Resistin promotes nasopharyngeal carcinoma metastasis through TLR4-mediated activation of p38 MAPK/NF-κB signaling pathway

Abstract

Background

Nasopharyngeal carcinoma (NPC) is a malignant tumor with a high risk of local invasion and early distant metastasis. Resistin is an inflammatory cytokine predominantly produced from the immunocytes in humans. Accumulating evidence suggested clinical association of circulating resistin with the risk of tumorigenesis, the relationship between blood resistin levels and the risk of cancer metastasis. In this study, we explored the blood levels and the role of resistin in NPC.

Methods

A hospital-based case control study was used to assess the association of circulating resistin level with the risk of NPC and clinicopathological characteristics. Wound-healing and Transwell assays were applied to confirm the effects of resistin on NPC cell invasion and migration. A mouse model for lung metastasis was used to explore the role of resistin in NPC tumor metastasis. We also investigated the underlying signaling mechanisms with various specific pharmacological inhibitors and biochemistry analysis.

Results

High resistin levels in NPC patients positively association with lymph node metastasis, and resistin promoted the migration and invasion of NPC cells in vitro. These findings were also replicated in the mouse model of NPC tumor metastasis. We further showed that activation of p38 MAPK pathway was critical for resistin-induced migration and invasion through interaction with TLR4 with NF-κB as the primary mediator of resistin induced epithelial-mesenchymal transition in NPC cells.

Conclusion

Taken together, our results suggests that resistin promotes NPC metastasis through activating the TLR4/p38 MAPK/NF-κB signaling pathway.

Background

Nasopharyngeal carcinoma (NPC) is a malignant tumor originating from the nasopharyngeal epithelium [1]. The incidence of NPC is familial and regional clustering in Southeast Asia and South China [2, 3]. Established risk factors for NPC include Epstein-Barr virus (EBV) infection, family history of NPC, and environmental factors [4–6]. In addition, the development of NPC is usually accompanied by chronic inflammation and metabolic dysregulation. Emerging evidence indicate that immune system and cytokines may play an important role in the diagnosis and prognosis of NPC [7–10]. Indeed, our own previous study found that the levels of macrophage inflammatory protein (MIP)‑1α and MIP‑1β increased the tumorigenic risk of NPC [11].

Inflammatory cytokines, such as adiponectin, resistin, and leptin, have been shown to correlate with the development, progression, and mortality of various types of cancer [12–14]. We have recently shown that null mutation of adiponectin increases the occurrence of endometrial cancer in the PTEN heterozygotic mutant mouse model [15]. Resistin is a peptide hormone predominantly synthesized and secreted from monocytes and macrophages in humans [16], and has been implicated in promoting insulin resistance, obesity, chronic low-grade inflammation, and tumor cell adhesion [17–19].

Recent studies have demonstrated that circulating resistin levels are significantly elevated in patients with breast, gastric, colorectal, lung and endometrial cancers [20–24], suggesting that serum resistin can be a potential diagnostic biomarker for cancers. Further studies have indicated circulating levels of resistin were positively correlated with increased tumor stage, size, and lymph node metastasis in various cancer subtypes [21, 24, 25], and that the treatment of resistin could promote tumor cell proliferation, angiogenesis, migration, and chemotherapy resistance in both animal models and cultured cells [26–30].

In this study, we initially examined clinical correlation of blood resistin levels with the risk of NPC in a case-control study, and further explored the correlation between resistin levels and the clinical characteristics of NPC patients. Mechanistically, resistin promoted the migration and invasion of NPC cells in vivo and in vitro. Furthermore, we investigated the underlying mechanisms of resistin induced epithelial-mesenchymal transition (EMT) in NPC cells.

Materials And Methods

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.

Results

Clinical correlation of serum resistin levels with the risk of NPC

Descriptive characteristics of study subjects with NPC and controls who provided serum are presented in Table 1. Serum resistin levels were higher in NPC patients compared with controls (4.12, 0.83 vs. 3.59, 0.87 ng/ml; P < 0.001) (Table 1). Sex corrected resistin levels were higher among cases compared with controls; these differences were statistically significant among men (4.18, 0.81 vs. 3.58, 0.87 ng/ml; P < 0.001), and women (4.38,1.24 vs. 3.46, 0.89 ng/ml; P = 0.003) (Table 1).

In multivariable logistic regression models, we observed high serum resistin levels were associated with increased NPC risk after we adjusted for established or suspected risk factors of NPC, including age, gender, EBV VCA-IgA and EBNA1 IgA. (Table 2). Analysis the differences of serum resistin levels with clinical characteristics, we observed different levels of serum resistin in NPC patients with different lymph node metastasis (Table 3). Moreover, serum resistin level was the significantly independent predictors for lymph node metastasis in NPC patients by multivariate logistic regression analysis after we adjusted for established or suspected risk factors of NPC, including age, gender, EBV VCA-IgA and EBNA1 IgA (Table 4).

Resistin does not affect the proliferation but promotes the migration and invasion in NPC cells

The manifestation of clinical correlation of resistin, we further explored whether resistin affected the activity of NPC cells. Co-culture of different concentrations of resistin for 48 hours with NPC cells did not affect cell proliferation based on the counting of cell numbers (Fig. 1A). In a separate assay, resistin treatment did not affect the proliferation of NPC cells in colony formation assay (Fig. 1B).

Interestingly, resistin treatment of the NPC cells enhanced the wound healing, migration, and invasion activities in a dose‐dependent manner (Fig. 2A-C). Epithelium-mesenchymal transition (EMT) plays an important role in tumor cell invasion and cancer metastasis [32]. Using Western blot assays, we found that resistin induced the expression of EMT-promoting transcription factors, ZEB1, Snail and Slug; however, the level of β-catenin was not altered by resistin treatment (Fig. 2D). The loss of E-cadherin expression is a hallmark of EMT [33]. Resistin treatment significantly suppressed the expression of E-cadherin in NPC cells as well as other epithelial markers, claudin-1 and ZO-1 (Fig. 2D). Conversely, the levels of N-cadherin and vimentin, the hallmarks of mesenchymal cells, were significantly increased after resistin treatment (Fig. 2D). Importantly, resistin also elevated the expression of matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9) (Fig. 2D), both of which are essential for cell motility and invasion. Thus, resistin can promote the migration and invasion by inducing EMT in the NPC cells.

TLR4 is necessary for resistin-induced NPC cell migration

Resistin is a type of cysteine-rich polypeptide hormone that functions through its purported receptor, Toll‐like receptor 4 (TLR4), which plays a critical role in the regulation of inflammation and is also involved in tumor cell proliferation, invasion, and metastasis [25, 28, 29]. The expression of TLR4 is widely observed in head and neck squamous cell carcinoma (HNSC) and NPC tissues (Fig. S1A, S1B), as well as in several NPC cell lines (Fig. S1C). Although there is no discernable difference in TLR4 expression in NPC as compared with the normal tissues (Fig. S1D), high levels of TLR4 expression were correlated with increasing tumor grade and nodal metastasis (Fig. S1E, S1F). Blockade TLR4 with pharmaceutical inhibitors LPS-RS Ultrapure, a specific TLR4 antagonist, suppressed cellular migration and invasion induced by resistin treatment (Fig. 3A-D). Similarly, knockdown of TLR4 expression significantly nullified resistin-induced elevation of N-cadherin, MMP-2, and MMP-9 expression as well as the reduction of E-cadherin in the NPC cells (Fig. 3E, Fig. S2). These results unequivocally demonstrated that resistin induce the migration and invasion of NPC cells through TLR4.

The p38 MAPK signaling pathway is involved in resistin-induced migration in NPC cells

We further examined the downstream signaling events of TLR4. While resistin did not affect the phosphorylation of AKT, it stimulated the phosphorylation of p38 mitogen-activated protein kinase (MAPK) (Fig 4A), but suppressed the level of ERK1/2 phosphorylation (Fig. 4A). Incubation of the NPC cells with a specific p38 inhibitor, SB203580, largely reversed resistin‐induced migration (Fig. 4B), whereas the inhibitors of ERK1/2, JNK and AKT showed no effect on the migration (Fig. 4B). Consistent with this observation, knockdown of p38 MAPK expression by siRNA prevented cell migration and invasion in resistin-treated NPC cells (Fig. 4C, 4D, Fig. S3). Reduction of p38 expression the resistin induced the changes of EMT-related proteins were inhibited by transfection with p38 MAPK siRNA (Fig. 4E). In analyzing the whole cell lysates from resistin-treated cells, we also found that blocking TLR4 activity through LPS-RS Ultrapure or siRNA transfection abolished resistin induced activation of p38 MAPK (Fig. 4F, 4G), further proving that TLR4 was required for resistin-induced activation of p38 MAPK in the NPC cells.

Resistin regulates expression of EMT related protein via NF-κB

The involvement of Nuclear factor-κB (NF-κB) in regulating the expression of EMT related protein has been well documented [34, 35]. Co-culture with resistin increased phosphorylation of p65 protein in a dose-dependent manner in the NPC cells (Fig. 5A). Resistin treatment promoted the phosphorylation of IκBα (Fig. 5B), which in turn leads to the degradation of IκBα and activation of NF-κB. Consistent with these results, the proportion of nuclear translocation of p65 and p50 proteins markedly increased following resistin treatment (Fig. 5C). Moreover, pre-treatment with NF-κB inhibitors, BAY-117083 and PDTC, completely suppressed resistin-induced EMT and migration of the NPC cell (Fig. 5D, 5E). Combined together, these results demonstrate that resistin promotes EMT of NPC cells largely through activation of NF-κB pathway.

We further delineated the molecular mechanisms underlying resistin-induced EMT alteration in the NPC cells, particularly with respect to NF-κB signaling. The transcriptional activation of NF-κB induced by resistin was suppressed by co-culture with LPS-RS Ultrapure, a specific inhibitor of TLR4 (Fig. 6A). Immunofluorescence staining revealed resistin induced nuclear translocation of p65 was abolished by LPS-RS Ultrapure treatment in the NPC cells (Fig. 6B). Importantly, impeding the activation of p38 MAPK via its pharmacological inhibitor also suppressed resistin mediated activation of NF-κB in NPC cells (Fig. 6A, 6C), and reversed resistin-induced phosphorylation of IκBα (Fig. 6D, 6E). Taken together, these results demonstrated that induction of cellular migration by resistin depends on TLR4/p38 MAPK/NF-κB signaling pathway.

Resistin promotes NPC tumor metastasis in animal models

To clarify whether intravenously administered resistin would exhibit a pharmacokinetic profile suitable for in vivo evaluation, we measured serum concentrations of resistin in nude mice after intravenous administration of 20 μg/kg of resistin. We found that the concentration of resistin was 20.79 ng/ml at 15min (Fig. 7A), consistent with the concentration of resistin promoting migration in vitro.

To understand the effects of resistin in metastasis in vivo, we established luciferase expressing 5-8F-Luc cells. Lung metastasis model was established through intravenously injecting 5-8F-Luc cells into nude mice, and tumor metastasis was monitored by bioluminescence imaging (Fig. 7B). Intravenous delivery of exogenous recombinant resistin protein significantly increased lung metastasis 6 weeks post-injection (Fig. 7C-E) with the lungs showing more and larger metastatic nodules in resistin-treated group than in the control group (Fig. 7F, 7H), accompanied by increased the wet weight of the lungs (Fig. 7G). Immunohistochemical staining showed that treatment with exogenous resistin markedly elevated the levels of phospho-p65 and vimentin (Fig. 7I, 7J). Taken together, these results unequivocally validated the concept that elevation of blood resistin could enhance the metastasis ability of NPC cells in the metastatic animal model.

Discussion

Using a case-control cohort of 100 patients and 100 controls, we revealed for the first time that high serum resistin levels were associated with an increased risk of NPC. Importantly, we showed that serum resistin level was positively correlated with lymph node metastasis in the NPC patients. Consistent with these clinical findings, resistin treatment promoted the invasion and migration of NPC cells in cultured cells, as well as metastasis in human NPC cell-derived animal model. Resistin promoted the invasion and metastasis of NPC cells through inducing EMT, a molecular event that was initiated by the interaction of resistin with its purported receptor, TLR4, and further mediated by the activation of p38 MAPK and NF-κB pathways.

Nasopharyngeal carcinoma is typically characterized by a heavy lymphocytic infiltration, suggesting that inflammation might be a potential risk factor for the progression of this cancer [11]. Indeed, a series of cytokines, such as leptin, adiponectin and visfatin have been found in tumor microenvironment and implicated in cancer cell growth, apoptosis, invasion, angiogenesis, and metastasis [13, 19]. Resistin is considered a cytokine predominantly produced and secreted by macrophages, dendritic cells, and monocytes in humans [17, 36]. The purported ortholog receptor of resistin, TLR4, is usually expressed has recently been identified on multiple tumor cells, including gastric cancer, breast cancer and lung adenocarcinoma [25, 28, 29]. Recent studies already suggested that the polymorphisms and high expression of TLR4 are linked to an increased risk of NPC [37–39]. Our results indicated that TLR4 was widely expressed in NPC, head and neck tumors, and its expression level was positively correlated with high grades of tumor and lymph node metastasis in HNSC, and that inhibition of TLR4 signaling prevented resistin-induced migration and invasion, and that TLR4 knockdown prevented abolished resistin-induced expression of multiple critical EMT proteins. These findings are consistent with published reports that activation of TLR4 could promote cancer cell proliferation, adhesion, EMT, invasion and migration [25, 28, 29]. Thus, TLR4 is the functional receptor of resistin signaling as well as the increased migration and invasion in NPC cells.

Intracellular signal pathway, such as MAPK and PI3K/AKT, are involved in mediating TLR4 functions [40, 41]. In the NPC cells, we only found the activation of p38 signal after resistin treatment, and that pretreatment with specific inhibitors of p38 MAPK largely reversed resistin-induced migration or invasion. Importantly, blockade of TLR4 signal reduced resistin-induced activation of p38 MAPK signaling, proving that TLR4/p38 MAPK signaling pathway is critical for resistin’s induction of migration and invasion. NF-κB proteins belong to a family of transcription factors, involved in cellular functions including inflammation, immune responses, cell proliferation and apoptosis [40]. Moreover, NF-κB is an important regulator of EMT process of tumor cells [34, 35]. Activation of NF-κB by cytokines from tumor microenvironment plays an important role in the invasion and metastasis of NPC cells [42]. Indeed, pharmacological inhibition of the NF-kB signaling pathways attenuated resistin-induced EMT related protein expression, a process that depended upon activation of TLR4/p38/NF-κB pathway (Fig. 8). These data, combined together, provide a mechanism underlying how high blood levels of resistin promotes metastasis of NPC.

Conclusion

In conclusion, the findings from this study demonstrated that serum resistin levels are positively correlated with the risk of NPC development and can potentially serve as an independent predictor of lymph node metastasis in NPC cases. We propose that resistin promoted NPC metastasis through induction of EMT by activating TLR4/p38 MAPK /NF-κB signaling pathway. Circulating levels of resistin may be put into consideration of predicting prognosis of NPC patients.

Abbreviations

NPC: nasopharyngeal carcinoma; EBV: Epstein-Barr virus; EMT: epithelial-mesenchymal transition; mRNA: messenger RNA; HNSC: head and neck squamous cell carcinoma; TCGA: The Cancer Genome Atlas; GEO: Gene Expression Omnibus (GEO); siRNA: small interfering RNA; MMP-2: matrix metalloproteinase 2; MMP-9: matrix metalloproteinase 9; NF-κB: Nuclear factor-κB; MAPK: mitogen- activated protein kinase; TLR4: Toll‐ like receptor 4.

Declarations

Ethics approval and consent to participate

This study was approved by the Institutional Review Board of Sun Yat-sen University Cancer Center (SYSUCC) (NO. YP2009051). The study was carried out in compliance with the ARRIVE guidelines. All animal experimental procedures were approved by the Experimental Animal Academic Ethics Committee of South China University of Technology (AEC2021059).

Consent for publication

Not applicable.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that there is no conflict of interest regarding the publication of this paper.

Funding

This work was supported by the Key Research and Development Program of Guangdong Province for "Innovative drug creation" (2019B020201015 to FL); the National Key R&D Program of China (2018YFA0800603 to AZZ); the National Natural Science Foundation of China (81630021 to AZZ, 82100064 to YM; 81872700, 82073625 to SC); The Guangdong Innovative Research Team Program (2016ZT06Y432 to AZZ, FL); The Startup R&D Funding of Guangdong University of Technology (50010102 to AZZ and FL); The Key Research and Development Program of Guangdong Province (2019B020227003 to FL). Dr Du was supported by The Key Project of Department of Education of Guangdong Province (2020ZDZX1048).

Authors’ contributions

FL, ZZ and AZZ designed the research. JD and SC performed collection and testing of clinical samples, epidemiological analysis. ZZ, QX and YL performed laboratory investigation and analysis data. SZ, ZZ and YM oversaw laboratory analyses and provided statistical support. FL, AZZ and ZZ wrote and reviewed the manuscript.

Acknowledgements

The NPC serum specimens were provided by the serum bank of SYSUCC in the case–control study and thank all the researchers and personnel who provided valuable suggestion. The experimental data were analyzed in Guangdong University of Technology. Clinical data were collected and stored in the Cancer Center of Sun Yat-sen University

Authors' information

¹The School of Biomedical and Pharmaceutical Sciences, Guangdong University of Technology, Guangzhou 510006, China. ² Department of Epidemiology and Health Statistics, School of Public Health, Guangdong Medical University, Dongguan 523808, China. ³ Department of Cancer Prevention Research, Sun Yat‐sen University Cancer Center, Guangzhou 510060, China

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Tables

Table 1. Characteristics of nasopharyngeal carcinoma cases and control subjects

Abbreviations: VCA-IgA: viral-caspid antigen–IgA; EBV: Epstein–Barr virus.

^a P values were calculated by Wilcoxon rank sum test and Chi-Square test.

Table 2. Analysis of multivariate logistic regression with risk for NPC^a

Abbreviations: OR: odd ratio; 95% CI: 95% confidence interval; VCA-IgA: viral-caspid antigen–IgA; EBV: Epstein–Barr virus.

^a Multivariate analysis were performed by Cox regression models.

^b P values were calculated by a univariate Cox proportional hazards regression model.

Table 3. Differences of serum resistin among NPC patients with different clinicopathological features

Abbreviations: LN, lymph node

^a P values were calculated by Wilcoxon rank sum test.

^b Stage was based on the AJCC TNM staging system.

Table 4. Analysis of multivariate logistic regression with risk for NPC LN metastasis as a dependent variable^a