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.


| INTRODUC TI ON
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive inflammatory disease of the interstitial lung, and transformation of myofibroblast from fibroblast is a hallmark of IPF. 1 During the development of IPF, myofibroblast not only expresses α-smooth muscle actin (α-SMA), but also produces extracellular matrix (ECM) production, such as type I collagen, leading to the IPF. 2 Epithelial-to-mesenchymal transition (EMT) is highlighted as an important, possible mechanism of IPF. During EMT, the epithelial cells lose epithelial characteristics and acquired the mesenchymal phenotype with increased proliferative and migratory ability. [3][4][5] It is well known that transforming growth factor-beta1 (TGF-β1), which is a pro-fibrotic factor, has a pivotal role in inducing EMT. 6 Therefore, novel therapeutic strategies should be focused on regulating TGF-β1-mediated EMT for effective management for IPF.
Metastasis-associated protein 1 (MTA1) is a member of the MTA family (including MTA1, MTA2 and MTA3), which is served as the constituents of the Mi-2/nucleosome remodelling and deacetylase protein complex. 7 To date, MTA1 has been discovered to play indispensable roles in cell proliferation, tumorigenesis and metastasis, and it is known as a tumour inhibitor in many cancers. 8,9 However, the implication of MTA1 in IPF is undefined. Proteins of the MTA family have been identified as critical regulators of the EMT process, especially MTA1. 10,11 For instance, MTA1 enhances cell invasion and migration by inducing EMT in several types of cancers. [12][13][14] We, therefore, have been suggested that MTA1 may involve in TGF-β1-indued EMT and play a potential role during the development of IPF.
Based on the above analysis, this study aimed to determine the expression of MTA1 in IPF and to investigate the regulation of MTA1 on EMT in bleomycin-induced fibrosis in vivo, as well as TGF-β1-treated alveolar epithelial cells in vitro. On the other hand, we have previously demonstrated that astragaloside IV (ASV), the active substances of traditional Chinese medicinal plant astragalus membranaceus, inhibited TGF-β1-mediated EMT. 15 Therefore, we also explored the regulation of ASV on MTA1 expression, to provide a potentially effective agent that targeting MTA1 in IPF.

| Cell proliferation
Cell counting kit-8 assay (MedChemExpress) was performed to evaluate cell proliferation. RLE-6TN cells (5000 per well) were plated into 96-well plates. After transfected with sh-MTA1 or sh-Scram, the CCK-8 reagent was added to each well (dilution, 1:10) and cells were incubated for 2 hours at 37°C. The cell viability was measured at 450 nm using a microplate reader.

| Transwell assay
Transwell chambers (8 μm pore size membranes) were used for cell migration assay. After transfection, 3 × 10 5 RLE-6TN cells were plated on the top chamber and incubated in medium without FBS. The medium with 10% FBS was placed in the lower chamber. After 24 hours in the incubator at 37℃, non-migrated cells were gently removed and cells on the lower chamber were fixed with 0.5% crystal violet. The number of migrated cells was calculated under the Nikon Eclipse Ti microscopy.

| Real-time quantitative PCR
Total RNA from pulmonary tissues or cells was isolated by TRIzol (Invitrogen). cDNA was obtained from 2 μg of total RNA by reverse transcription by using PrimeScript reagent Kit (Takara). RT-qPCR was performed on a LightCycler96 Real-Time PCR System (Roche, Basel, Switzerland) with the SYBR Green PCR Master Mix (Takara). GAPDH was used as internal controls. The relative expression of target genes was calculated by the 2 −ΔΔCt method. 16 The primers were shown in Table 2.
Next membranes were incubated with horseradish peroxidase-conjugated secondary antibody (ab6721, Abcam) for 1 hour at 24°C. All blots were imaged using an enhanced chemiluminescence detection kit (Beyotime). β-actin (sc-70319, Santa Cruz) was considered as the internal control.

| Pulmonary fibrosis model
A total of 40 rats were randomly assigned to 4 groups: control group (received a single intratracheal instillation of 50 mL saline), IPF group (received a single intratracheal instillation of 50 mL saline containing 5 mg/kg bleomycin), sh-MTA1 group (MTA1 knockdown adenoassociated virus was intraperitoneally injected to knockdown MTA1) and sh-Scram group (adeno-associated virus containing Scrambled shRNA was intraperitoneally injected). All rats were sacrificed after treatment for 28 days, and the lung tissues were collected followed by fixation and embedding. Procedures involving animals and their care were conducted following a protocol approved by the Ethics Committee of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine.

| Histology and immunohistochemistry
The paraffin-embedded tissues were cut into 4-μm thick sections.
The sections stained with haematoxylin and eosin (H&E) staining and Masson's trichrome staining following the manufacturers' instructions. For immunohistochemistry, sodium citrate was used for antigen retrieval, and 0.3% hydrogen peroxide was used to block endogenous peroxidase. The slides were then incubated with the anti-MTA1 antibody (ab87275) at 1:300 dilution overnight at 4℃. The second day, sections were incubated with horseradish peroxidaseconjugated goat anti-rabbit IgG. After that, the slides were counterstained with haematoxylin, followed by dehydration in graded alcohols and xylene. Immunopositive cells were quantified using ImageJ software.

| Statistical analysis
Data analysis was performed using SPSS version 22.0 software (SPSS Inc). The Student's t test or one-way analysis of variance

| MTA1 is increased in bleomycin-induced fibrosis rats and TGF-β1-treated RLE-6TN cells
Firstly, H&E staining was used to validate that bleomycin could induce pulmonary fibrosis in our study. To determine the profile of MTA1 in IPF, immunohistochemistry was performed and the results in Figure 1A showed that compared with the control one, the expression levels of MTA1 were obviously up-regulated in the rats with pulmonary fibrosis. To exactly measure MTA1 levels, we performed Western blot and RT-qPCR analysis and the results revealed that MTA1 was significantly increased in bleomycin-induced fibrosis, both at protein levels ( Figure 1B and 1C) and mRNA levels ( Figure 1D). In addition, to investigate the change of MTA1 upon TGF-β treatment, as well as the cellular localization of MTA1 in alveolar cells, we treated RLE-6TN cells with 10 ng/mL TGF-β1. As displayed in Figure 1E, the addition of TGF-β1 increased MTA1 levels significantly in a time-dependent manner and the levels of MTA1 reached the peak at 48 hours following TGF-β1 treatment.
Immunofluorescence analysis then illustrated that MTA1 was mostly located in the nucleus of alveolar cells, and slightly in the cytoplasm.
Accordingly, TGF-β1 treatment increased the levels of α-SMA, which promote us to investigate the potential role of MTA1 in regulating EMT ( Figure 1F).

| Overexpression of MTA1 induces EMT of RLE-6TN cells
To evaluate the function of MTA1 in IPF, we firstly overexpressed MTA1 in vitro. As shown in Figure 2A, and α-SMA ( Figure 2F). Furthermore, the results of the CCK-8 assay showed that MTA1 overexpressing promoted cell viability ( Figure 2G) and Transwell assay showed that its overexpression facilitated cell migration ability ( Figure 2H).

| Inhibition of MTA1 reverses TGF-β1-induced EMT by regulating snail
To date, the mechanism underlying the regulation of MTA1 on EMT is unclear, we therefore further investigate the regulation of MTA1 on Snail expression, an important factor in regulating EMT. Luciferase reporter assay in our study showed that after overexpression of MTA1, the transcriptional activity of the Snail promoter was significantly up-regulated ( Figure 3A,B). A previous

| ASV suppresses MTA1 expression by regulating TGF-β1/smad3 signalling
As depicted in Figure 5A, a series of concentrations of ASV had no effects on MTA1 mRNA expression. However, ASV could inhibit MTA1 protein expression in a concentration-dependent manner in TGF-β1-treated RLE-6TN cells ( Figure 5B,C). We then explored how ASV influenced the expression of MTA1. As a previous study had shown that ASV effectively attenuated the progression of IPF by regulating TGF-β1/smad signalling, 15,17 we therefore tested whether ASV regulated MTA1 through TGF-β1 signalling. Interesting, the expression of MTA1, as well as the ratio of p-smad3/smad3, was suppressed in si-smad3 group when compared with that in si-NC group. Besides, knockdown of smad3 further decreased MTA1 expression in ASVtreated cells ( Figure 5D-F), indicating that TGF-β1/smad3 signalling may be involved in the regulation of ASV on MTA1 in alveolar cells.

| D ISCUSS I ON
In the present study, we characterized the pro-fibrotic property of MTA1 in IPF and elucidated the molecular mechanism underlying We demonstrated that MTA1 was significantly up-regulated in IPF. Although how TGF-β1/smad3 regulates MTA1 expression still needs to be clarified, the data in this study further a new potential molecular mechanism of the protective of ASV on IPF.
Overall, our study demonstrated that MTA1 inhibition could reverse the formation of the pulmonary EMT process by suppressing the expression of Snail. Thus, MTA1 inhibition represents a promising therapeutic target for patients with IPF that deserves further study and evaluation. Project of Shandong Province.

CO N FLI C T S O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

AUTH O R CO NTR I B UTI O N S
XC and QQ designed the study and performed the statistical analy-