Immunohistochemical staining and scoring
All of the tissue samples were fixed in phosphate-buffered neutral formalin, embedded in paraffin, and then cut into 5 μm thick sections. Immunohistochemistry (IHC) was performed on the sections using antibodies specific to MAOA (1:200, Proteintech, 10539-1-AP), MAOB (1:100, Proteintech, 12602-1-AP), Ki67 (1:500, Cell Signaling Technology,#9027),PCNA (1:5,000,Cell Signaling Technology, #13110), NDRG1(1:200, Proteintech,26902-1-AP). Briefly, tissue sections were deparaffinized, rehydrated with graded ethanol, incubated with 0.3% hydrogen peroxide for 30 minutes, and blocked with 10% BSA (Sangon, Shanghai, China). Slides were first incubated with the antibody at 4 °C overnight and then labelled with the HRP secondary antibody (Thermo Fisher Scientific, Inc., US) at room temperature for 1 h. Positive staining was visualized with DAB substrate liquid (Gene Tech, Shanghai) and counterstained with haematoxylin. All sections were observed and photographed with a microscope (Carl Zeiss, Germany). Scoring was conducted according to the ratio and intensity of positively stained cells, and the scores were determined independently by two senior pathologists. Scoring by the pathologists was performed in a blinded manner[16, 17].
Cell culture and reagents
The human gastric cancer cell lines BGC-823, HGC-27, SGC-7901, MGC-803, MKN-45 and AGS were all maintained in the Shanghai Cancer Institute, Renji Hospital. All cells were cultured in RPMI-1640 medium (Beijing Solarbio Science & Technology Co., Ltd.) or F-12 Medium (Gibco; Thermo Fisher Scientific, Inc.) according to ATCC protocols and supplemented with 10% (v/v) foetal bovine serum (FBS) and 1% antibiotics (100 μg/ml streptomycin and 100 U/ml penicillin) at 37 °C in a humidified incubator at 5% CO2. The cell medium was replaced every 2-3 days, and 1X PBS was used for cell washing prior to the medium being replaced[16, 18].
Reverse transcription-quantitative polymerase chain reaction (RT‒qPCR)
The thermocycling conditions were as follows: 60 °C for 34 sec and 95 °C for 15 sec for 40 cycles. Total RNA was extracted from gastric cancer tissues or gastric cancer cell lines using TRIzol reagent (Thermo Fisher Scientific, Inc.) and reverse transcribed using a PrimeScript RT‒PCR kit (Takara Bio., Inc., Otsu, Japan) according to the manufacturer’s protocols. RT‒qPCR was performed with SYBR Premix Ex Taq (Takara Bio., Inc.) using a 7500 Real-time PCR system (Thermo Fisher Scientific, Inc.). The 2-ΔΔCq method (15) was used to quantify relative gene expression, which was normalized to β-actin[16, 18]. The primers used in this study can be found in Supplementary Table 1.
Western blotting
Total protein was extracted using total protein extraction buffer (Beyotime Institute of Biotechnology, Haimen, China), and the protein concentration was measured using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.). Proteins (30 μg per lane) were separated using 10% SDS‑PAGE and transferred onto a nitrocellulose membrane. Following blocking with 1% BSA at room temperature for 1 h, the membrane was probed with MAOA (1:1000,Proteintech, 10539-1-AP),mTOR (1:1,000, Cell Signaling Technology, #2983),p-mTOR (1:1,000, Cell Signaling Technology, #2971),Akt (1:1000, Cell Signaling Technology, #4685),p-Akt (1:2,000, Cell Signaling Technology, #4060),PI3K (p85)(1:1,000, Cell Signaling Technology,#13666),p-PI3K(p85) (1:1,000, Cell Signaling Technology, #17366),NDRG1(1:2000, Proteintech,26902-1-AP),PDK1(1:1000, Immunoway, YT3645),PFKL (1:1000, Immunoway, YT3685),LDHA(1:1,000, Cell Signaling Technology,#3582),PKM2(1:1000, Immunoway, YT3777),β-Tubulin (1:5000,Immunoway, YM3030),GAPDH(1:5000,Immunoway, YM3029),β-actin(1:5000,Immunoway, YM3028) primary antibodies overnight at 4 °C, and a rabbit immunoglobulin G (H+L) secondary antibody (dilution, 1:10,000; cat. no. 31460; Thermo Fisher Scientific, Inc.) at room temperature for 1 h. Proteins were visualized using the Molecular Imager ChemiDoc XRS+ System (Bio-Rad Laboratories, CA, USA)[18].
TCGA database, GEO database and K-M plotter analysis
For 450 gastric cancer samples in the TCGA database (https://cancergenome.nih.gov/), NE synthesis and degradation enzyme mRNA expression was analysed in 415 tumour tissue samples and 35 normal tissue samples. In addition, 32 paired tumour tissues and normal tissues were also analysed for NE synthesis and degradation enzyme mRNA expression. Neurotransmitter synthesis and degradation enzymes were also analysed in normal and tumour tissues in the GEO database (https://www.ncbi.nlm.nih.gov/geo/). The relationship between neurotransmitter synthesis and degradation enzyme expression levels and survival prognosis for gastric cancer patients was analysed on the K-M plotter website (https://kmplot.com/analysis/).
ECAR and OCR measurements in a Seahorse experiment
In vitro cell metabolic alterations were monitored with the Seahorse XF96 Flux Analyser (Seahorse Bioscience) according to the manufacturer’s instructions. BGC-823 or SGC-7901 cells were seeded on an XF96-well plate, followed by PBS and specific treatment for 24 hr. For assessment of the real-time glycolytic rate (ECAR), an indicator of net proton loss during glycolysis, cells were incubated with unbuffered medium followed by a sequential injection of 10 mM glucose, 1 mM oligomycin (Sigma‒Aldrich), and 80 mM 2-deoxyglucose (2-DG, Sigma‒Aldrich, D8375). Mitochondrial respiration (OCR) was assessed using sequential injections of 1 mM oligomycin, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP, Sigma‒Aldrich, C2920), and 2 mM antimycin A and rotenone (Sigma‒Aldrich). To achieve maximal OCR, antimycin A and rotenone were used to inhibit mitochondrial respiration by blocking complex III (ubiquinone: cytochrome b-c complex). Both ECAR and OCR measurements were normalized to total protein content and reported as pmoles/min for OCR and mph/min for ECAR[16, 19].
Glucose uptake and lactate production measurement
Briefly, cells were seeded onto 6-well plate culture dishes and starved for 24 hr after they had adhered. Then, the cells were treated with the indicated antagonists for 2 hr, followed by specific treatment for an additional 24 hr. The culture medium was collected for the measurement of glucose and lactate concentrations, and cells were harvested for protein lysates. The glucose uptake assay was performed using a glucose assay kit (Sigma‒Aldrich). Glucose consumption was calculated by deducting the measured glucose concentration in the medium from the original glucose concentration. Lactate production in the medium was detected using the Lactate Assay Kit (BioVision, Mountain View, CA) according to the manufacturer’s instructions. The results were normalized on the basis of the total protein concentration of each sample[16, 19].
Patients and tissue microarray (TMA) construction.
This was a retrospective analysis of 587 patients with primary GC who underwent gastrectomy at the Department of Gastrointestinal Surgery, Ren Ji Hospital, (School of Medicine, Shanghai Jiao Tong University, Shanghai, China) between January 2006 and December 2011. The final follow‑up date was December 31, 2015 for all cases examined. The mean age of the patients was 61.6 years (range, 22‑89 years), including 401 males and 186 females. A total of 251 cancer‑associated mortalities occurred. All patients received standard treatments, including D2 radical resection [excluding 6 cases with Tumor‑Node‑Metastasis (TNM) stage IV] and first‑line adjuvant chemotherapy (for patients with advanced gastric cancer) according to the National Comprehensive Cancer Network guidelines (https://www.nccn.org/). For all cases examined. OS time was defined as the interval between the gastrectomy and patient death or survival.
Formalin‑fixed paraffin‑embedded (FFPE) tissue blocks were collected from the Pathology Department of Ren Ji Hospital. TNM staging was performed based on the American Joint Committee on Cancer (8th Edition) staging system. For each case, the diagnosis was confirmed by two senior pathologists through a review of H&E‑stained slides. Representative FFPE blocks were selected for construction of the TMA using a tissue arrayer of 5‑μm thickness.
Statistical analysis
The statistical analysis was performed using SPSS 22.0 software (IBM, USA). All of the data are expressed as the mean ± standard deviation. Student’s t test or one-way ANOVA was used to compare the means of two or three groups. The correlation between MAOA expression and clinicopathological characteristics was calculated using the chi-square test or Fisher’s exact test. A Kaplan‒Meier survival curve was used to evaluate overall survival, and the log-rank test was used to compare differences between curves. A P value of < 0.05 was considered significant (*P < 0.05, ** P < 0.01, and *** P < 0.001).