Human BC cell lines (MCF-7, MDA-MB-468, LCC9 and T-47D) and the normal human breast MCF-10A cell line were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences. All cells were maintained in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.), supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin and streptomycin (Sigma-Aldrich; Merck KGaA) in a humidified atmosphere of 5% CO₂ at 37°C. All chemicals were purchased from Sigma-Aldrich (Merck KGaA).

The lentivirus plasmids containing the short hairpin RNA (shRNA) against FNDC1 (shFNDC1) or a negative control (shNC) were synthesized by Suzhou GenePharma Co., Ltd. shRNAs were subcloned into pRNA-H1.1 (Thermo Fisher Scientific, Inc.). The plasmids were transfected into 293T cells using Lipofectamine^® 2000 (Thermo Fisher Scientific, Inc.) according to the manufacturer's guide. Briefly, 24 h prior to the transfection, 2.5×10⁶ cells were seeded into a 10 cm dish until confluency of 50–70% the next day. A total of 4 µg lentiviral plasmid, 4 µg each 3rd generation viral packaging vectors (pMDL, pRSV and pVSV-G) and 20 µl Lipofectamine 2000 in 600 µl serum-free DMEM was incubated for 15–20 min at room temperature and added into the cells. A total of 72 h later, the supernatant was collected. Prior to the viral transduction, cells were harvested upon reaching 70–80% confluence. Lentiviral vectors were added at an MOI of 50 into the plate. The medium was replaced with fresh medium 24 h after transfection. Then, cells were treated with puromycin (2 µg/ml) for 3 days and the transfection efficiency was evaluated by western blotting.

Cell viability was assayed by the Cell Counting Kit-8 (CCK-8) (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. In brief, the cells were seeded onto 96-well plates at the density of 5×10³ cells/well. After infections for different times (24, 48, 72 and 96 h), 10 µl CCK-8 solution was added to each well and incubated for another 2 h. The absorbance was measured at 450 nm by a microplate reader Elx808 (BioTek Instruments, Inc.).

Following transfection for 24 h, the cells were seeded into the 6-well plates at a density of 500 cells/well in triplicate. After culturing for two weeks, the cells were washed with PBS, fixed with methanol for 0.5 h at room temperature and stained with crystal violet for 2 h at room temperature (0.1%; Beyotime Institute of Biotechnology). The colonies were visualized and counted under a CKX31 inverted light microscope at ×100 magnification (Olympus Corporation).

A wound healing assay was performed with minor modifications to a previously reported method (9). As serum-free medium caused excessive apoptosis and cell detachment, 10% FBS-supplemented medium was used (9,10). In brief, cells were seeded onto 6-well plates in 10% FBS medium until they reached 80–90% confluency. The monolayers were scratched using 200 µl sterile pipette tips, and the cells were washed with PBS three times to remove the debris and 10% FBS-supplemented fresh medium was added. A total of 24 h later, images were captured under a CKX31 inverted light microscope at ×100 magnification (Olympus Corporation).

For the cell invasion assay, 3×10⁴ cells were seeded into the upper chamber of Transwell assay inserts (8 µm; Corning Inc.). The Transwell chamber was precoated with Matrigel (Thermo Fisher Scientific, Inc.) before cell seeding. The chambers were inserted into a 24-well plate. The upper chambers were filled with 200 µl serum-free medium (RPMI-1640; Gibco; Thermo Fisher Scientific, Inc.), while the bottom chambers were filled with 500 µl complete medium (RPMI-1640; Gibco; Thermo Fisher Scientific, Inc.). After incubation at 37°C for 24 h, the cells were fixed at room temperature in 4% paraformaldehyde (Beyotime Institute of Biotechnology) for 10 min and stained at room temperature with 0.1% crystal violet (Beyotime Institute of Biotechnology) for 10 min. Nonmigrating cells in the upper chambers were wiped off. The migrated cells were counted in three randomly selected fields and photographed at a ×100 magnification with a CKX31 inverted light microscope at ×100 magnification (Olympus Corporation).

The total cellular proteins were obtained using RIPA lysis buffer (Beyotime Institute of Biotechnology), supplemented with a proteinase and phosphatase inhibitor cocktail (Sigma-Aldrich; Merck KGaA). The protein concentration was assayed by Bradford kit (Beyotime Institute of Biotechnology) according to the manufacturer's guide. Twenty micrograms of total protein were loaded onto a 12% SDS-PAGE and then transferred onto a PVDF membrane (EMD Millipore). The membranes were blocked with skimmed milk for 1 h at room temperature, after which the membrane was incubated with primary antibodies against the following overnight at 4°C: FNDC1 (cat. no. PA5-56603; dilution, 1:1,000; Thermo Fisher Scientific, Inc.), GAPDH (cat. no. G9545; dilution, 1:10,000, Sigma-Aldrich; Merck KGaA), MMP-2 (cat. no. 40994; dilution, 1:1,000; Cell Signaling Technology, Inc.), MMP-9 (cat. no. 13667; dilution, 1:1,000; Cell Signaling Technology, Inc.), vimentin (cat. no. 5741; dilution, 1:1,000; Cell Signaling Technology, Inc.), N-cadherin (cat. no. 13116; dilution, 1:1,000; Cell Signaling Technology, Inc.), E-cadherin (cat. no. 14472; dilution, 1:1,000; Cell Signaling Technology, Inc.), p-ERK (cat. no. 4370; dilution, 1:1,000; Cell Signaling Technology, Inc.), ERK (cat. no. 4696; dilution, 1:1,000; Cell Signaling Technology, Inc.), p-JNK (cat. no. 9255; dilution, 1:1,000; Cell Signaling Technology, Inc.), JNK (cat. no. 9252; dilution, 1:1,000; Cell Signaling Technology, Inc.), p-p38 (cat. no. 4511; dilution, 1:1,000; Cell Signaling Technology, Inc.), p38 (cat. no. 8690; dilution, 1:1,000; Cell Signaling Technology, Inc.), p-Akt (cat. no. 4060; dilution, 1:1,000; Cell Signaling Technology, Inc.) and Akt (cat. no. 4685; dilution, 1:1,000; Cell Signaling Technology, Inc.). Subsequently, the membrane was washed three times with PBS and incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies (both 1:2,000; anti-mouse secondary antibody; cat. no. 7076; and anti-rabbit secondary antibody; cat. no., 7074), which were obtained from Cell Signaling Technology, Inc. at room temperature for 1 h. The membrane was visualized using an ECL Prime Western Blotting kit (cat. no. 21342; Beyotime Institute of Biotechnology). All western blots were repeated three times. Band intensities were quantified using NIH ImageJ (version 2.0; imgaej.niv.gov/).

The bioinformatics data for FNDC1 expression in BC were retrieved from The Cancer Genome Atlas (TCGA) (portal.gdc.cancer.gov) and the Genotype-Tissue Expression (GETx) database (gtexportal.org/home/) using the Gene Expression Profiling Interactive Analysis (GEPIA) tool ver.2002 (gepia.cancer-pku.cn). The associations of FNDC1 expression with overall survival (OS) and disease-free survival (DFS) were analyzed by data mining in the Kaplan-Meier plotter (kmplot.com). The median FNDC1 expression was applied as the cut-off. Hazard ratios with a 95% confidence interval (CI) and log-rank P-values were calculated.

Statistical analyses were performed with SPSS 12.0 (SPSS, Inc.). The data are expressed as the mean ± standard deviation. The paired Student's t-test was used for comparisons between BC and normal tissues. In in vitro experiments, unpaired Student's t-test was used to compare the difference between two groups, and differences between multiple groups were analyzed using one-way analysis of variance followed by Tukey's post hoc test. Survival probability was calculated using the Kaplan-Meier method and the log-rank test was used to compare survival curves. P<0.05 (two-tailed) was considered to indicate a statistically significant difference.

According to the data extracted from TCGA database, the expression of mRNA levels of FNDC1 was significantly upregulated in BC tissues when compared with normal tissues that were collected from the same patients (Student's t-test; Fig. 1A), although there was no significant difference in expression of FNDC1 among the different BC stages (Fig. 1B). The OS curve, which was constructed using the Kaplan-Meier method from TCGA database, demonstrated that there is no significant difference between the BC patients with high or low expression of FNDC1 (after dividing the patients' survival times by the median time, with a follow-up threshold of 250 months; P=0.23; log-rank test; Fig. 1C). Furthermore, DFS curves did not show an association between the survival of patients with BC and the expression of FNDC1 (after dividing the patients' survival time by the median time, with a follow-up threshold of 250 months; P=0.61; log-rank test; Fig. 1C). These findings unveil the complexity of FNDC1 in the tumorigenesis of BC.

The biological functions of FNDC1 in BC cells were investigated. The protein levels of FNDC1 in normal human breast MCF-10A cells and four breast cancer LCC9, MCF-7, MDA-468 and T-47D cell lines were assayed. As shown in Fig. 2A, the expression of FNDC1 was highest in MCF-7 and MDA-468 cells; therefore, these two cell lines were selected for the subsequent studies. Subsequently, the expression of FNDC1 was successfully silenced in two BC cell lines (MCF-7 and MDA-MB-468; Fig. 2B). A CCK-8 assay demonstrated that the silencing of FNDC1 significantly inhibited the viability of BC cells, compared with to the control group (Fig. 2C). Colony formation assays indicated that the colony numbers of BC cells were markedly decreased following knockdown of FNDC1 (Fig. 2D). Wound healing and Transwell invasion assays demonstrated that the silencing of FNDC1 significantly repressed the migration and invasion of BC cells (Fig. 2E and F). To reveal the molecular mechanisms, western blot analysis was performed. It was revealed that the silencing of FNDC1 led to the downregulation of MMP-2/9, vimentin and N-cadherin and the upregulation of E-cadherin in BC cells (Fig. 2G). Taken together, these results suggested that the silencing of FNDC1 inhibits the tumorigenesis of BC cells.

Whether FNDC1 has any effects on signaling pathways was investigated. To begin with, the status of the MAPK signaling pathways was investigated. It was demonstrated that ERK, JNK and p38 were not affected following the silencing of FNDC1 in BC cells (Fig. 3A). Subsequently, the PI3K/Akt signaling pathway was investigated. It was found that the phosphorylation levels of PI3K and Akt were decreased following the silencing of FNDC1 (Fig. 3B). Therefore, the silencing of FNDC1 led to the inhibition of PI3K.

To verify whether the PI3K/Akt signaling pathway is required for the oncogenic effects of FNDC1, BC cells were transfected with shFNDC1 and then the cells were incubated with the Akt activator SC79 24 h after transfection. It was revealed that SC79 reactivated the Akt signaling pathway following the silencing of FNDC1 in the two BC cell lines, as indicated by increased p-Akt/Akt ratios (Fig. 4A). In addition, it was found that SC79 reversed the effects of the silencing of FNDC1 on cell viability (Fig. 4B), migration (Fig. 4C) and invasion (Fig. 4D). Furthermore, the effects of the silencing of FNDC1 on MMPs and EMT markers were also reversed by the treatment of SC97 in BC cells (Fig. 4E). Taken together, these results suggested that the effects of the silencing of FNDC1 on the tumorigenesis of BC cells relied on the inactivation of the PI3K/Akt signaling pathway.