Metastasis-associated protein 1 promotes epithelial-mesenchymal transition in idiopathic pulmonary fibrosis by up-regulating Snail expression

Abstract

Idiopathic pulmonary fibrosis (IPF) is a progressive and usually fatal lung disease that lacking effective interventions. It is well known that aberrant activation of transforming growth factor-beta1 (TGF-β1) frequently promotes epithelial-mesenchymal transition (EMT) in IPF. Metastasis-associated gene 1 (MTA1) has identified as an oncogene in several human tumours, and aberrant MTA1 expression has been related to the EMT regulation. However, its expression and function in IPF remain largely unexplored. Using a combination of in vitro and in vivo studies, we found that MTA1 was significantly up-regulated in bleomycin-induced fibrosis rats and TGF-β1-treated alveolar type Ⅱ epithelial (RLE-6TN) cells. Overexpression of MTA1 induced EMT of RLE-6TN cells, as well as facilitates cell proliferation and migration. In contrast, knockdown of MTA1 reversed TGF-β1-induced EMT of RLE-6TN cells. The pro-fibrotic action of MTA1 was mediated by increasing Snail expression through up-regulating Snail promoter activity. Moreover, inhibition of MTA1 effectively attenuated bleomycin-induced fibrosis in rats. Additionally, we preliminarily found astragaloside IV (ASV), which was previously validated having inhibitory effects on TGF-β1-induced EMT, could inhibit MTA1 expression in TGF-β1-treated RLE-6TN cells. These findings highlight the role of MTA1 in TGF-β1-mediated EMT that offer novel strategies for the prevention and treatment of IPF.

Keywords: astragaloside IV; epithelial-mesenchymal transition; idiopathic pulmonary fibrosis; metastasis-associated protein 1; snail.

Figure 1

MTA1 is up‐regulated in bleomycin‐induced rats and TGF‐β1‐treated RLE‐6TN cells. A, Haematoxylin and eosin (H&E) staining was used to show the histologic change in bleomycin‐induced IPF; the expression of MTA1 was analysed by immunohistochemistry, and positive rate of MTA1 was presented as a histogram. B‐C, Expression of MTA1 protein in pulmonary tissues from control or bleomycin‐induced rats was analysed by Western blot analysis. D, RT‐qPCR was performed to detect the expression of MTA1 mRNA in pulmonary tissues. E, Western blot analysis was used to determine the change of MTA1 expression in RLE‐6TN cells upon 10 ng/mL TGF‐β1 treatment for various time. F, Immunofluorescence was used to detect the expression of α‐SMA and MTA1 in RLE‐6TN cells with or without 10 ng/mL TGF‐β1 treatment for 48 h. *P < .01 vs control

Figure 2

Overexpression of MTA1 results in fibrogenesis in RLE‐6TN cells. A‐B, Overexpression of MTA1 was achieved by transfected with pcDNA3.1 carrying MTA1 gene, and the results were validated by RT‐qPCR and Western blot analysis. C‐E, Relative expression of Col 1α, Col 3α and CTGF in RLE‐6TN cells with or without overexpression of MTA1, as detected by RT‐qPCR. F, Relative expression of Snail, collagen1 and α‐SMA in RLE‐6TN cells with or without overexpression of MTA1, as detected by Western blot analysis. G, Cell proliferation of RLE‐6TN cells was evaluated by CCK‐8 assays after overexpression of MTA1. H, Cell migration was evaluated by Transwell assay after overexpression of MTA1. *P < .05 vs. pcDNA3.1

Figure 3

MTA1 regulates EMT of RLE‐6TN cells by targeting snail. A, The schematic diagrams of pGL3‐snail used in a luciferase reporter assay. B, Overexpression of MTA1 via plasmid transfection resulted in significant up‐regulation of snail promoter activity in RLE‐6TN cells. C, Relative expression of MTA2 and MTA3 protein in RLE‐6TN cells transfected with pcDNA3.1 or MTA1 under TGF‐β1 treatment. D, Relative expression of MTA1, snail and EMT‐related proteins, including collagen1 and α‐SMA, was determined in RLE‐6TN cells with MTA1 overexpression and/or snail silencing. * P < .05 vs control; **P < .05 vs TGF‐β1 + pcDNA3.1; ^# P < .05 vs MTA1 + si‐NC; ^## P < .05 vs pcDNA3.1

Figure 4

Inhibition of MTA1 or astragaloside IV (ASV) treatment reverses TGF‐β1‐induced EMT. A, Relative expression of MTA1 protein in RLE‐6TN cells transfected with sh‐MTA1 or control vector (sh‐Scram). B‐E, Relative expression of EMT‐related proteins, including snail, collagen1 and α‐SMA, was determined in RLE‐6TN cells with MTA1 silencing or ASV treatment. F, CCK‐8 assay was performed to assess the cell proliferation of RLE‐6TN cells transfected with sh‐MTA1, sh‐Scram or ASV treatment. G, Transwell assay was performed to detect cell migration of RLE‐6TN cells with MTA1 silencing or ASV treatment. *P < .05 vs control; ^# P < .05 vs TGF‐β + sh‐Scram

Figure 5

Astragaloside IV (ASV) suppresses MTA1 expression via TGF‐β1/smad3 signalling. A, Relative expression of MTA1 mRNA in RLE‐6TN cells with different concentrations of ASV, as detected by RT‐qPCR. B‐C, Relative expression of MTA1 in TGF‐β1‐treated RLE‐6TN cells with different concentrations of ASV, as detected by Western blot analysis. D‐F, Relative expression of MTA1, p‐smad3 and smad3 protein in TGF‐β1 or ASV‐treated RLE‐6TN cells with or without knockdown of smad3. *P < .05 vs control; ^# P < .05 vs TGF‐β1; ^# P < .05 vs TGF‐β1 + si‐NC; ^$ P < .05 vs TGF‐β1 + si‐NC + ASV

Figure 6

Inhibition of MTA1 attenuates bleomycin‐induced IPF in rats. A‐B, Haematoxylin and eosin (H&E) and Masson staining were performed to show the histologic change and alteration of collagen accumulation in IPF rats with or without MTA1 silencing. (A and C) Immunohistochemistry showed the change of the positive rate of α‐SMA in IPF rats with or without MTA1 silencing. D‐F, Relative expression of Col 1α, Col 3α and CTGF in pulmonary tissues with or without MTA1 silencing was detected by RT‐qPCR. G, Relative expression of Snail, collagen1 and α‐SMA in pulmonary tissues with or without MTA1 silencing was detected by Western blot analysis. *P < .05 vs control; ^# P < .05 vs IPF + sh‐Scram