MCF-7 is a widely studied epithelial cancer cell line derived from breast adenocarcinoma, with the characteristics of differentiated mammary epithelium. MCF-7 cells express estrogen receptor-α (ER-α), as well as androgen, progesterone and glucocorticoid receptors, which make them valuable tools in medical research. Although MCF-7 cells are easy to propagate, they are generally a slow-growing population, with a doubling time of 30–40 h. MDA-MB-231 is a highly aggressive, invasive and poorly differentiated triple-negative breast cancer (TNBC) cell line. Similar to other invasive cancer cell lines, the invasiveness of the MDA-MB-231 cells is mediated by proteolytic degradation of the extracellular matrix. In a 3D culture, this cell line displays endothelial-like morphology and is distinguished by the stellate projections that often bridge multiple cell colonies.

MCF-7 and MDA-MB-231 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were grown in 25-cm² cell culture flasks with RPMI-1640 medium (HyClone; GE Healthcare Life Sciences) or L15 (HyClone; GE Healthcare Life Sciences) supplemented with 10% fetal bovine serum (FBS; HyClone; GE Healthcare Life Sciences), 2 mM L-glutamate, 100 U/ml penicillin G, and 100 U/ml streptomycin at 37°C in 5% CO₂ and 95% air. The cells were then seeded into 6- or 24-well culture plates (Corning, Inc.) with the cell confluence of ~70% prior to transfection.

According to the design principle of mammalian eukaryotic cell RNA interference, shRNA sequences were designed for the different targets of mucin-like protein 1. Each interference sequence was synthesized into a single chain of sense and antisense, and a double chain was formed after annealing. The primers were as follows: 5′-CCGGGTGTGTCCCTGAGATGGAATCCTCGAGGATTCCATCTCAGGGACACACTTTTTG-3′ (sense) and 5′-AATTCAAAAAGTGTGTCCCTGAGATGGAATCCTCGAGGATTCCATCTCAGGGACACAC-3′ (antisense).

The two chains were annealed into double chains, connected by T4DNA ligase and the AgeI/EcoRI double enzyme cutting carrier PLKO1 (Fig. 1), converted by DH5a competent cells, and single clones were selected. The contrast scramble plasmid was provided by Genesent, and its hairpin structure was as follows: 5′-CCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGG-3.

SBEM cDNA was provided by Genesent, and the lentivirus overexpression vector was pCDH-CMV-MCS-EF1-Puro. The plasmid map is shown in the Fig. 2. The cloning primers were as follows: 5′-TAGAGCTAGCGAATTATGAAGTTCTTAGCAGTCC-3′ (sense) and 5′-AGATCCTTCGCGGCCTCAGGGACACACTCTACCA-3′ (antisense).

MCF-7 and MDA-MB-231 cells with stable SBEM knockdown or overexpression were generated by lentiviral vectors carrying either sh-SBEM, a SBEM overexpression construct, or the respective negative controls (Genesent), in accordance with the manufacturer's instructions. Cells were placed into 60-mm dishes at 3×10⁵/dish and allowed to grow overnight. Lentivirus expression plasmid (8 µg) was mixed with 20 µl Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) and transfected into the cells according to the manufacturer's instructions. Twenty-four hours after transfection, the cells were trypsinized, diluted, and placed into 96-well plates. Transfected cells were then selected with 2 µg/ml puromycin and 200 µg/ml G418. Western blotting was conducted to detect the knockdown and overexpression effects of SBEM.

To determine the regeneration and repair abilities of breast cancer cells, 4×10⁵ MCF-7 and MDA-MB-231 cells were seeded in 6-well plates and incubated at 37°C with 5% CO₂ overnight. Artificial wounds were created using a 10-µl pipette tip (0 h) to generate a gap in the confluent cell layer with confluence percentage of ~80%. The cells were washed with PBS twice and incubated with serum-free medium at 37°C with 5% CO₂ as a control. At 0 and 8 h, phase-contrast images were captured using a microscope at a magnification of ×400 (CX41-32C02PH, Olympus Corporation).

The detailed procedures of cell migration and invasion assays were conducted as previously reported (13). Briefly, 3×10⁵ cells were suspended in serum-free medium and seeded into the upper layer of Transwell membrane with an 8-µm pore size in a 24-well plate (Corning, Inc.). The membranes were coated with Matrigel (1:8; BD Biosciences) for invasion assays, or left uncoated for migration assays. Medium containing 10% FBS was placed in the bottom chamber as an attractant. After 24 h, the cells were fixed in 4% paraformaldehyde for 15 min at room temperature and stained with 0.1% crystal violet solution for 15 min. The invading cells were then examined and counted in 10 randomly selected fields under a light microscope at a magnification of ×400. The mean number of invading cells was then calculated.

Total RNA of 5×10⁶ MCF-7 cells was extracted using the Trizol RNA extraction protocol (cat. no. 10606ES60, Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Reverse transcription of mRNA to cDNA was performed in 20 µl reaction volumes with random priming using an RT-PCR Kit (cat. no. RR047A, Takara Biomedical Technology Co., Ltd.). The sequences of primers used in this study are listed in Table I, and the real-time PCR reaction system is provided in Table II. The primers were synthesized by Beijing AuGCT Biotech Co. Ltd. Quantitative PCR was performed using SYBR Green PCR Kit (cat. no. RR820A, Takara Biomedical Technology Co. Ltd.) and Fast Real-Time PCR System (ABI 7900HT, Applied Biosystems; Thermo Fisher Scientific, Inc.). The detailed process was similar to that previously described (4). The increase of fluorescence was detected due to the exponential accumulation of PCR products and the 2^−ΔΔCq method was used to calculate the relative quantity of gene expression in each sample (14).

The proteins were quantified using the BCA method. An amount of 10 µl protein plus 10 µl PBS were loaded and 200 µl working liquid was added in 96-well plates. The absorbance value at 560 nm was measured by enzyme-labeling instrument. The concentration of proteins was calculated on the basis of the standard curve. For western blotting, 1×10⁶ cells were plated in 100-mm Petri dishes for 24 h. The cells were then washed with cold PBS and lysed with 200 µl of cold lysis buffer [150 mmol/l NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 50 mmol/l Tris-HCl (pH 7.2), 0.2 mmol/l sodium vanadate, 1% phenylmethylsulfonyl fluoride and 0.2% aprotinin].

The samples were kept on ice for 20 min and then spun at 12,000 × g at 4°C for 20 min, and the protein concentration of the supernatant was determined. Cell lysates were fractionated on 10% SDS-PAGE, and protein was transferred onto nitrocellulose membranes (Pall Life Sciences). The membranes were blocked with 5% skimmed milk powder dissolved in TBST at room temperature for 2 h. The membranes were then probed with primary antibodies against SBEM (dilution 1:1,000, cat. no. HPA-039093; Sigma-Aldrich; Merck KGaA), N-cadherin (dilution 1:500, cat. no. 13116S; Cell Signaling Technology, Inc.), E-cadherin (dilution 1:500, cat. no. 3195S; Cell Signaling Technology, Inc.), Twist (dilution 1:500, cat. no. 69366S; Cell Signaling Technology, Inc.), vimentin (dilution 1:500, cat. no. 5741S; Cell Signaling Technology, Inc.) and claudin-1 (dilution 1:500, cat. no. 13995S; Cell Signaling Technology, Inc.). The expression of GAPDH as control was determined using anti-GAPDH (dilution 1:500, cat. no. 5174S; Cell Signaling Technology, Inc.). After hybridization at 4°C overnight, HRP-labeled IgG (dilution 1:500, cat. no. 074S; Cell Signaling Technology, Inc.) was added and incubated at 37°C for 2 h. The membrane was washed with TBST (0.1% Tween-20) for 3 times, and ECL chemiluminescence system (Applygen Technologies Inc.) was used for coloration. The immunoreactive bands were detected using an Odyssey Infrared Imaging System (Gene Company Ltd.). The intensity of each band was measured with Odyssey 3.0 software (Li Cor Inc.).

All data are presented as mean ± standard deviation. Statistical analysis was performed using Statistical Package for the Social Sciences version 14.0 (SPSS, Inc.). All statistical tests were two-sided. The comparison between two groups of samples adopted the t-test, and P-value ≤0.05 was considered to indicate a statistically significant difference.

In accordance with the manufacturer's instructions, MCF-7 and MDA-MB-231 cells with stable SBEM knockdown or overexpression were generated by lentiviral vectors carrying sh-SBEM, a SBEM overexpression construct, or the negative controls, respectively. The microscopic observations of cell morphology are presented in Figs. 3 and 4. MCF-7 cells displayed typical epithelioid characteristics. They were small and polygonal in shape. MDA-MB-231 cells are mostly spindle-like with narrow strip in shape, and some of the cells adopted a more rounded morphology.

In order to test and verify the knockdown and overexpression of the SBEM gene, western blotting was used to detect SBEM protein expression in MCF-7 and MDA-MB-231 cells. Compared with the scramble group, SBEM protein expression decreased significantly after cells were transfected with sh-SBEM. Compared with the vector group, SBEM protein expression was increased significantly after the cells were transfected with the overexpression plasmid. The difference was statistically significant in both MCF-7 and MDA-MB-231 cells (P<0.05). Therefore, MCF-7 and MDA-MB-231 cell lines with stable SBEM knockdown or overexpression were successfully established (Fig. 5).

Wound healing was observed after 8 h. The results of the MCF-7 cells demonstrated that the cell repair rate in sh-SBEM group was (6.8%) much lower than that in the scramble group (39.6%). The cell repair rate in the SBEM overexpression group was higher (48.1%) than that in the vector group (27.0%) (Fig. 6 and Table III). The results of the MDA-MB-231 cells demonstrated that the cell repair rate in the sh-SBEM group (4.8%) was markedly lower compared with that in the scramble group (31.0%). The cell repair rate in the SBEM overexpression group was higher (69.9%) compared with that in the vector group (26.2%) (Fig. 7, Table III). These results indicated that SBEM knockdown obviously inhibited and SBEM overexpression obviously promoted the migration ability of the breast cancer cells.

The numbers of cells crossing the basement membrane were recorded for 24 h. In the MCF-7 cells, the cell numbers were 20±1.15 in the sh-SBEM group, and 75.6±2.01 in the scramble group (P<0.05); the cell numbers were 81.2±1.47 in the SBEM-overexpressing group, and 25.5±1.08 in the vector group. The difference between the two groups was statistically significant (P<0.05; Fig. 8). In MDA-MB-231 cells, the cell numbers were 48.5±1.35 in sh-SBEM group, and 161.7±1.25 in the scramble group; the cell numbers were 241.7±1.15 in the SBEM-overexpressing group, and 118±1.63 in the vector group. The difference between the two groups was statistically significant (P<0.05; Fig. 9). The results demonstrated that SBEM knockdown obviously inhibited and SBEM overexpression obviously promoted the invasion ability of breast cancer cells.

In order to confirm the association of the SBEM gene with the process of EMT, the real-time PCR and western blotting were used to detect the expression of EMT-related markers and regulators in SBEM-overexpressing MCF-7 cells. The results revealed that the levels of N-cadherin, Twist and vimentin were elevated, while those of E-cadherin and claudin-1 were decreased following SBEM overexpression. The relative mRNA expression levels, protein bands and relative band intensities are presented in Figs. 10 and 11.