leading to production of collagens and pulmonary fibrosis. Moreover, miR-26a downregulation results in upregulation of Smad4, which increases nuclear translocation of p-Smad3 and further suppresses expression of miR-26a. Once the miR-26a/p-Smad3 loop is activated, it is perpetuated on its own and finally aggravates collagen deposition and other extracellular matrix proteins involved in fibrosis. CTGF, connective tissue growth factor; miR, microRNA; TGF, transforming growth factor. Molecular Therapy 2014 22, 1122-1133DOI: (10.1038/mt.2014.42) Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions