All available data on TGF-β1-treated breast cancer cells or normal mammary gland cells from the Gene Expression Omnibus (GEO) database were investigated and an mRNA microarray GSE54491 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE54491) was identified. GSE54491 employed an Affymetrix mouse gene 1.0 ST array [transcript (gene) version] to identify mRNAs that were differentially expressed between TGF-β1-treated and TGF-β1-untreated normal murine mammary gland (NMumG) cells. MDA-MB-231 cells were selected for treatment with 10 ng/ml TGF-β1 to assess protein expression, according to previous studies (14,21). After 72 h, the expression of SMS1 and SMS2 was examined by western blot analysis.

MDA-MB-231, MCF-7 and BT549 cells were purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). These cells were cultured in Dulbecco's modified Eagle's medium (DMEM; HyClone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Tianjin Haoyang Biological Products Technology Co., Ltd., Tianjin, China) and incubated at 37°C in a humidified atmosphere (90% relative humidity) containing 5% CO₂. To choose a suitable breast cancer cell line and determine the optimal concentration of TGF-β1, western blotting was performed in order to detect the expression levels of SMS1 in the three breast cancer cells (MDA-MB-231, MCF-7 and BT549). Finally, MDA-MB-231 cells were selected and treated with different concentrations (0, 5, 10, 15 and 20 ng/ml) of TGF-β1 (cat. no. 10804-HNAC; Sino Biological, Inc., Beijing, China). After 72 h, the expression levels of SMS1, E-cadherin and vimentin (EMT markers) were investigated by western blotting.

To construct an SMS1 overexpression cell model, SMS1-overexpressing plasmids [pcDNA3.1(+)], which were constructed by Magus Technology (Shanghai, China), were transfected into MDA-MB-231 cells. Transfection was conducted according to the manufacturer's protocol. First, 12 h prior to transfection, 1×10⁶ cells were seeded into wells of a 6-well plate (Beaver Nano-Technologies Co., Ltd., Suzhou, China) that contained antibiotic-free medium. At the time of transfection, the cell confluency was 60–70% (22). The SMS1-overexpressing plasmid (4 µg; SMS1 group) or a negative plasmid (4 µg; Control group) was diluted with 50 µl DMEM (FBS-free and antibiotic-free medium) or 5 µl Entranster^™-D-4000 (Engreen Biosystem New Zealand Ltd., Auckland, New Zealand) and 50 µl DMEM. After 5 min, the dilutions were mixed together and incubated at 37°C for 20 min and subsequently dispensed into each well. DMEM was replaced with DMEM containing 10% FBS after 6 h (23). The cells were cultured for 24 h, and the Control and the SMS1 groups were treated with 10 ng/ml TGF-β1 at 37°C, as at that concentration, vimentin and E-cadherin expression had significantly altered. Therefore, the following four groups were created: Control, SMS1, TGF-β1 and SMS1+TGF-β1. After 72 h, all cells were harvested for subsequent experiments.

A total of 2×10⁵ MDA-MB-231 cells were seeded in 12-well tissue culture plates. After transfection and 10 ng/ml TGF-β1 treatment (36 h), the cells were maintained in serum-free medium at 37°C for 8 h. Using a sterile 200-µl pipette tip to gently swipe along the midline of the cell well, the cells were scraped from the well and were washed with PBS three times. Subsequently, the MDA-MB-231 cells were treated with TGF-β1 at the above concentration for 24 h. Finally, the wound closure was measured with a phase contrast inverted microscope (Olympus IX71; Olympus Corporation, Tokyo, Japan; magnification, ×4).

The cells were treated as above. Briefly, cells were starved for 8 h. Single-cell suspension (1×10⁵ cells) was added to serum-free medium with or without 10 ng/ml TGF-β1 on a top well of a 24-well Transwell plate (cat. no. 3413; Corning Incorporated, Corning, NY, USA) precoated with Matrigel (cat. no. 356234). After 36 h, the cells invading the lower chamber containing medium was supplemented with 10% FBS were stained with 0.1% crystal violet solution for 5 min at room temperature (CAS:548-62-9; Shanghai Macklin Biochemical Co., Ltd., Shanghai, China) and counted using a phase contrast inverted light microscope (Olympus IX71; Olympus Corporation, Tokyo, Japan; magnification, ×10).

Proteins were extracted using radioimmunoprecipitation assay buffer (cat. no. ROO20; Beijing Solarbio Bioscience & Technology Co., Ltd., Beijing, China), and the protein concentration was measured using a bicinchoninic acid assay (cat. no. CW0014; Beijing Kangwei Century Biotechnology Co., Ltd., Beijing, China). Equal amounts of cleared lysates (~50 µg protein) were separated by 10–12% SDS-PAGE and subsequently transferred onto polyvinylidene fluoride membranes (Immobilon-P; EMD Millipore, Billerica, MA, USA). Equal transfer was validated by staining with Ponceau red (cat. no. CW0057S; Beijing Kangwei Century Biotechnology Co., Ltd.) for 30 min at room temperature. The membranes were blocked with 10% skimmed milk or 10% bovine serum albumin (BSA; cat. no. A8020; Beijing Solarbio Science & Technology Co., Ltd.) in TBS for 1 h at room temperature and subsequently incubated with primary antibodies in TBS containing 0.05% Tween-20, 2% BSA and 0.05% sodium azide overnight at 4°C (24). The following antibodies were used at the indicated dilutions: SMS1 (cat. no. sc-133135; 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), vimentin (cat. no. 10366-1-AP; 1:2,000; ProteinTech Group, Inc., Chicago, IL, USA), E-cadherin (cat. no. 20874-1-AP; 1:2,000; ProteinTech Group, Inc.), phospho (p)-Smad2 (cat. no. AF8314; 1:2,000; Affinity Biosciences, Shanghai, China), Smad2 (cat. no. WL03369; 1:1,500; Wanleibio Co., Ltd., Shanghai, China) and TβR1 (cat. no. AF5347; 1:2,000; Affinity Biosciences), and a GAPDH antibody (cat. no. HRP-60004; 1:12,000; ProteinTech Group, Inc.). Subsequently, the membranes were incubated at room temperature for 1.5 h with secondary horseradish peroxidase-conjugated anti-rabbit antibodies (cat. no. SA0000I-2; 1:8,000; ProteinTech Group, Inc.) or anti-mouse antibodies (cat. no. SA0000I-I; 1:8,000; ProteinTech Group, Inc.) in TBS containing 0.05% Tween 20. Signals were determined using an enhanced chemiluminescence reagent (cat. no. CW0049M; Beijing Kangwei Century Biotechnology Co., Ltd.) and an autoradiography system (Image Lab; version 5.1; Bio-Rad Laboratories, Inc., Hercules, CA, USA) (14). Each assay was repeated at least three times.

A total of 2×10⁴ cells were seeded on coverslips in a 24-well plate. Followign the treatment, the cells were gently washed with PBS. Subsequently, they were fixed in 4% paraformaldehyde for 20 min at room temperature, followed by permeabilization using 0.5% Triton X-100 (cat. no. T8200; Beijing Solarbio Science & Technology Co., Ltd.) in PBS for 20 min at room temperature. The coverslips were subsequently blocked in 5% BSA in PBS for 60 min at room temperature. Subsequently, the cells were incubated with anti-TGF-βRI (cat. no. AF5347; 1:100; Affinity Biosciences), anti-vimentin (cat. no. 10366-1-AP; 1:200; ProteinTech Group, Inc.) and anti E-cadherin (cat. no. 20874-1-AP; 1:50; ProteinTech Group, Inc.) antibodies overnight at 4°C and subsequently washed in PBS. The cells were exposed to secondary antibody conjugated with fluorescein isothiocyanate (cat. no. BA1105; 1:50; Wuhan Boster Biological Technology, Ltd., Wuhan, China) or TRITC (cat. no. E032420-01; 1:250; Earthox Life Sciences Millbrae, CA, USA) for 60 min at room temperature. DAPI was used to stain the nuclei (cat. no. AR1177; Wuhan Boster Biological Technology, Ltd.) for 5 min at room temperature, and the cells were washed with PBS. Finally, an inverted fluorescence (Olympus IX71; Olympus Corporation; magnification, ×20) was used to acquire the data of vimentin and E-cadherin and a confocal microscope (Leica SP8; Leica Microsystems, Ltd., Milton Keynes, UK; magnification, ×40) was used to acquire the data of TGF-βRI. Images were analyzed by Image J software (Ver. 2.1; National Institutes of Health, Bethesda, MD, USA).

All data are presented as the mean ± standard deviation. Unpaired t-test was used for single comparisons. For multiple comparisons, one-way analysis of variance followed by Tukey's or Games-Howell post-hoc test was used. All experiments were repeated at least three times. P<0.05 was considered to indicate a statistically significant difference.

The GSE54491 dataset was downloaded from National Center for Biotechnology Information GEO DataSets. For the GSE54491 dataset, normal murine mammary gland (NMumG) cells transformed by overexpression of endothelial growth factor receptor (NME) cells were cultured in the presence of TGF-β1 (5 ng/ml) for 4 weeks, at which point TGF-β1 supplementation was discontinued and the cells were allowed to recover for an additional 4 weeks (Post-TGF-Rec). Total RNA was prepared from unstimulated cells (Pre-TGF) at similar passage numbers and compared by microarray analysis. In the present study, the two groups were analyzed in triplicate, including three Pre-TGF and three Post-TGF-Rec samples. The results showed that the expression of SMS1 decreased by 0.48-fold (P<0.001; n=3; Fig. 1A), whereas, the expression of SMS2 was not significantly different (P=0.731; n=3; Fig. 1B), in the Post-TGF-Rec group compared with the Pre-TGF group. However, TβRI (Fig. 1C) was increased by 0.32-fold (P<0.001; n=3). To validate the bioinformatics results, the expression of SMS1 and SMS2 in MDA-MB-231 cells following treatment with 10 ng/ml TGF-β1 was measured. The results confirmed that the expression of SMS1 was significantly decreased (Fig. 1D; P<0.05; n=3), whereas SMS2 was not significantly different between groups (Fig. 1D; P>0.05; n=3); these results were consistent with the bioinformatics data.

To examine whether the expression of SMS1 is different in breast cancer cells, the expression of SMS1 in three breast cancer cells, including MDA-MB-231, MCF-7 and BT549 was investigated. The results demonstrated that the expression of SMS1 was the lowest in MDA-MB-231, followed by MCF-7 and BT549 (Fig. 2A; P<0.05, n=3). Therefore, in the present study, MDA-MB-231 was selected.

MDA-MB-231 cells were cultured with different concentrations of TGF-β1 (0, 5, 10, 15 and 20 ng/ml) for 72 h and the expression of SMS1, E-cadherin and vimentin was examined. An increase in vimentin and a decrease in E-cadherin expression were evident when MDA-MB-231 cells were treated with 10 ng/ml TGF-β1 (P<0.05 and P<0.01, respectively; n=3; Fig. 2B-D, respectively). Similarly, the decrease in SMS1 expression was evident when MDA-MB-231 cells were treated with 10 ng/ml TGF-β1 (P<0.05 and P<0.01, respectively; n=3; Fig. 2E).

Protein expression levels of the EMT markers E-cadherin and vimentin were measured to evaluate the influence of the overexpression of SMS1 on the TGF-β1-induced EMT process (Fig. 3A; P<0.05 and P<0.01, respectively, n=3). Following treatment with TGF-β1, the expression of E-cadherin was decreased by 0.39-fold, and vimentin was increased by 2.3-fold compared with the control group (Fig. 3B; P<0.05 or P<0.01; n=3). In contrast, overexpression of SMS1 together with TGF-β1 treatment blocked TGF-β1-induced EMT, in which the expression of E-cadherin was decreased by 0.23-fold and vimentin was increased by 0.92-fold. The immunofluorescence and confocal microscopy imaging results verified these results to some extent (Fig. 3C and D).

Previous studies have demonstrated that the Smad-dependent signaling pathway is induced by TGF-β1, including the activation of TβRI and phosphorylated Smad2 and Smad3 (10,13). To further investigate the mechanism of SMS1 in regulating the TGF-β1 induced EMT process, the effect on Smad-dependent signaling pathway proteins was investigated. Western blot analysis revealed that TGF-β1 treatment induced the expression of TβRI and the phosphorylation of Smad2, which were increased by 0.77 and 0.43-fold, respectively. The expression of Smad2 demonstrated no significant difference (Fig. 4B; P>0.05, respectively; n=3). However, SMS1 overexpression downregulated TβRI expression and blocked Smad2 phosphorylation (Fig. 4A and B; P<0.05 and P<0.01, respectively, n=3). The expression of TβRI was investigated by immunofluorescence. The results demonstrated that the overexpression of SMS1 altered the expression of TβRI on the cell membrane, which may be associated with the endocytosis of TβRI (Fig. 4C) (16,17).

To further clarify the role of SMS1 in the TGF-β1-induced EMT in MDA-MB-231 cells, changes in the migration and invasion abilities of cells treated with or without TGF-β1 following transfection with an SMS1 plasmid, were investigated. Treatment with TGF-β1 significantly increased the cell migration abilities in MDA-MB-231 cells by 1.1-fold, as demonstrated by wound healing analysis. In addition, the increased migration abilities of MDA-MB-231 cells were significantly inhibited (by 0.32-fold) by transfection with an SMS1 plasmid (Fig. 5A and B; P<0.01; n=3). Furthermore, treatment with TGF-β1 for 72 h significantly increased the invasion abilities of MDA-MB-231 cells by 1.9-fold in a Matrigel invasion assay. Overexpression of SMS1 significantly suppressed the TGF-β1-induced invasion abilities of MDA-MB-231 cells by 0.47-fold (Fig. 5C and D; P<0.05 and P<0.01, respectively; n=3).