Human specimens
After all patients signed the informed consent, 7 fresh luminal B breast cancer tissues and the matched adjacent non-tumor breast tissues were collected during surgery at the People’s Hospital of Zhengzhou University. The fresh tissues were collected and immediately snap-frozen in liquid nitrogen and stored at -80°C until further use. All breast cancer tissue specimens were pathologically tested by two separate pathologists and confirmed to be luminal B breast cancer. None of the patients had received preoperative chemotherapy, endocrine therapy, radiotherapy or any other therapies. Approval was given by Medical Ethics Committee of Henan Provincial People’s Hospital.
Cell lines and culture
Normal human breast epithelial cell line MCF-10A and luminal B breast cancer cell lines ZR-75-1, ZR-75-30, and BT-474 were purchased from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences. The human luminal B breast cancer cell line MDA-MB-415 cell line was purchased from Procell.
MCF-10A cells were cultured in the MEGM kit (Lonza/Clonetics) supplemented with 10 ng/ml cholera toxin. BT-474 cells were maintained in RPMI-1640 (Biological Industries) medium supplemented with 10% fetal bovine serum (FBS) (Biological Industries), 10 µg/mL insulin (Yeasen Biotechnology, Shanghai, China), and 1% penicillin-streptomycin solution (P/S) (Sangon Biotech, Shanghai, China). MDA-MB-436 cells were kept in Leibovitz’s L-15 medium (Procell, Wuhan, China), supplemented with 10 µg/mL insulin, 10 µg/mL glutathione, 15% FBS, and 1% P/S. All cells were placed in an incubator at 37°C with 5% CO2.
RNA Isolation and real-time PCR
Total RNA was extracted from luminal B breast cancer tissues, matched adjacent non-tumor breast tissues, luminal B breast cancer cell lines, and human normal breast epithelial cell line using the Ultrapure RNA Kit (CW0581, ComWin Biotech Co., Ltd, China), following the manufacturer’s instructions. The purity and concentration of the total RNA were detected by Nano Drop 2000. RNA was reversely transcribed into cDNA by the reverse transcription kit (Vazyme Biotech Co., Ltd) according to the manufacturer’s protocol. Real-time PCR was performed using the Vazyme Real-Time PCR Kit. 2−ΔΔCT method was applied to calculate the relative expression of target genes, with GAPDH as the internal reference. The primer sequences used are displayed in Table 1.
Table 1
Primer sequences and siRNAs used in this study
Gene | Primer sequence |
GAPDH-F | 5’-AAGACCTTGGGCTGGGACTG−3’ |
GAPDH-R | 5’-ACCAAATCCGTTGACTCCGA−3’ |
PCAT7-F | 5’-AAACAAGCCAACCGCACAAT−3’ |
PCAT7-R | 5’-CCTGCTTGCTGTGTTACTGC−3’ |
siRNA1 | 5’-GUGGCAGAUACCACCUUAAATT−3’ |
siRNA2 | 5’-CCCGUCUUUACUAAAUAUATT−3’ |
siRNA3 | 5’-GUGCCAAGGAGACUCAAUATT−3’ |
NC | 5’-UUCUCCGAACGUGUCACGUTT−3’ |
Small interfering RNA (siRNA) synthesis and transfection
GenePharma was entrusted with designing and synthesizing specific interference siRNA sequences targeting PCAT7 (siRNA1, siRNA2 and siRNA3), as well as the negative control sequence (si-NC). The sequences used are displayed in Table 1. Cells were seeded in 6-well plates with 3×103 cells per well and cultured at 60%−80% confluence. Then the cells were transfected using siRNA-Mate (GenePharma, Shanghai, China). Real-time PCR was used to determine the knockdown efficiency at 48 h after transfection. And the most effective siRNA was used for the following experiments.
Colony formation assay
After being transfected for 48 h, the cells were inoculated in 6-well plates. After 14 days of incubation, the plates were photographed under the microscope and the cells were washed with phosphate buffered saline (PBS). Then the cells were fixed with 4% paraformaldehyde for 30 min, followed by washing with PBS twice and staining with 0.1% crystal violet for 30 min. The plates were photographed and the number of colonies was counted.
Wound healing assay
Transfected BT−474 cells were inoculated in 6-well plates, when cells were grown to approximately 80% confluence, a 200 µL plastic tip was used to make horizontal lines. Then, the well were washed twice by PBS to remove floating cells and cellular fragments. Photos were taken at 0 and 48 h to measure the distance.
Transwell invasion assay
After being transfected for 24 h, the cells were digested and washed with PBS twice. Next, cells were re-suspended in serum-free medium and seeded in the upper chamber. The lower chamber was filled with complete medium containing 20% FBS. After being cultured for 48 h, the upper chambers were washed twice with PBS. Then, cotton swabs were used to wipe off the non-invaded cells in the upper chamber. The invaded cells were fixed for 1 hour with paraformaldehyde. Next, the cells were stained with 0.1% crystal violet for 30 min. Finally, a microscope was used to count the number of stained cells in randomly selected fields.
Cell cycle essay
Transfected cells were collected 24 h later. Then the cells were digested, centrifuged at 1500 rpm for 5 min, and washed twice with pre-cold PBS. Next, the cells were fixed with 75% pre-cold ethanol at 4°C for 4 h. After that, the fixed cells were washed with PBS once and incubated with 400 µL of propidium iodide (PI) and 100 µL of RNase for 30 min at 4°C in the dark. Then the cell cycle was evaluated by flow cytometry.
Western blotting
After being transfected for 48 h, the cells were washed three times with PBS. Then cellular proteins were extracted. Protein samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PCDF) membranes. After being blocked with skim milk (5%), the membranes were incubated overnight with primary antibodies at 4°C. Then the membranes were incubated with secondary antibodies. The chemiluminescent substrate kit was used to determine the expression of targeted proteins.
Statistical analysis
Data Analysis was performed using GraphPad Prism 9.0 (GraphPad Software, Inc., La Jolla, CA, USA). Experimental data was represented as the mean ± standard deviation. Independent samples t-tests were performed to compare differences between two group. P < 0.05 was considered to be statistically significant.