Cell lines and cell culture
The ERβ-positive human breast cancer cell lines MCF-7 and MDA-MB-231 were obtained from the Scientific Research Center of the Fourth Hospital of Hebei Medical University. Briefly, MCF-7 and MDA-MB-231 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (BI, Israel) and 4.5 g/L glucose, 2 nM L-glutamine, 5000 IU/L penicillin, 5 mg/L streptomycin, 125 U/L Fungizone and 2.2 g/L sodium bicarbonate. All cells were cultured at 37°C in a humidified atmosphere of 5% CO2.
Breast cancer samples from 107 patients treated from January to December 2010 at the Fourth Hospital of Hebei Medical University were obtained, and the patient follow-up data from January 2010 to December 2018 were collected. According to the guidelines of relevant institutions, samples were collected after informed consent was obtained from the patients and after approval by the Institute Research Ethics Committee of the Fourth Hospital of Hebei Medical University. None of enrolled patients received neoadjuvant therapy, radiation therapy, or chemotherapy before surgery, and all had complete follow-up data.
Cells in the logarithmic growth phase were seeded in a 6-well culture plate, and transfection was initiated when the cells grew to approximately 70-80% confluence. The transfection step was performed according to the instructions of the Lipofectamine 2000 reagent (Invitrogen, USA). In all, 4 µg of plasmid and 10 µl of cationic transfection reagent Lipofectamine 2000 were diluted in 250 µl of serum-free DMEM medium and mixed. After 20 minutes, the mixture was added to 1.5 ml of serum-free medium. After 4 h of transfection, the DMEM culture medium containing 10% fetal bovine serum was changed, and then, 48 h after transfection, protein extraction and related detection were performed.
In all, ≥1 × 106 cells were added to 500 µl of RIPA lysate, mixed thoroughly, placed at 30°C for 4 minutes, and centrifuged at 12,000 r/min for 20 minutes at 4°C, after which the supernatant was collected to determine the protein concentration by Coomassie blue. An equal amount of total protein was added to each well and was transferred to a PVDF membrane after SDS-PAGE. The membrane was blocked with 5% BSA at 37°C for 1 h. Next, the membrane was incubated with primary antibodies including those against ERβ (1:1000, Affinity, USA), P-AKT473 (1:1000, Cell Signaling Technology, USA), β-actin, MMP2, and MFN2 (1:1000, Proteintech, China) overnight at 4°C. Then, the membrane was incubated with an HRP-labeled secondary antibody (1:5000, Proteintech, China) for 2 h at room temperature, after which the proteins were visualized using enhanced chemiluminescence (TIANGEN, China). Protein quantification was performed using ImageJ software.
The slides were deparaffinized and incubated for 10 min with 3% H2O2 in water. Then, the slides were incubated with 10% goat serum at 37°C for 20 minutes, after which the slides were incubated overnight with an anti-MFN2 antibody (1:100, Proteintech, China). Subsequently, the slides were incubated with the appropriate secondary antibody for 30 min at room temperature. For quantification, 5 random images (400×) per experimental group were captured with a microscope (Leica, Germany). The final weighted expression score (0–2) was obtained by calculating the intensity values of IHC staining and the percentage of positive cells. Based on previous research, 0-1 points indicated low expression, while 2 points indicated high expression.
Cells were plated on coverslips in 6-well plates, fixed in 4% paraformaldehyde and washed with PBS. After permeabilization with 0.5% Triton X-100 in PBS and blocking with 10% goat serum, the cells were incubated with a primary antibody against MMP2 (1:100, Proteintech, China) at 4°C overnight. Then, the cells were washed 3 times with PBS and incubated with a secondary fluorescent antibody at room temperature for 2 h. After 3 washes in PBS, cells on the coverslips were analyzed by confocal microscopy.
Wound healing assay
First, 6-well plates were seeded with 1×106 cells until the cells were 100% confluent. The cell layer was then carefully scratched with a sterile 200 μL pipette tip to generate a wound, which was then washed and cultured in complete medium without FBS. At 0 h and after 24 h, the wounds were photographed under a light microscope, and the wound closure percentage (%) was evaluated using ImageJ software.
All data in this study were obtained independently three times and are presented as the mean±SD. Statistical differences between the results of each group were evaluated using Student’s t-test or one-way analysis of variance (ANOVA). A P-value <0.05 was considered statistically significant.