Patient samples and ethical approval
The serum samples of 150 patients with NPC and 150 healthy controls undergoing routine health examinations were consecutively collected from the serum bank of Sun Yat-sen University Cancer Center (SYSUCC). The patients were selected based on the criteria as previously described [11]. The TNM staging for patients with NPC was defined according to the staging system described in the seventh edition of Union for International Cancer Control (UICC), and NPCs were classified by the World Health Organization (WHO) classification. All diagnoses of NPC were proven by biopsy. This study was approved by the Institutional Review Board of Sun Yat-sen University Cancer Center (SYSUCC) (NO. YP2009051).
Animal study
All animal experimental procedures were approved by the Experimental Animal Academic Ethics Committee of South China University of Technology (AEC2021059). All experimental methods were carried out in compliance with the ARRIVE guidelines. 5-week-old male nude mice were purchased from the GemPharmatech (Nanjing, Jiangsu, China), were kept in the Laboratory Animal Center of South China University of Technology (Guangzhou, Guangdong, China), and maintained in specific pathogen-free conditions with stationary temperature of 23-25°C and 12-h light/dark cycles.
To establish a tumor metastasis model in animals, 5-8F cells were transfected with lentivirus-vector expressing system LV-luciferase (Genechem, Shanghai, China)) and selected for stabilized expressing clones by series dilution selection. The 5-8F-Luc cells were pretreated with or without resistin (25 ng/ml) for 48 h, 1×106 cells were washed and resuspended in 100 μl PBS. Subsequently, cells were injected into the lateral tail vein of nude mice. Mice were injected intravenously with 20 μg/kg resistin for 2 weeks, first for three consecutive days, and then replaced with injection every other day. Tumor cell metastasis was monitored using the IVIS Lumina series Ⅲ imaging system (Xenogen, Alameda, CA, USA). After 6 weeks, the mice were sacrificed, the lungs were separated, weighted, and photographed. Subsequently, the lungs were fixed and embedded in paraffin, and processed for hematoxylin and eosin (HE), and immunohistochemistry staining.
Cell culture and regents
The human NPC cell lines CNE-2 and S18 were kindly gifted by Professor Chaonan Qian at SYSUCC. HNE2 and 5-8F cell line were obtained from the Central South University Advanced Research Center (Changsha, Hunan, China). C666-1 cell line were obtained from the American Type Culture Collection (ATCC, VA, USA). CNE-2, C666-1 and S18 cells were cultured in Dulbecco's modified eagle medium, HNE2 and 5-8F cells were cultured in RPMI-1640 medium, all supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA), 1% penicillin-streptomycin (Hyclone, Logan, UT, USA). Cells were maintained in a humidified atmosphere of 5% CO2 at 37°C.
Recombination human resistin was dissolved in deionized water to prepare a working stock solution of approximately 0.01 mg/ml (PeproTech, Rocky Hill, NJ, USA). LPS-RS Ultrapure was purchased from InvivoGen (San Diego, CA, USA). SB203580, Pyrrolidinedithiocarbamate ammonium (PDTC) and BAY 11-7082 were purchased from MedChem Express (Monmouth Junction, NJ, USA).
Cell viability and proliferation assays
NPC cells were cultured in 96-well plates, treated with different concentrations of resistin for 48 h. After incubation CCK-8 solution according to the instructions (Sangon Biotech, Shanghai, China), absorbance was 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 carried out as described previously [31].
Wound-healing assay
The cells were seeded in 12-well plates, cell confluence was 100% after adherent. Monolayer cells were washed with phosphate-buffered saline (PBS) and scraped with a plastic 200 µL pipette tip, and then incubated with fresh medium treated with resistin. The “wounded” were photographed by microscope at 0h and 24h. The relative migration rates were calculated by cell-covered area (0h) / cell-covered area (24h).
Migration and invasion assays
24‐well Transwell inserts (BD Biosciences, San Jose, CA, USA) coated with or without growth factor‐reduced Matrigel (Corning Incorporated, Corning, NY, USA) were used for migration and invasion assays. NPC cells were suspended in 200ul serum-free medium treated with or without resistin, added to the upper chamber of a Transwell chamber in duplicate, and incubated for 24 h at 5% CO2 at 37° C, allowing them to migrate into the lower chamber containing medium with 20% FBS. For signaling blockade, cells were pre-incubated with inhibitor for 2 h. After 24 h of incubation, membrane-trapped cells were fixed, stained with crystal violet, and counted using a light microscope.
Transient transfection with small interfering RNA (siRNA)
TLR4, p38 MAPK and scrambled control siRNAs were synthesized by RiboBio (Guangzhou, Guangdong, China), and transfected with transfection reagent kit (RiboBio) according to manufacturer’s protocol. The siRNA sequences used for this study are listed in Table S1.
RNA extraction and qRT-PCR
Total RNA was isolated from cell by using Trizol reagent (Sigma-Aldrich, St. Louis, MO, USA). cDNA was reversely transcribed using the HiScript II Q RT kit (Vazyme Biotech, Nanjing, China). Quantitative real-time PCR (qRT-PCR) analysis was performed in a qTOWER3 G real-time PCR system (Analytik Jena) by using ChamQ Universal SYBR qPCR Master Mix (Vazyme) according to the manufacturer’s instructions. The relative expression levels of mRNA were normalized to the expression of β-actin by using the 2-ΔΔCT method. Primers were synthesized by Sangon Biotech (Shanghai, China), primer sequences are provided in Table S2.
Immunoblotting analysis
Cells were scraped and lysed with Radio-Immunoprecipitation (RIPA) lysis buffer containing a protease inhibitor (Beyotime Biotechnology, Shanghai, China), quantified with bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Nuclear and cytoplasmic protein extraction was analyzed using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime) according to the manufacturer’s instructions. Then the equivalent proteins were separated by SDS-PAGE, and transferred on polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membranes were blocked with 5% nonfat dried milk blocking buffer for 1h at room temperature; followed by overnight incubation at 4°C with the primary antibody. Membranes were washed with Tris–HCl buffer containing Tween 20 and then incubated the secondary antibody for 1h at room temperature. Blots were detected with ECL detection system (Thermo) using ChemiDoc XRS+ system (Bio-Rad). The antibodies used were as follows in Table S3.
Immunofluorescence staining
NPC cells were plated onto glass bottom cell culture dish (Wuxi NEST Biotechnology Co., Ltd, Jiangsu, China). After incubation with resistin, cells were fixed with 4% paraformaldehyde, permeabilized in 0.2% Triton X-100 PBS buffer, and then blocked with Immunol Staining Blocking Buffer (Beyotime), incubated with primary antibody rabbit anti-p65 (CST; 1:400) overnight at 4°C, followed goat anti-rabbit Alexa 555 fluorescent secondary antibody incubation for 2h at room temperature. After washing with PBS, cells were mounted with anti-fade mounting medium with DAPI (Beyotime). The images were obtained using fluorescent microscope.
Dual-luciferase reporter assay
The pNFκB-luc and pRL-TK plasmids were purchased from Beyotime. NPC cells were seeded in BeyoGold™ 96-Well White Opaque Plates (Beyotime), transfected with Lipofectamine™ 3000 Reagent (Invitrogen, Carlsbad, CA, USA). Reporter enzyme activity was determined with a dual-luciferase reporter assay system (Beyotime), according to manufacturer’s instructions. Luminescence signal was determined with a Varioskan LUX multimode microplate reader (Thermo). Relative luminescence units = Firefly luciferase activity / Renilla luciferase activity.
Immunohistochemistry staining
The sections were deparaffinized, rehydrated and performed microwave heating antigen retrieval in citrate antigen retrieval solution. The sections were blocked with 3% H2O2 for 15 min, incubated with Immunol Staining Blocking Buffer (Beyotime) for 1 h. And then incubated with primary antibodies mouse anti-p-p65 (CST; 1:50), rabbit anti-Vimentin (CST; 1:50), and rabbit anti-E-cadherin (CST; 1:100) at 4°C overnight. After washing, followed by goat anti-mouse-HRP or anti-rabbit-HRP (Jackson ImmunoResearch, West Grove, PA, USA) incubation for 1 h. Sections were incubated with developing solution (diaminobenzidine, DAB) and counterstained with hematoxylin (ZSGB-Bio, Beijing, China).
Statistical analysis
Data are presented as mean ± SD, were analyzed by Student’s t test or by analysis of variance (ANOVA) with Sidak's multiple comparisons test using GraphPad Prism 7.0 (GraphPad Software, La Jolla, CA, USA). A value of P < 0.05 was considered statistically significant.