Patients and clinical samples
Fifty-four human breast cancer samples and adjacent normal tissue were obtained from patients who had an initial surgery from January 2018 to December 2019 from the pathology department of First Affiliated Hospital, Hainan Medical University, China. The inclusion criteria were female, aged from 18 to 80 years, who did not undergo chemotherapy or endocrine therapy and were neither pregnant nor lactating. The exclusion criteria were the lack of complete histopathological data and patients with other systematic dysfunctions. This study was approved by the Ethics Committee of Hainan Medical University (Hainan, China). The clinicopathological signatures of the cohort are shown in Table 1. Tumor differentiation and tumor stages were graded and classified according to the Edmondson-Steiner grading system and TNM staging system, respectively.
Immunohistochemistry
After fixation of the specimens in 10% formaldehyde for 24 h, the samples were dehydrated and embedded in paraffin. The sections were rehydrated in a graded ethanol series following cutting and dewaxing of the sections (4 μm) in xylene. Heat-induced epitope retrieval was performed using citrate buffer (pH=6, ZSJB-Bio, Beijing, China) and a microwave oven. To block endogenous peroxidase activity, the samples were incubated with 3% hydrogen peroxide for 10 min after antigen retrieval. The samples were incubated in rabbit monoclonal Sp1 antibody (1:200, ab124804, Abcam, Cambridge, UK) overnight at 4 °C, then in horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG polymer (PV-6001, ZSJB-Bio, Beijing, China) for 20 min and treated with the DAB detection kit (ZSJB-Bio, Beijing, China) to visualize positive staining. The slides were observed and captured by microscopy (CX41, Olympus, Japan).
The immunohistochemical expression was scored from 0 to 3 (score 0: negative; scores of 1 – 3: positive), and calculated as follows. The positive tumor cells were scored as 0, 1, 2, or 3 based on the percentage of positive cells in the tumor tissue (< 5%, 5%–25%,26%–50%, and > 50%, respectively) and the signal intensity was scored as 0, 1, or 2, which indicated no staining, canary, and claybank staining, respectively. Lastly, the final score was obtained by multiplying the positive cell score and signal intensity score.
Cell line and cell culture
The MCF-7 human breast cancer cell line was obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China) and cultured in Dulbecco’s minimum essential medium (DMEM, Solarbio, Beijing, China) supplemented with 10% fetal bovine serum (FBS, Sijiqing, Hangzhou, China) at 37 °C in a humidified atmosphere containing 5% CO2.
Cell transfection
MCF-7 cells (1 × 105 cells/mL) were inoculated and grown in six-well plates. After the MCF-7 cells reached a density of approximately 80%, 5 μg/mL of shRNA targeting Sp1 (Genesil, Wuhan, China) or negative control oligonucleotides were transfected using Lipo2000 transfection reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.
Immunocytochemistry
After transfection for 72 h, 1 × 105 cells/mL of MCF-7 cells were seeded onto coverslips and grown overnight in six-well plates. The cells were treated with 4% paraformaldehyde for 10 min, 0.5% TritonX-100 for 20 min, 3% H2O2 for 10 min, and sheep serum for 10 min. Then, the cells were incubated with HRP-conjugated goat anti-rabbit IgG polymer (PV-6001, ZSJB-Bio, Beijing, China) for 20 min, and rabbit Sp1 monoclonal antibody (1:200, ab124804, Abcam, Cambridge, UK) overnight at 4 °C, and then treated with the DAB detection kit (ZSJB-Bio, Beijing, China) to visualize positive staining. The cells onto coverslips were observed and captured by microscopy (CX41, Olympus, Japan).
Western blot analysis
The Epiquik whole-cell extraction kit (Epigentek, NY, USA) was used to extract the total cellular protein from the MCF-7 cells. After transfection of the cells for 72 h, they were rinsed in cold phosphate-buffered saline (PBS) and 20 μg of protein extract was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were then blocked with 5% nonfat milk powder, and incubated overnight with rabbit Sp1 monoclonal antibody (1:1,000, ab124804, Abcam, Cambridge, UK). Specific reactive bands were detected using HRP-labeled anti-rabbit IgG secondary antibody (1:1,000, Beyotime, Shanghai, China) for 1 hour at room temperature. Visualization of the immunoreactive bands was performed with a DAB detection kit (ZSJB-Bio, Beijing, China). Anti-GAPDH antibody was used as a loading control (1:1,000, Goodhere, Hangzhou, China).
Quantitative real-time PCR
The expression of Sp1 and the reference gene GAPDH was determined by quantitative real-time PCR (qPCR). Total RNA was isolated from the MCF-7 cells after transfection for 48 h using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The reverse transcription of 2 μg of RNA into cDNA was done using a reverse transcriptase kit (Beyotime, Shanghai, China). qPCR was performed using the SYBR Green kit (Tiangen, Beijing, China) in a 20-μL total reaction volume. Stratagene Mx3000P software (Agilent Technologies, Inc., Santa Clara, CA, USA) was employed to analyze the Sp1 expression levels, which are presented as fold-changes calculated based on the 2-ΔΔCt normalization method. All the above experiments followed the instructions of the manufacturers. The primer sequences for Sp1 were forward, TGGTGGGCAGTATGTTGT and reverse, GCTATTGGCATTGGTGAA, and those for GAPDH were forward, GGAGTCAACGGATTTGGT and reverse, GTGATGGGATTTCCATTGAT.
Cell proliferation assay
MTT cell proliferation assays were performed according to the manufacturer’s instructions (Beyotime, Shanghai, China) with minor modification. Briefly, 1 × 104 MCF-7 cells per well were inoculated in a 96-well plate after transfection for 24 h, 48 h, and 72 h. After 24 h, 10 μL of MTT (5 mg/mL) was added to the 96-well plate and incubated for 4 h. Then, 100 μL Formanzan dissolved liquid were added and incubated for 4 h. And plates were read by a microplate reader (Bio-Rad, Hercules, CA, USA) at 570 nm absorbance.
Cell migration assay
A 24-well Transwell chamber covered with an 8-μm pore membrane (Corning, Lowell, MA, USA) was used to perform the cell migration assay. After transfection for 48 h, MCF-7 cells were trypsinized in serum-free media and added to the upper chamber along with DMEM medium supplemented with 20% FBS. After 24 h, the cells on the upper surface of the insert were removed, and the cells on the lower surface of the insert were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet. The numbers of migrated cells were counted in at least five random fields for each slide using a microscope (CX41, Olympus, Japan) at 100× magnification.
Statistical analysis
Statistical analyses were carried out using SPSS21.0 (IBM, Armonk, NY, USA). The chi-squared test was used to analyze the correlation of Sp1 expression levels with the clinicopathological variables of the patients. Differences between the two groups were analyzed by the independent-samples T-test or the general linear model (repeated measures). A P-value of < 0.05 was considered statistically significant.