Animal breeding and subcutaneous transplantation
All animal experimental procedures were approved by the Experimental Animal Academic Ethics Committee of Guangdong Pharmaceutical University (gdpulacspf2017064).
Adiponectin deficient mice were generously provided by Professor Philipp Scherer of the University of Texas Southwestern (Dallas, TX, USA). Male nude mice were purchased from the GemPharmatech (Nanjing, Jiangsu, China), and crossed with APN−/− female mice to generate three genotypes of nude mice: APN+/+, APN+/−, and APN−/−. Mice were kept in the Laboratory Animal Center of Guangdong Pharmaceutical University (Guangzhou, Guangdong, China), and maintained in specific pathogen-free conditions with stationary temperature of 23–25 °C and 12-h light/dark cycles.
1 × 106 CNE-2 or 5-8F cells were resuspended in 100 µL PBS and subcutaneously injected into the right armpit region of five- to six-week-old male nude mice. Tumors were measured using digital Vernier calipers every day, with tumor volume calculated using the formula [sagittal dimension (mm) × cross dimension (mm)]2/2 and expressed in cm3. All animals were sacrificed, tumor tissues were collected, imaged, and weighed.
For AdipoRon administration, four days after injection NPC cells, the mice were randomly allocated into two groups (Vehicle and AdipoRon groups) of 6 mice per group. In the AdipoRon group, mice were intragastrically administered 50 mg/kg AdipoRon suspended in corn oil every other day. In the Vehicle group, mice were administered solvent alone in corn oil.
Cell culture and regents
The CNE-2 and S18 cell lines were kindly gifted by Professor Chaonan Qian at SYSUCC. HNE2, 5-8F, C666-1 and 6-10B cells were from the Central South University Advanced Research Center (Changsha, Hunan, China). HNE2, 5-8F and 6-10B cells were cultured in RPMI-1640 medium, CNE-2 and S18 cells were cultured in Dulbecco's modified eagle medium containing 4.5 mg/mL glucose, all supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA), 100 U/mL penicillin and 100 ug/mL streptomycin (Hyclone, Logan, UT, USA). Cells were maintained in a humidified atmosphere of 5% CO2 at 37 °C. The cell line was authenticated via deoxyribonucleic-acid profiling using short tandem repeat analysis.
Recombination human full-length adiponectin was dissolved in deionized water to prepare a working stock solution of approximately 0.5 mg/mL (BioVendor, Brno, Czech Republic). AdipoRon was dissolved in DMSO to prepare a working stock solution of approximately 50 mM (Selleck Chemicals, Houston, TX, USA). Compound C was purchased from MedChem Express (Monmouth Junction, NJ, USA), and was prepared as a stock concentration at 10 mM in DMSO and stored at − 80 °C.
Cell viability and proliferation assays
Cell viability was measured using cell counting kit-8 (CCK-8) (Sangon Biotech, Shanghai, China). Cells were cultured in 96-well plates, with six duplicate wells in each group, and pre-treated in 100 μL medium with or without different concentrations of inhibitors for 1 h, followed by solvent alone, AdipoRon or APN for the indicated period. After incubation, CCK-8 solution was added to each well followed by a further 2 h incubation under 5% CO2 at 37 °C. Absorbance was automatically measured at 450 nm with a microplate reader (Infinite F50, Tecan Group Ltd., Mannedorf, Switzerland). The relative cell viability was calculated as the percentage of untreated cells.
Cell proliferation was measured using plate clone formation and 5-ethynyl-2'-deoxyuridine (EdU) assays. CNE-2 cells were plated in 12-well plates and treated with human recombinant adiponectin or AdipoRon. Then, the culture medium was replaced with fresh medium containing adiponectin every 3 days. After 7 days’ treatment, the medium was removed, and cell colonies were fixed and stained with crystal violet (Sangon Biotech). Images were taken with a digital camera, colonies contained more than 50 cells in each well were counted. The EdU assay were preformed according to manufacturer’s instructions (RiboBio, Guangzhou, Guangdong, China). The EdU-positive rate was calculated as EdU-positive cells/Hoechst-stained cells × 100%. The assays were repeated in triplicate.
Transient transfection with small interfering RNA
The small interfering RNA (siRNA) oligos against AdipoR1, AdipoR2 and scrambled control siRNA were commercially synthesized by RiboBio (Guangzhou, Guangdong, China), and transfected with riboFECT CP transfection reagent (RiboBio, Guangzhou, Guangdong, China) according to the manufacturer’s protocol. The siRNA duplexes used for this study are listed in Additional file 1: Table S2. Two days after transfection, the cells were subjected to total RNA isolation and viability assays.
Cell cycle assay
CNE-2 cells were incubated in serum-free medium overnight, and then cells were treated with adiponectin or AdipoRon. Cells were collected, washed, and suspended in cold PBS. Cells were then fixed in 70% cold ethanol at 4 °C overnight. After fixation, the cells were washed with PBS twice, resuspended in 0.2 mL PI/RNase staining buffer (BD Biosciences, San Jose, CA, USA) for 30 min at room temperature. The cell cycle distribution was determined by the DxP Athena flow cytometry system (Cytek Biosciences, Fremont, CA, USA), and the percentages of different phases of cell cycle were determined using ModFit LT 5.0 (Verity Software house, Topsham, ME, USA).
Cell apoptosis assays
PE Annexin V apoptosis detection kit (BD Biosciences, #559763) was used to determine cell apoptosis. Cells treated with the indicated drug concentrations. After treatment, we harvested the cells, washed them twice with PBS, and stained them using Annexin V-PE and 7-AAD for 15 min in the dark, followed by analysis using the DxP Athena flow cytometry system (Cytek Biosciences). The upper right quadrant represents late apoptotic cells, and the lower right quadrant represents early apoptotic cells. The assessment of the apoptosis rate was the sum of early and late apoptosis.
RNA extraction and qRT-PCR
Total RNA was extracted from cell by using Trizol reagent (Sigma; T9424). The quantity and quality of RNA were determined using a ScanDrop2 nano-volume spectrophotometer (Analytik Jena), and reversely transcribed based on the HiScript II Q RT kit (Vazyme; R223) according to the manufacturer’s instructions. Amplification and real-time detection were performed on a qTOWER3 G real-time PCR system (Analytik Jena) by using ChamQ Universal SYBR qPCR Master Mix (Vazyme; Q711) in 20 μL reaction. The relative expression levels of each targeted gene were normalized by subtracting the corresponding mouse β-actin threshold cycle (CT) values by using the ΔΔCT comparative method. Three biological replicates per group were used for qPCR. Primers were synthesized by Sangon Biotech (Shanghai, China). Sequences of all primers used are provided in Additional file 1: Table S3.
Immunoblotting analysis
Cells were collected and homogenized in RIPA lysis buffer containing a protease inhibitor (Beyotime Biotechnology, Shanghai, China). The protein concentration was determined using bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Then the equivalent proteins were separated by SDS-PAGE, and transferred on Immobilon-p Transfer Membrane (Millipore, Billerica, MA, USA) with the wet electrical transfer method using Mini Trans-Blot (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked with 5% nonfat dried milk in TBS containing 0.1% Tween-20 for 1 h at room temperature; followed by the primary antibody incubation overnight at 4 °C and the secondary antibody for 1 h at room temperature. The bands were detected with ECL detection system according to the manufacturer’s protocol (Thermo Fisher Scientific) using ChemiDoc XRS + system (Bio-Rad). The gray intensities of bands were measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA) and were normalized for β-actin. The antibodies used were as follows: mouse anti-β-actin (A5316) and mouse anti-GAPDH (G8795) (Sigma-Aldrich, St. Louis, MO, USA); rabbit anti-p21 (#2947), rabbit anti-p27 (#3686), rabbit anti-CDK2 (#2546), rabbit anti-CDK4 (#12790), rabbit anti-Cyclin B1 (#12231), rabbit anti-Cyclin D1 (#2978), rabbit anti-ERK1/2 (#4695), rabbit anti-p-ERK1/2 (#4370), rabbit anti-LKB1 (#3047), rabbit anti-p-LKB1 (#3482), rabbit anti-AMPKα (#5831), and rabbit anti-p-AMPKα (#2535) (Cell Signaling Technology, Danvers, MA, USA); mouse anti-AdipoR2 (sc-514045) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); rabbit anti-AdipoR1 (ab126611) (Abcam, Cambridge, MA, USA); Goat anti-mouse-HRP and goat anti-rabbit-HRP (Jackson ImmunoResearch, West Grove, PA, USA). The densitometry of the bands was quantified using ImageJ software.
Immunohistochemistry staining
IHC was carried out as described previously [26]. The sections were deparaffinized, rehydrated and performed antigen retrieval with microwave method in 10 mM citrate buffer. The sections blocked with 3% H2O2 for 15 min, incubated with 5% normal goat serum in PBST for 1 h at 37 °C. Then sections were incubated with primary antibodies mouse anti-AdipoR2 (Santa Cruz; 1:50), rabbit anti-AdipoR1 (Abcam; 1:100), rabbit anti-Ki-67 (#9027, CST) and rabbit anti-CD31 (#77699, CST) at 4 °C overnight. After washing, followed by horseradish peroxidase-conjugated secondary antibody incubation for 1 h. Sections were incubated with developing solution (diaminobenzidine, DAB) and counterstained with hematoxylin (ZSGB-Bio, Beijing, China). Goat anti-mouse-HRP and goat anti-rabbit-HRP (Jackson ImmunoResearch) were used as the secondary antibodies.
Bioinformatics analyses of AdipoR1 and AdipoR2 expression
Messenger RNA (mRNA) expression data for 566 head and neck squamous cell carcinoma (HNSC) samples were downloaded from The Cancer Genome Atlas (TCGA) data portal (https://xenabrowser.net/datapages/). According anatomic neoplasm subdivision, including 44 tonsil, 9 oropharynx, 143 oral tongue, 87 oral cavity, 3 lip, 128 larynx, 10 hypopharynx, 7 hard palate, 66 floor of mouth, 22 buccal mucosa, 29 base of tongue and 18 alveolar ridge tumors.
Microarray gene expression profiling data including GSE12452 (10 normal controls and 31 NPC samples) [27], GSE53819 (21 normal controls and 18 NPC samples) [28], GSE61218 (21 normal controls and 18 NPC samples) [29], GSE64634 (4 normal controls and 12 NPC samples) [30], GSE103611 (48 NPC samples) [31], GSE132112 (95 NPC samples) [32], and GSE13597 (3 normal controls and 25 NPC samples) [33]. The RNA-seq data of NPC samples including GSE102349 (113 NPC samples) [34] and GSE68799 (4 normal controls and 42 NPC samples). These data were downloaded from the Gene Expression Omnibus (GEO) database.
Statistical analysis
Data were expressed as mean ± SD. Statistical analyses were performed using GraphPad Prism 7.0 (GraphPad Software, La Jolla, CA, USA). The statistical significance between groups was assessed by Student’s t test or by analysis of variance (ANOVA) with Sidak's multiple comparisons test. A value of P < 0.05 was considered statistically significant.