2.1 TCGA data sets
The TCGA-BRCA cohort data of 104 normal patients and 1079 BRCA patients and all relevant clinical date were downloaded from The Cancer Genome Atlas (TCGA, https://tcga-data.nci.nih.gov/tcga/). The expression profiles of GABRQ were analyzed by these datasets. The downloaded original data pre-procession and bioinformatics analysis were conducted by the R studio software (3.51).
2.2 Patients and specimens
The specimens containing 16 paired paraffin embedding BRCA tissues and adjacent non-tumor tissues were obtained from the First Affiliated Hospital of Zhengzhou University (ZZU cohort). The study was approved by the Institutional Review Board of the First Affiliated Hospital of Zhengzhou University.
2.3 TMA cohorts
We analyze another BRCA TMA(the tissue microarray) cohort, which contains 44 GABRQ high expression BRCA tissue specimens and 42 GABRQ low expression BRCA tissue specimens. The TMA cohort was purchased from Outdo Biotech (Shanghai, China).
2.4 Cells and culture
The BRCA cell lines (MCF-7、MDA-MB-231、T47D、BT-474、BT-549) and normal breast cell lines used in this study were purchased from the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 100 U/ml penicillin/streptomycin (Corning, NY, United States) in a humid incubator (5% carbon dioxide, 95% air) at 37°C .
In brief, 5-µm-thick TMA sections were deparaffinized and then hydrated, blocking endogenous peroxidases and antigen retrieval. TMA sections were incubated with primary antibody overnight at 4°C after blocking for one hour at room temperature. Subsequently, TMA sections were probed with secondary biotinylated goat anti-rabbit antibody, and then detected by SignalStain® DAB (Cell Signaling Technology, Danvers, MA) and counterstained with Hematoxylin QS (Vector Laboratories). Two experienced pathologists, who were blinded to the clinicopathologic data, independently evaluated immunostaining. Sections were scored for the GABRQ staining patterns as follows: 1+, weak staining; 2+, moderate staining; 3+, strong staining; and 4+, intense staining. Simultaneously, scores of 3 + and 4 + were definitized as high expression and the other scores were deemed as low expression for statistical analysis.
2.6 Real-time quantitative PCR (RT-qPCR)
Different BRCA cell lines tissue and normal tissues were selected for RT-qPCR assay. Total RNA was extracted utilizing TRIzol reagent (Life Technologies) according to the manufacturer instructions. TransScript First-Strand cDNA Synthesis SuperMix (TransGen) was used to reverse-transcribe cDNA. The RT-qPCR assay was performed using PowerUp SYBR Green Kit (ABI) and QuantStudio 6 System (ABI). Data were analyzed by using the comparative Ct method (2 − ΔΔCt). β-Actin was served as the internal control.
2.7 Western blot
RIPA buffer was utilized to extract total protein from cultured cells. Following extraction, BCA assays (Beyotime) were performed to quantify all proteins. An equal amount of protein samples was separated by 12% SDS-PAGE and then transferred to the nitrocellulose membranes (Millipore). The membranes were blocked with 5% non-fat milk/PBS for 1 hour. Then, the membranes were incubated by primary antibodies at 4°C overnight. After washing the membranes with PBST three times, the membranes were further incubated with secondary antibodies for 2 hours. The membranes were developed using enhanced chemiluminescence solution (Beyotime) and exposed to the photographic film for visualization.
2.8 Cell proliferation assay
For colony formation assays, MCF-7 or T47D cells treated with si-GABRQ-2 were placed in 6-well plates and incubated for 2 weeks, then, all cells were fixed, stained, photographed and analyzed. Furthermore, an EDU kit (RiboBio, Guangzhou, China) was used to further assess the cell proliferation ability after GABRQ knockdown. Images were taken with a microscope at 100× (Olympus, Tokyo, Japan) for further analysis. The ratio of EDU-stained cells (red fluorescence) and DAPI-stained cells (blue fluorescence) were used to evaluate the cell proliferation activity.
2.9 Cell invasion assay
Cell invasion assay was performed with Matrigel Invasion Chambers (BD Biosciences, San Jose, CA, USA). Here,1×104 transfected cells were cultured on the upper chamber of the transwell insert in serum-free medium, and the bottom chamber is filled with DMEM with 10% FBS. After 24 hours, the invasive cells were stained with 0.5% crystal violet, imaged and counted.
2.10 Tumor Xenografts
Mice assays were approved by the Animal Health Committee of Zhengzhou University. The male nude mice (4–6 weeks) were purchased from Beijing Vital River Laboratory (Beijing, China). Cells transfected with GABRQ knockdown lentivirus (Sh-GABRQ) and empty lentivirus control (Sh-NC) were subcutaneously implanted into the lower flank of nude mice. Tumor growth was examined every week. Mice were euthanized at 5 weeks post-implantation. The tumor tissues were weighed and extracted for further IHC staining.
2.11 Statistical analysis
GraphPad Prism software (version 7.0, United States) and SPSS (Version 23.0, IBM, Seattle, WA, United States) were carried out for statistical analyses. Differences between two groups were analyzed by Student’s t-test or the Mann–Whitney U test. Overall survival (OS) and disease-free survival (DFS) was calculated with Kaplan- Meier curves and log-rank tests. The correlation was performed by Spearman rank analysis. A p-value < 0.05 was considered to be statistically significant. All data were presented as the mean ± standard deviation (SD).