Cell culture and antibodies
NPC cell lines HONE1 and HNE2 were maintained in our laboratory. The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Invitrogen, Shanghai, China), penicillin (100 U/ml), and streptomycin (100 µg/mL) in a humidified incubator under 5% CO2 at 37 °C. The antibodies used are listed in Table S3.
Constructs and reagents
The p-IRES-Flag-BPIFB1 plasmid was constructed previously; p-CMV3-GLUT1 was purchased from Sino Biological (Beijing, China). GLUT1 siRNAs were purchased from GenePharma (Shanghai, China). The Neofect Reagent (Neofect biotech Co., Ltd. China) was used for plasmid transfection and Hiperfect (Qiagen, Hilden, Germany) was used for siRNA transfection according to the manufacturer’s protocol. Reagents 2-deoxy-D-glucose(2-DG) and 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose(2-NBDG) were purchased from MedChemExpresss (MCE USA); SP600125 and anisomycin were from Selleck Chemicals (Houston, TX, USA).
NPC clinical samples
In total, 26 non-tumor NPE tissues and 52 NPC samples were collected at the Second Xiangya Hospital of Central South University (Changsha, China). The study was approved by the Joint Ethics Committee of the Central South University Health Authority and informed consent was obtained from all participants. The diagnoses of all specimens were confirmed via histopathological examination.
RNA isolation and quantitative real-time PCR
Total RNA was isolated using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s protocol. cDNA synthesis was performed using a reverse transcription kit (Vazyme, Nanjing, China) according to the manufacturer’s instructions. SYBR®Green (Vazyme) was used for qRT-PCR analysis on the MiniOpticon system (Bio-Rad, Hercules, CA, USA). After the reactions were completed, relative gene expression levels were calculated using the 2–ΔΔCt method, and β-actin was used as the endogenous control. The primer sequences used are listed in Table S4.
Total proteins were lysed using RIPA buffer (Beyotime Biotechnology, Shanghai, China) containing a protease/phosphatase inhibitor cocktail (Roche Applied Sciences, Mannheim, Germany), separated by 10% SDS-PAGE, and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membrane was blocked with 5% non-fat milk with TBST for 1 hour at room temperature and incubated with the primary antibodies overnight at 4 °C. After washing, the membrane was incubated with HRP-labeled secondary antibodies (CUSBIO, Wuhan, China) for 1 h at room temperature. The proteins were then detected using ECL reagent (Millipore).
Measurement of glucose uptake, lactate production, and 2-NBDG uptake
Cells were seeded in 12-well plates (105/well); after incubation for 8 h, the medium was replaced with fresh complete medium, and the cells were incubated for another 24 h. Glucose and lactate levels were measured using the Automatic Biochemical Analyzer (AU680, Beckman Coulter International, Brea, CA, USA). The relative glucose consumption rate and lactate production rate were normalized to the cell numbers in the samples.
For the 2-NBDG uptake assay, cells were treated with fresh glucose-free medium containing 100 µM 2-NBDG for 45 min and the mean fluorescence intensity was immediately measured using flow cytometry.
For the glycolysis assay, a Glycolysis Stress Test Kit (Agilent, USA) was used to measure the extracellular acidification rate according to the manufacturer’s instructions and protocols (Seahorse Bioscience, North Billerica, MA, USA).
Tube formation assay
For the tube formation assay, 50 µl of Matrigel (BD) was plated in 96-well plates and incubated at 37 °C for 1 h to allow Matrigel polymerization. Then, 3 × 104 cells per well were seeded onto the Matrigel layer and incubated at 37 °C for 4 h. Randomized fields were captured using microscope. The numbers of capillary-like structures were quantified using ImageJ software. The data are presented as the average numbers of tubes ± standard deviation (SD).
Luciferase reporter assay
All luciferase reporter constructs were generated from the PGL3-basic vector (Promega, Madison, WI, USA). The wild type and mutant sequences of GLUT1 promoter, named PGL3-GLUT1-WT and PGL3-GLUT1-mut, were amplified and cloned into the PGL3-basic vector. The primers used are shown in Table S4. Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega). The relative luciferase signal was presented as firefly luciferase activity normalized to the Renilla luciferase activity. The AP1-luc reporter was purchased from Beyotime Biotechnology (Beijing, China).
Cells were fixed with 1% formaldehyde for 10 min at room temperature, and the fixation was stopped with 0.125 M glycine; the cell lysis buffer was then added and the samples were sonicated to generate 200–1000 bp fragments. The resulting cell lysates were immunoprecipitated using H3K27 antibody (active motif, USA) and analyzed via qRT-PCR. The primers used are shown in Table S4.
Female 4-week-old, nu/nu-BALB/c athymic nude mice were randomly divided into four groups (five mice each). First, 5 × 106 HONE1 cells transfected or co-transfected with the empty vector, BPIFB1 overexpression vector, or GLUT1 overexpression vector were injected subcutaneously into respective mice. Tumor growth was monitored every 3 days. Tumor size was assessed by measuring the largest perpendicular diameters, and the tumor volume was calculated as follows: V = 1/2 × (length) × (width) × (width). After 33 days of subcutaneous inoculation, the mice were euthanized by cervical dislocation and the tumor tissue was excised. The formed tumor masses were removed and weighed. All animal protocols were approved by the Institutional Laboratory of Animal Care and Use Committee at Central South University(2020syaw0896).
Immunohistochemistry and CD31/PAS staining
Immunohistochemistry was performed on formalin-fixed paraffin-embedded sections of clinical NPC tissues and mouse xenograft tissues. Briefly, the tissues were deparaffinized and rehydrated, and the samples were subjected to EDTA-mediated high-temperature antigen retrieval; the samples were then incubated overnight at 4 °C with the primary antibodies. The staining was scored according to the staining intensity and the distribution of stained cells. Distribution was evaluated as none (0), ≤ 10% (1), 10%–50% (2), 50%–80% (3), and > 80% (4). Intensity was evaluated as none (0), faint (1), moderate (2), strong (3), or very strong (4). The sections were reviewed by two pathologists. The final staining scores were calculated as the product of staining intensity multiplied by the percentage of stained cells. To detect VM, a CD31/PAS staining Kit (Solarbio, Beijing, China) was used according to the manufacturer’s instructions. The VM structure criteria were negative for CD31 but positive for PAS (CD31-/PAS+). The numbers of positive cells were counted from ≥ 4 randomly chosen fields.
Statistical analyses were performed using GraphPad Prism 7.0 software. Differences between groups were analyzed using the Student’s t-test when there were only two groups, or using one-way ANOVA when there were more than two groups. Pearson’s correlation coefficient was used to determine the correlations of BPIFB1 with GLUT1, VEGFA, VE-cadherin, and MMP2. A two-tailed value of p < 0.05 was considered statistically significant (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).