Gene expression profiling interactive analysis (GEPIA database)
GEPIA [21, 22] (http://gepia2.cancer-pku.cn/#survival) provides website analysis functions, including the gene expression analysis of tumor/normal, differential analysis of cancer types or pathological stages and survival analysis. The expression and relapse-free survival analysis (RFS) analysis of GALNT7 in BC were analyzed by GEPIA.
TCGA database analysis
mRNA expression in human BC subtypes was analyzed by TCGA Research Network (http://cancergenome.nih.gov). The original data from the TCGA database was normalized and analyzed by the edgeR analysis method . To analyze the overall survival of patients with BC subtypes, patient samples were analyzed by Kaplan-Meier analysis.
Tissue specimens were collected from 128 participants, which involved 59 adjacent tissues and 69 luminal BC cases. Paraffin-embedded BC tissues were obtained from the Pathology Department of the Affiliated Hospital of Southwest Medical University. Firstly, paraffin-embedded tissue slides were dewaxed and blocked with normal goat serum (ZSGB-BIO, China) for nonspecific staining treatment at temperature for 30 min, after which they were treated with the primary antibody rabbit anti-SPDEF (1:200) (ABclone, USA) overnight at 4 °C. Subsequently, the slides were treated with biotinylated anti-rabbit IgG secondary antibody and next with horseradish peroxidase-conjugated streptavidin complex (ZSGB-BIO, China). Finally, the slides were incubated with adiaminobenzidine chromogen (DAB) solution and counterstained with hematoxylin to develop the colour reaction. Patient Consent Forms were obtained according to protocols approved by the Institutional Review Board of the Affiliated Hospital of Southwest Medical University. The H-score, or histochemical score was used to conduct quantitative assessment in immunohistochemistry (IHC). The staining intensity is scored on a scale of 0 to 3 (with 0 being no staining, 1+ being weak staining, 2+ being moderate staining, and 3+ being strong staining). The percentage of cells stained at each intensity level is then determined. H-score = (Percentage of cells stained at 1+ intensity) + (2 x Percentage of cells stained at 2+ intensity) + (3 x Percentage of cells stained at 3+ intensity).
Survival analysis in Kaplan-Meier plotter database
To analyze the survival rates of patients with luminal BC, the prognostic significance of mRNA expression was evaluated using Kaplan-Meier Plotter [24, 25] (http://kmplot.com/analysis), which contained gene expression data and survival information of luminal BC patients.
Normal human mammary cells MCF10A and human breast cancer cell lines, MCF7, T47D and BT-474 cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). MCF10A were cultured in special medium (Procell, China). MCF7 and T47D cells were maintained in DMEM high glucose medium (Hyclone, USA) supplemented with 10% fetal bovine serum (Gibco, USA). BT-474 cell were maintained in RPMI-1640 (Gibco, USA) supplemented with 20% fetal bovine serum (Gibco, USA), 10 μg/mL Insulin (Beyotime, China) and 2mM L-glutamine (Procell, China). Cells were incubated at 37 °C in an atmosphere of 5% CO2.
RNA extraction and quantitative reverse transcription PCR (RT‐qPCR) analysis
Total RNA was extracted from cells using the Trizol (Takara, Japan) and chloroform. RNA concentration was measured using NanoDrop OneTM (Thermo Scientific, USA). And RNA converted into cDNA using PrimeScript RT reagent Kit (Takara, Japan). Quantitative PCR (qPCR) was conducted using the TB Green™ Premix Ex Taq™ II (Takara, Japan) in the Bio‐Rad CFX96 system according to the manufacturer’s protocol. The sequences of PCR primers are listed in Table S1.
Stable knockdown of genes by lentivirus infection
Stable knockdown of SPDEF and over-expression of GALNT7 in breast cancer cells was achieved with infection of sh-SPDEF (GeneBiogist, China) in the presence of 8 µg/ml polybrene (Genepharma, China) for 24 h. 48 h post the infection, the efficiency of infection was determined by RT-qPCR. Corresponding negative control (NC) cell lines were established by infection of viruses expressing empty vectors.
The proteins were extracted by Protein Extraction Kit (NCM Biotech, China). 50 µg protein sample was separated on 10% polyacrylamide gel (NCM Biotech, China) and transferred to PVDF membrane (Bio‐Rad, USA). Blocking the membrane in 5% notfat milk dissolved in TBST for 1-hour at room temperature. Then the membrane was hybridized in primary antibodies overnight at 4 °C on a shaker. The following primary antibodies were used: anti-SPDEF (1:7500 dilution, ABclone, USA), anti-GALNT7 (1:2500 dilution, TianQiShun, China) and anti-GAPDH (1:7500 dilution, Proteintech, USA). The membrane was incubated with the secondary antibodies diluted in TBST. Finally, the ECL system was used for HRP-conjugated secondary antibody (Bio-Rad, USA).
Cell proliferation assay
Cellular proliferation was detected using a CCK-8 colorimetric assay kit (Beyotime, China). Luminal BC cells (2 × 103/well) were seeded in 96-well plates to culture. CCK8 (10 μl) reagent was added to each well and the absorbance of each well at 450 nm was measured every 24 h with the multimode microplate reader (EnSpire, Singapore). Cell proliferation activity was continuously detected for 6 days.
Wound scratch assay
The scratch test was used for cell migration assay. 3 × 105 cells were seeded into a 6-well plate and cultured in medium with 10% FBS. When the cells grew to 80% confluence, the scratch was scored monolayer cells with sterile pipette tip, and continue to incubate with serum-free medium.
Transwell migration and invasion assays
Transwell chambers were used to execute cell invasion assay with Matrigel (60 μl, 1:10 dilution in serum-free medium, Corning, USA) and migration assay without Matrigel. Liminal BC cells (5 × 104/well) were seeded in the upper chamber in 100 μl of serum-free medium, at the same time, 600 μl medium with 10% FBS was added to the lower chamber as a chemoattractant. After incubation at 37 °C for 24 h, the upper surface of the membrane was gently removed with a swab. While the cells invaded the lower surface, the membrane was stained with 0.1% crystal violet. The stained cells in 3 randomly selected fields were counted.
Transient overexpression assay
hGALNT7 pReceiver‐M98 and negative control were purchased from GeneCopoeia (GeneCopoeia, USA). Transient overexpression assay was performed and optimized using Lipo8000TM (Beyotime, China) and MEM medium (Gibco, USA) according to the manufacturer’s protocol.
Gene set enrichment analysis (GSEA)
Clinical characteristics and differential analysis of stemness indices
The stemness indices, mRNAsi (mRNA expression-based stemness index) and mDNAsi (DNA methylation-based stemness index) were uesd to describe the similar features between cancer cells and stem cells and it might be considered as the stemness indices of Cancer Stem Cells (CSCs) . TCGA luminal BC samples were divided into two groups according to mRNA expressions and the mRNAsi and mDNAsi indices of the two groups were evaluated based on R analysis.
Sphere formation assay
Luminal BC cells were plated at 1 × 103 cells/well in low adhesion 6-well plates and were maintained in DMEM/F12 medium (Gibco, USA), supplemented with 2% B27 (Gibco, USA), 20 ng/ml Recombinant Human EGF (Peprotech, USA) and 20 ng/ml Recombinant Human bFGF (Peprotech, USA). After the cells were incubated for 12 days, the size and number of tumor spheres were calculated.
Colony formation assay
MCF7 or T47D cells were digested with trypsin, suspended as a single cell and counted using the Bullboar counting plate. 1 × 103 cells were seeded in 6-well plates and incubated for 1 week at 37 °C. Then, cells were washed with PBS, fixed with 4% Methanol for 30 min (mins) and stained for 10 min with 0.1% crystal violet.
Subcutaneous tumor transplantation assay
Single-cell suspensions at designated concentrations were combined with equal volume of Matrigel Matrix (R&D Systems, USA). Then the mixture was injected subcutaneously into each side of the hind leg of BALB/c nude mice. After tumor cell transplantation, the tumor dimensions were measured every other day and the volume was calculated using V = (length × width^2)/2. When the volume reached about 1 cm , the mice were sacrificed and the neoplasm was excised for weighting. All protocols were approved by the Institutional Animal Care and Use Committee of Southwest Medical University and conducted with humane animal care.
The expression of the ALDH1A1 marker was evaluated using flow cytometry, adhering strictly to the manufacturer’s protocol. We utilized the primary anti‐ALDH1A1 antibody at specific working dilutions (0.2 μg of 60171-1-Ig, Clone:1A10A2, sourced from Proteintech, USA). This was followed by the application of the secondary antibody, Fluorescein (FITC)–conjugated Affinipure Goat Anti-Mouse IgG(H + L), at a dilution ranging between 1:20 and 1:100 (also provided by Proteintech, USA).
Drug sensitivity assays
CCK-8 colorimetric assay was used to detect the sensitivity of cells to paclitaxel in vitro. Cells were resuspended in a final concentration of 2 × 103 cells/well, seeded into 96 well plates and subjected to paclitaxel after preincubation for 48 h. After 24 h of incubation withpaclitaxel, CCK-8 reagent (10 µl/well) was added and the plates were further incubated for 2 h. Subsequently, absorbance was determined at 450 nm by microplate reader.
Chromatin immunoprecipitation (ChIP) assay
ChIP assay was performed to detect the molecular interactions of SPDEF with the promoter of GALNT7 according to the manufacturer’s instruction (Beyotime, China). The primers of ChIP -qPCR listed in Table S2.
JASPAR software (http://jaspar.genereg.net/) was used to predict the presumptive binding sites of SPDEF that identified the GALNT7 promoter region. The first three matching sequences were selected to verification. Luciferase reporter vectors containing the wild-type (WT) or mutant sequences (Mut) towards the SPDEF binding of the GALNT7 promoter region were constructed (Tsingke, China). The pGL3-basic vector was included as the control. For luciferase activity assays, luminal BC cells were seeded in 24-well plates and cultured 24 h before transfection. Then cells were co-transfected with pGL3-basic or WT/Mut vector, and SPDEF over-expression plasmid/empty vector by EndoFectinTM-Max transfection reagent (GeneCopoeia, USA). The activity of Renilla plasmid was measured using the Dual-Luciferase Reporter Assay Kit (GeneCopoeia, USA).
Enzyme-linked immunosorbent assay (ELISA)
Serum samples were collected from 64 participants, including 33 healthy control and 31 luminal BC patients without therapy, respectively. The study was carried out in accordance with the Institutional Review Board of the Affiliated Hospital of Southwest Medical University, and signed consent forms were obtained from each participant. GALNT7 in human serum were detected by ELISA kit (Ruixin Biological Technology Co., Ltd, Quanzhou, China). CEA and CA125 were quantified by Cobas e602 Electrochemiluminescence System (Roche, Switzerland).
All data were analyzed by to the Shapiro-Wilk and Kolmogorov-Smirnov Test to check for normality, as well as the Levene’s test to verify the homogeneity of variances. Subsequent difference analyses were conducted by the t-test, chi-square test, Welch’s t-test, Wilcoxon or Mann-Whitney tests as appropriate using GraphPad Prism 7 and R, and presented as mean ± SEM. The P values less than 0.05 were considered statistically significant, *P < 0.05, **P < 0.01, ***P < 0.001. Correlation analysis was calculated with the Pearson correlation coefficient formula. Each experiment was replicated 3 times to ensure the reliability and reproducibility of the results.