Bioinformatics analysis
The differential gene expression in tumor and normal tissues and the correlation between SMC1A, FOXM1 of patients with BC were analyzed by GEPIA (http://gepia.cancer-pku.cn/) tool [18]. The protein expression of SMC1A was obtained from CPTAC (Clinical Proteomic Tumor Analysis Consortium) and The Human Protein Atlas (https://www.proteinatlas.org/). The genes related SMC1A from LinkedOmics (http://www.linkedomics.org) [19]. Gene set enrichment analysis (GSEA) tool was used to conduct KEGG pathways. Kaplan-Meier Plotter (http://kmplot.com/analysis/index.php) was used to assess the prognosis of patients with BC.
Cell lines and cultures
Two human BC cell lines (MDA-MB-231and MDA-MB-468) were purchased from the Chinese Academy of Sciences Shanghai Institute of Life Sciences. All cells were cultured at 37°C in a humidified atmosphere of 5% CO2 in the appropriate medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) solution.
Plasmid constructions and Lentiviral Production
The lentiviral vector (pLKO.1-TRC Cloning Vector) carrying shRNA targeting human SMC1A was obtained from Sigma Aldrich (shRNA#1, sense, 5'- CGACAGATTATCGGACCATTT-3', shRNA#2, sense, 5'- GCCGGGACTGTATTCAGTATA-3'). The cDNA of FOXM1 was cloned to pCDNA3.1 vector and pCDNA3.1 vector was used as a control. All plasmids were identified by sequencing. Lentivirus was produced in HEK-293T cells and harvested at 48 h after transfection. Lentiviral particles with shRNAs or Negative Control (SCR) were added into MDA-MB-231and MDA-MB-468 cells, the medium with lentivirus particles was replaced with refresh growth medium after 24 h of infection. Stable cell lines were selected out in 2 μg/mL puromycin (Sigma Aldrich, St. Louis, MO, USA) for 4 days.
Cell proliferation assay
Cell Counting Kit-8 (CCK-8) (Dojindo, Japan) assay was used to evaluate cell proliferation ability. MDA-MB-231 and MDA-MB-468 cells with or without SMC1A silencing were seeded into 96-well plates with the number of 2 × 103. Subsequently, 90 μl basic medium with 10 μl CCK-8 replaced the medium. After incubation at 37 °C for 2 h, the absorbance at 450 nm was measured with a microplate reader (BioTek Instruments, USA).
Colony formation assay
MDA-MB-231 and MDA-MB-468 cells with or without SMC1A silencing were plated into each well of a 6-well culture plate (600 cells/well), with the medium refreshed every 3 days. Cells were fixed with 4% paraformaldehyde for 15 min and visualized by 0.5% (w/v) crystal violet (Sigma-Aldrich) staining when clones were visible. The number of colons was quantified by Image J software.
Flow cytometry
For cell apoptosis, the apoptotic rate of tumor cells was stained using Annexin V Apoptosis Detection Kit II (BD Biosciences) according to according to the manufacturer’s protocol. Finally, the apoptotic cells were analyzed by flow cytometer (BD Biosciences, CA, USA).
For cell cycle analysis, MDA-MB-231 and MDA-MB-468 cells with or without SMC1A silencing were stained by PI-staining solution after fixing in 75% ethanol at 4 °C overnight, and then the percentages of cells in the G0-G1, S, and G2-M phase were counted using a flow cytometer.
In vivo experiments
All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Tongji University and conducted under the National Institutes of Health. BALB/c nude mice aged 4 weeks were purchased from the Shanghai SLAC Laboratory Animal Co., Ltd (Shanghai, China). Approximately 1 × 107 MDA-MB-231 cells transduced with SCR or SMC1A shRNA#1 were subcutaneously injected into the right flank of nude mice. Tumor size was examined every week. The volume was calculated using the formula V= length × width2/2. All mice were sacrificed 4 weeks later, and the xenografts were dissected and weighed.
Western Blotting Analysis
Western blotting was carried out as previously described [20]. In brief, a total of 50 µg of protein was isolated by 10% SDS-PAGE and then transferred into PVDF membranes (Millipore, Billerica, MA, USA). The membranes were incubated with rabbit anti-SMC1A (ab21583, Abcam), rabbit anti-STMN1 (11157-1-AP, Proteintech), rabbit anti-FOXM1 (13147-1-AP, Proteintech), rabbit anti-AKT (#491, Cell Signaling Technology) or rabbit anti-Phospho-Akt (Ser473) (#4060, Cell Signaling Technology) at 4 °C overnight after blocking. The signals were visualized using ECL chemiluminescent regents (Thermo Fisher Scientific, Inc.) by ImageQuant™ LAS 4000 mini (GE Healthcare).
Immunohistochemical (IHC) analysis
Immunohistochemistry was performed as our previous described [21].
Statistical analysis
The data was expressed as mean ± standard deviation (SD). All experiments were repeated at least three independent experiments. All statistical analyses were performed using GraphPad Prism software (version 5; GraphPad Software Inc., La Jolla, CA, USA). Student’s T-test was used to compare two groups, and one-way ANOVA was used to compare three or more groups. A p-value < 0.05 was considered to indicate significance.