Patient information and clinical sample collection
In total, 30 paired ER+ BC tissue samples and adjacent normal tissues were obtained from patients who underwent surgery at the First Affiliated Hospital of Sun Yat-sen University (Guangzhou, China) between January 2013 and December 2014. Samples before/after endocrine therapy including criterion: patients who underwent pathological sampling and receive standard endocrine therapy and breast tumor was eventually excised by surgical operation. All BC tissue samples were confirmed by pathology. This study was approved by the Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University and conformed to the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. Clinical samples were collected from patients after written informed consent was obtained.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted with TRIzol reagent (Invitrogen, NY, USA) according to the manufacturer’s protocol. The relative RNA level was normalized to β-actin, and all experiments were performed in triplicate. The primer sequences used are listed below:
MTFR2 F: 5’-GAAACTGGATCCCAATGTGAA-3’ and
R: 5’-GAATAAGGTTAAGCTTCGTGCAA-3’.
Cell culture and cell transfection assay
Human mammary cancer cell lines (T47D and MCF-7) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Life Technologies, NY, USA), 1% penicillin G, and streptomycin. CRISPR-Cas9 targeting RNA was obtained from Shanghai Generay Biotech Co., Ltd. The transfection assay was performed using Lipofectamine 3000 (Invitrogen, CA, USA) according to the manufacturer’s protocol when the cells reach approximately 50-70% confluence.
Immunoblotting
Cells were washed twice with PBS and then lysed in cold RIPA buffer with protease inhibitors. Total protein (30-60 μg) was separated by 12% SDS-PAGE and transferred to a nitrocellulose membrane. After being blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 hour at room temperature, the membranes were then incubated overnight with the corresponding primary antibodies. After being incubated with the secondary antibody, the proteins were detected using ECL (EMD Millipore, MA, USA). β-Actin was used as an internal control.
CRISPR/Cas9-mediated gene knockout
The target sequences of gRNA were designed using the online tool at http://crispr.mit.edu/. CRISPR-lentivirus were harvested and used to infect cells for 4-6 hr in the incubator. After five days culture. K.O. stable cell lines were harvested.
Cell proliferation assay
Cell Counting Kit-8 (CCK-8, Dojindo, Tabaru, Japan) was used to measure cell proliferation. Cells were seeded into 96-well plates. After treatment with TMX and Let, the relative absorption of CCK-8 was measured and normalized.
Colony formation assay
To determine long-term effects on proliferation, the cells were seeded in a six-well plate. After being treated with TMX and Let, the cells were incubated for 14 days, the colonies were stained with crystal violet (Sigma-Aldrich, St. Louis, MO, USA), and the number of colonies was counted.
Xenograft mouse model
Four-week-old BLAB/c nude mice were randomly divided into groups and received injections of 5×105 cells. Tumor volume was measured using a Vernier caliper. For the AAV injection assay, specific cells were injected subcutaneously, AAVs containing empty vector and specific shRNA were injected into the tumor every 3 days, and the tumor volume was measured, the shRNA sequence was previously described (14).
Statistical analysis
All data analyses were performed with SPSS 20.0 statistical software. The χ2 test was used to analyze the relationships between categorical variables. The differences between groups were compared by Student’s t-tests. Cox regression and Kaplan-Meier analyses were used to analyze overall survival (OS), and p<0.05 was considered to be a significant difference from the control.