Mcf 10a

Human breast carcinoma cell lines (MCF-7) and human normal breast cells (MCF-10A) were used for in vitro experiments, which were purchased from Procell Life Science&Technology (Wuhan, China). MCF-7 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM; Gibco, Carlsbad, USA) supplemented with 10% fetal bovine serum (FBS) and 10 μ/ml penicillin G/streptomycin, and MCF-10A were maintained in 10A cell-specific culture medium (Procell Life Science&Technology). Both cell lines were maintained at 37°C in a humidified atmosphere containing 5% CO2.
Get rid of the culture medium from the petri dish and wash it three times with phosphate-buffered saline (PBS). Cells were collected by scraping off after the addition of cell lysis buffer (Beyotime, Shanghai) and then mixed and place at a standstill for 30 minutes. Collect cell sample by extracting supernatant after centrifugation.

The MCF-7, MDA-MB-231 and MCF10A cell lines were purchased from Procell (Procell, Wuhan, China), and were originally obtained from American Type Culture Collection. All cell lines were cultured in special culture medium purchased from Procell. Special medium for MDA-MB-231 (Procell, CM-0150A): Leibovitz's L-15(PM151010) + 10% FBS (164210-50) + 1% P/S(PB180120). Special medium for MCF-7 (Procell, CM-0149): MEM (contain NEAA)(PM150410) + 10 μg/mL Insulin(PB180432) + 10% FBS(164210-50) + 1% P/S(PB180120). Special medium for MCF-10A(Procell, CM-0525): DMEM/F12(PM150312) + 5% HS(164215) + 20 ng/mL EGF + 0.5μg/mL Hydrocortisone + 10 μg/mL Insulin(PB180432) + 1% NEAA(PB180424) + 1% P/S(PB180120). Cells were maintained as monolayer cultures in a humidified atmosphere of 5% CO₂ at 37 °C. When cultured cells reached 90% confluence, they were passaged after detachment using Recombinant Trypsin EDTA Solution (Biological Industries, Israel). All cell lines used were confirmed to be free of mycoplasma, and used for experiments when they entered an exponential growth stage.

MDA-MB-231, MDA-MB-468, SKBR3 and MCF-10A cells were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China). MDA-MB-231 cells were incubated in DMEM culture medium (Gibco, 11,965,092) with 10% foetal bovine serum (FBS). MDA-MB-468 cells were incubated in RPMI 1640 culture medium (Gibco, 11,875,093) with 10% FBS. SKBR3 cells were cultured in special culture medium (Procell, CM-0211) containing McCoy's 5A, 10% FBS and 1% penicillin/streptomycin. MCF-10A was cultured in special culture medium (Procell, CM-0525) containing DMEM/F12, 5% Horse serum, 20 ng/mL EGF, 0.5 μg/mL Hydrocortisone, 10 μg/mL Insulin, 1% NEAA and 1% P/S. Total RNA was isolated with the TRIzol reagent (Invitrogen). Complementary DNA was synthesized from 1 μg total RNA using MightyScript Plus First Strand cDNA Synthesis Master Mix (Sangon Biotech). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed with a PowerUp SYBR Green Master Mix (Thermo Fisher, A25742) after RNA extraction and reverse transcription from all four cell lines. Relative mRNA levels were calculated using the comparative Ct method (ΔCt). The primer sequences are listed in Supplementary Material S1.

A total of 66 pairs of BC tissue samples and adjacent normal tissue samples were obtained from BC patients at the Nanyang Second General Hospital, and all tissues were stored at -80℃. The clinical characteristics of these patients were shown in Table 1. All patients had not undergone any chemotherapy and radiotherapy before surgery. The experiment here got approval from the Ethics Committee of Nanyang Second General Hospital.Table 1

Clinical characteristics of patients with breast cancer (n = 66)

Characteristic	Cases
Age
< 50	38
≥ 50	28
Histologic grade
I	3
II	45
III	18
Lymph node metastasis
Yes	35
No	31
T stage
T1/T2	52
T3/T4	14
N stage
N0/N1	39
N2/N3	27

BC cell lines (MDA-MB-231 and MCF-7), human embryonic kidney cell line 293 T and human normal mammary epithelial cell line (MCF10A) were obtained from Procell (Wuhan, China). MDA-MB-231 cells were maintained in Leibovitz's L-15 medium (Procell); MCF-7 cells were maintained in Minimum Essential Medium (MEM) medium (Procell); MCF10A cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium (Procell); 293 T cells were cultured in DMEM medium (Procell). All mediums were added with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Rockville, MD, USA) and 1% penicillin–streptomycin liquid (Gibco). All cells were cultured at 37℃ at 5% CO₂.