Keywords

Nasopharyngeal carcinoma, m6A, FTO, Immune cells infiltration, GSEA, KEGG

Abbreviations

m6AN6: Methyladenosine; NPC: Nasopharyngeal Carcinoma; FTO: Fat Mass and Obesity Associated; GSEA: Gene Set Enrichment Analysis; KEGG: Kyoto Encyclopedia of Genes and Genomes; VHL: von Hippel-Lindau; GEO: Gene Expression Omnibus

Introduction

Nasopharyngeal carcinoma is a malignant epithelial tumor of the nasopharynx, which is common in Southeast Asia and is highly invasive and metastatic. Mainly distributed in Guangdong, Fujian, Hong Kong, and other places. There are considerable geographic differences, with incidence rates as high as 30 per 100,000 in endemic regions such as southern China and Southeast Asia [1]. The main reasons are EB virus infection, genetic and nutritional factors. Among them, Epstein-Barr virus infection, especially the resulting epigenetic changes, is closely related to the pathogenesis of nasopharyngeal carcinoma [2].

Epigenetic modification refers to reversible and heritable changes in gene function, such as DNA methylation, RNA methylation, and histone acetylation, without changes in genomic DNA sequences [3]. Research shows that epigenetic regulation plays a significant role in embryonic development, differentiation, and disease occurrence in mammals. N6-methylladenine (m6A) is one of the most common and most frequently occurring apparent modifications in mRNA sequences. This modification process is dynamically reversible [4]. It is catalyzed by a methyltransferase complex consisting of METTL3, METTL14, and WTAP called "encoder" and can be "erased" by the demethylases FTO and ALKBH5 called "decoders" [5]. With the rapid development of high-throughput sequencing technology and the gradual deepening of epigenetics research, the function and role of m6A methylation in various biological processes have attracted human attention [6]. The study of m6A has gradually emerged as one of the biggest frontier research hotspots in the life sciences field. Numerous studies have shown that m6A regulates gene expression by regulating mRNA structure, splicing, stability, and translational efficiency, regulates microRNA processing and other pathways, and participates in biological processes such as repair of DNA damage, stem cell differentiation, and tumorigenesis is involved [5].

In 2011, the FTO gene was found to have m6A demethylase activity [7], which made m6A modification a dynamic reversible modification similar to DNA methylation and histone methylation.

Research has shown that FTO contributes to the self-renewal and immune escape of cancer stem cells, also demonstrating the potential of FTO for targeted therapy [8]. Yi Niu, et al. [9] found that FTO is up-regulated in human breast cancer and is significantly associated with lower survival in breast cancer patients. Yiren Xiao, et al. [7] discovered a synthetic lethal interaction between FTO and the tumor suppressor VHL (von Hippel-Lindau) in renal cell carcinoma cells. Taketo K, et al. [10] found that the m6A transcriptome, one of the writers, promotes chemotherapy and radiotherapy resistance in pancreatic cancer cells. Rui Su, et al. [11] also confirmed that the m6A methylase FTO plays a critical role in the occurrence and development of cancer. R-2-hydroxyglutarate (R-2HG) inhibits the proliferation and survival of cancer cells with high FTO expression through targeting FTO/m6A/MYC/CEBPA signalling.

In this study, the differential expression of m6A in nasopharyngeal carcinoma and adjacent tissues was analysed by bioinformatics, and FTO was selected as the protective gene m6A demethylase as the research object. Found that FTO expression correlated with disease progression and also increased immune cell infiltration in nasopharyngeal carcinoma. In functional studies, it was determined that FTO is lowly expressed in nasopharyngeal carcinoma cells and enhances the invasive and migrate abilities of nasopharyngeal carcinoma cells. Finally, we carried out Gene set enrichment analysis (GSEA), FTO expression was significantly correlated with the KEGG genome closely related to cancer development, including focal adhesions, TGF-β signaling pathway, and WNT signaling pathway. Our data demonstrate the significant correlation of FTO expression with the KEGG genome closely associated with cancer development, and the protective role of FTO during NPC progression, and suggest that FTO may be a promising target for future NPC therapy.

Materials and Methods

NPC datasets collecting and processing

Public RNA-sequencing and microarray data of NPC were collected from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/). In total, 216 NPC samples and 36 normal samples from five datasets were enrolled in this study: GSE102349 (RNA-sequencing date, 113 samples), GSE64634 (microarray data, 16 samples), GSE68799 (RNA-sequencing date, 46 samples), GSE538196 (microarray data, 36 samples), GSE12452 (microarray data, 41 samples). Of these six datasets, clinical data were available for GSE102349 (progression-free survival and clinical stage) and GSE12452 (clinical stage) datasets. For RNA-sequencing data, Fragments per Kilobase Million (FPKM) values were converted to Transcripts per Million (TPM) to match microarray data using the "limma" package in R software (version 4.1.0). Then expression matrix of five datasets was subjected to quantile normalization to remove the batch effect and merged into one meta-cohort via the "sva" package in R software (version 4.1.0).

Identification of differentially expressed m6A regulators between NPC and normal samples. A total of 22 m6A regulators, including 8 writers (CBLL1, KIAA1429, METTL3, METTL14, RBM15, RBM15B, WTAP, and ZC3H13), 2 erasers (ALKBH5 and FTO), and 12 readers (ELAVL1, FMR1, HNRNPA2B1, HNRNPC, IGF2BP2, IGF2BP3, LRPPRC, YTHDC1, YTHDC2, YTHDF1, YTHDF2, and YTHDF3) were collected from recent publications. The differential expression analysis of these m6A regulators was performed utilizing the "limma" package in R software (version 4.1.0). Heatmap and boxplot were drawn to visualize the results by "heatmap" and "ggplot2" packages respectively. Pearson correlation analysis was used to calculate the correlation coefficient between m6A regulators expression and visualized by a network diagram using the "graph" package.

Survival and clinical analysis

GSE102349 dataset containing 113 samples with progression-free survival (PFS) information was used for survival analysis. In brief, NPC patients of this dataset were divided into two groups according to the median cutoff of each m6A regulators expression. Then, the univariate Cox regression analysis was used to determine hazard ratios (HRs) and 95% confidence interval (95% CI) of various m6A regulators in PFS. m6A Regulators with HR greater than 1 are considered risk factors for death, less than 1 indicates vice-versa. For FTO, the survival analysis results were visualized individually by Kaplan–Meier (KM) curves with a log-rank test. 144 samples with clinical stage information from GSE102349 and GSE12452 datasets were used to study the correlation between FTO expression and clinical stage. The GraphPad Prism 8 software was used to count and visualize the expression distribution of FTO in clinical stages I-IV.

Immune cells infiltration analysis

To quantify the immune cell infiltrated in NPC samples, we used previously described and validated techniques: single sample Gene Set Enrichment Analysis (ssGSEA). ssGSEA calculates an enrichment score for a gene signature (genes list) by comparing the ranks of the genes in the signature with the ranks of all other genes in the expression matrix [12]. We utilized this algorithm to calculate enrichment scores for immune signatures in each sample by the application of the "GSVA" package. The immune signatures were collected from publication [13,14] including plasmacytoid DCs, inactivated DCs, activated DC, DCs, NK cells, macrophages, mast cells, neutrophils, B cells, CD8+ T cells, T helper cells, Tfh, Th1, Th2, Treg, TIL, cytolytic activity, and check-point. 2.5 KEGG pathway-based enrichment analysis with Gene Set Enrichment Analysis (GSEA).

To study the possible downstream pathways of FTO, we used GSEA to test for KEGG pathways that were significantly associated with FTO expression by software (version 4.1.0) downloaded from the website (http://www.gsea-msigdb.org/gsea/downloads.jsp). The KEGG pathway signatures identified using this approach were collected from MsigDB databases (http://www.gsea-msigdb.org/gsea/downloads.jsp). Weighted enrichment scores were calculated with gene expression lists ranked by Pearson. The minimum gene set size was set to 15 genes, the maximum gene set size was set to 500 genes, and the number of permutations was set to 1000. Pathway gene sets with nominal P-value < 0.005 and an FDR < 0.1 were considered statistically significant. Then a bubble plot and network connectivity (κ-score) ≥ 0.5 were drawn for visualization using the "ggplot2" package and Cytoscape plug-in "EnrichmentMap" respectively.

Cell lines and cell culture

Five cell lines were used in this study. CNE-1, CNE-2,5-8F were derived from nasopharyngeal carcinoma and kept in our laboratory (Central laboratory of Shenzhen hospital, university of Hong Kong). Nasopharyngeal carcinoma cell lines C666-1 and NP69 (immortalized nasopharyngeal carcinoma cell lines) were purchased from the Cell Bank of Shanghai Academy of Sciences. Human nasopharyngeal carcinoma cell lines (CNE-1, CNE-2, 5-8F, C666-1) were cultured in RPMI-1640 (cat. no.C11875500BT; Gibco) medium containing 10% fetal bovine serum (cat. no. 10270-106;Gibco). The NP69 cell line was maintained in keratinocyte/serum-free medium (cat. no.10725078; Gibco) supplemented with bovine pituitary extract. All cell lines were grown in a humidified incubator at 37 °C supplemented with 5% CO2 until the logarithmic growth phase was reached before harvesting [15]. And then cells were gathered for use in the next experiments.

Plasmid construction, RNAi, and stable transfection

The FTO overexpression vector was developed, the complete human FTO (NM-001363894) gene was inserted into the GV492 vector by Geenchem (Shanghai, China), and the mock vector without FTO sequence was used as a control. The human FTO gene was inserted into the lentiviral vector GV492 (Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin). The synthetic human FTO gene overexpression vector was ligated to GV492 (Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin). Nasopharyngeal carcinoma cells were transfected with a lentiviral vector, and puromycin (2 ug/ml) was used to screen out stable and successfully transfected cell lines. All transfection steps were performed according to the instructions. The expression levels of mRNA and FTO protein after transfection were detected by qRT-PCR and Western blot, respectively.

NPC sample collection

Nasopharyngeal carcinoma tumor specimens from patients with nasopharyngeal carcinoma were obtained from Shenzhen Hospital of Hong Kong University, Shenzhen, China. All samples were collected from NPC patients who visited the hospital from 2019 to 2021. Between the ages of 31 and 76, 47 were male and 25 were female. The patient had never received any treatment such as chemotherapy or radiation therapy before surgery. All patients were aware of the whereabouts and purpose of the specimens and signed informed consent. This study was approved and performed under the supervision of the Ethics Committee of the University of Hong Kong Shenzhen Hospital.

Quantitative real-time PCR

Total RNA was extracted from the cells using TRIzol reagent (cat. no.15596026; Invitrogen) according to the manufacturer's instructions and then stored at -80 °C in RNAse-free H2O. For RT-qPCR, total RNA was used for first-strand cDNA synthesis using a NovoScript Plus ALL-in-one 1st Strand cDNA Synthesis Supermix (gDNA Purge) (oncoprotein). The mRNA level of the ribosomal protein S18 was used as an internal control. The primer sequences for FTO were: Forward, 5'-ACTTGGCTCCCTTATCTGACC-3'; Reverse, 5'-TGTGCAGTGTGAGAAAGGCTT-3'. qPCR was performed with QuantiNova SYBR Green PCR Kit, in triplicate. The reaction conditions were 95 °C for 2 min, followed by 40 cycles of 95 °C for 5 sec, 55 °C for 10 sec, and 60 °C for 20 sec. All samples were run in triplicate. The relative expression levels of FTO were measured using the 2-∆∆Cq method.

Immunohistochemistry

For IHC assay, tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned the slicer then cuts it into 4 um slices warm the slide to 72 °C, add 150 ul of Dond Dewax Solution, incubate for 30 min, rinse with alcohol and wash once with Bond Wash Solution, add 150 ul of BOND Epitome Retrieval Solution 2, incubate at 100 °C for 20 min, then use 150 ul of Dond Dewax Solution again to remove the slide. Wax 3 min. Incubate 150 ul primary antibody FTO (cat. no.27226-1-AP; 1:1000 dilution; Proteintech, USA) at room temperature, wash three times with 150 ul Dond Wash Solution, incubate with 150 ul post-primary at room temperature for 8 min, 150 ul Dond Wash Solution wash three times, 2 min each time. Add 15OUL Horseradish Peroxidase and incubate for 8 min at room temperature, wash 3 times with Dond Wash Solution, then wash once with Deionized Water, add 150 ul Peroxide Block to block for 5 min at room temperature, rinse 3 times with Dond Wash Solution, and then wash once with Deionized Water. Mixed DAB Refine 150 UL was prepared on-site with DAB Part1 and DAB Part B prepared in advance, incubated with Mixed DAB Refine for 6 minutes at room temperature, then washed 3 times with Dond Wash Solution. Add 150ul Hematoxylin and incubate for 8 min at room temperature. Finally, between two Deionized Water rinses, a Bond Wash Solution was added to return to blue. Finally, dehydrated by ethanol, Dewax, and fixed on glass slides with neutral gum.

DAB shows immune complexes. Microscope photography (BOND III, Leica, Wetzlar, Germany).

Cell scratch assay

When the cells grow to fuse into a monolayer state, a blank area is artificially created on the fused monolayer cells, called scratch and the cells at the edge of the scratch will gradually enter the blank area to heal the scratch. It stimulates the process of cell migration in vivo to a certain extent and takes pictures every day to observe the speed of scratch healing.

Transwell invasion

Cell invasion assay was performed using Boyden chamber (cat. no.3422; Corning). A layer of Matrigel (cat. no.354234; Corning) was plated in the upper chamber and cells (5 × 10^4) were suspended in 0.2 ml medium without fetal bovine serum and placed in the upper chamber, and the lower chamber was loaded with 0.5 ml of culture medium containing 10% fetal bovine serum as a chemoattractant. After 24 hours, the cells were moved to the bottom surface of the filter and stained with 2.5% crystal violet (cat. no.G1072; Solarbio). The experiment was repeated three times. For each sample, 5 fields of view were randomly calculated and the mean determined.

Immunoblot

Proteins were extracted from cell lines with RIPA lysis buffer and the protein levels of target molecules were determined. Proteins were separated by SDS-PAGE. Proteins were transferred to PDVF membranes. After blocking with 5% BSA for 1 hour at room temperature, the membrane was incubated with a primary antibody overnight. Then, the membrane was washed three times with TBST solution for 5 min each and incubated with the secondary antibody for 1 hour at room temperature. The proteins were finally visualized using the ECL kit.

Soft agar clone proliferation assay

For anchorage-independent growth assay. Ten thousand FTO-transfected cells and negative control cells were plated on soft agar plates (1.0% agar for the lower layer and 0.7% for the upper agar) [16]. Plates were grown in a 37 °C incubator for 9 days, with medium changes every 3 days. Keep a layer of growth medium on top of the agar to prevent drying out [17]. Randomly observe and take pictures under the microscope, and count the cell colonies larger than 0.1 mm.

In vivo tumor formation

For the cell proliferation experiment in vivo, a total of 10 6-week-old male nude mice [18] were divided into two groups. 5 were injected with FTO-overexpressing nasopharyngeal carcinoma cells, The remaining mice were used as controls. All animal experimental procedures were approved by the Ethics Committee of Shenzhen Hospital of Hong Kong University and were conducted in a professional venue provided by TOPBIOTECH (Guangdong, China). First, 3 × 10^6 5-8F OE NPC cells and 5-8F NPC cells were counted, the cells were kept at 4 °C during transportation, and cells of different groups were injected into the subcutaneous anterior axillary fat pad of mice according to the group [19]. The nude mice were routinely fed for 2 weeks, and the tumor formation was observed the size of the tumor was measured every 3-4 days until the mice were sacrificed on the last day, and the tumors were obtained and measured and photographed.

Statistical analysis

Statistical analyses were conducted using GraphPad Prism (version 8.0.0) software. The results are expressed as the mean ± SD. For analysis, we used the Student t-test and One-way ANOVA, and P < 0.05 was considered statistically significant.

Result

The relationship between the differential expression of m6A-related enzymes in nasopharyngeal carcinoma tissues and paracancer tissues

To examine the expression of genes involved in m6A modification, we performed an analysis of the NPC transcriptome sequencing data set from the GEO database. To find differences in m6A regulator expression levels between NPC tumor tissues and non-tumor tissues, we examined a total of 22 m6A regulators [20], including 2 erasers, 8 writers, and 12 readers, these samples are all from the NPC cohort of the GEO database (252 samples). Regulators such as METTL3, ZC3H13, RBM15, YTHDC2, HNRNPC, HNRNPA2B1, and IGF2BP3 showed a remarkable upregulation, while WTAP, YTHDC1, YTHDF1, YTHDF2, YTHDF3, FMR1, LRPPRC, IGF2BP2, and FTO were underexpressed in tumor tissue compared to tumor-free nasopharynx tissue (Figure 1A, Figure 1B and Figure 1D). The relative expression of the various m6A regulators was closely linked, as visualized in the cyclized plot showing p < 0.05 (Figure 1C). We did a univariate COX regression model, and the analysis showed that among the many m6A regulators, only HNRNPC, METTL3, RBM15, and FTO are a few protective factors for NPC, and most of the others are risk factors, indicating a poor prognosis. These results underscore the great diversity in expression patterns of the m6A regulator in NPC and suggest that this diversity has crucial implications for NPC prognosis and progression.

Expression of FTO is associated with prognosis, clinical staging, and immune features

Clinical correlation analysis showed (Figure 2A and Figure 2B), the FTO expression of clinical-stage IV patients is higher than those of clinical stage II or III, which means high FTO expression is associated with higher disease progression. To investigate the effect of FTO on immune infiltration of NPC, we performed GSVA enrichment analysis with immune cells and functional gene sets. As shown in (Figure 2C), compared with the low-FTO group the high-FTO group had a higher level of immune cell infiltration (pDC, CD8+T, T helper, tfh, Treg, Macrophages, Neutrophils) and immune activation signatures (Cytolytic activity). These results suggested that FTO promoted immune cell infiltration and activation in the nasopharyngeal carcinoma microenvironment.

FTO expression levels are reduced in NPC tissues and cells

To test the experimental idea, we detected the content of FTO in nasopharyngeal carcinoma cells and tissues from protein level and RNA level, respectively.in subsequent studies, IHC was used to detect FTO protein levels in nasopharyngeal carcinoma tissues and Paracancer tissues, and qPCR and western blot were performed to examine the mRNA and protein levels of FTO in NPC cells and immortalized nasopharyngeal epithelial cells (NP69). Immunohistochemical results showed that the level of FTO protein was decreased in nasopharyngeal carcinoma tissue (Figure 3A and Figure 3B). Furthermore, we have detected FTO protein and mRNA levels in normal NP69 cells and nasopharyngeal carcinoma cell lines (CNE1, CNE2, 5-8F, C666-1), and FTO protein and mRNA levels in NP69- cells were higher than those in nasopharynx carcinoma cells (Figure 3C and Figure 3D). Taken together, these results suggest that FTO is downregulated in NPC.

Up-regulation of FTO inhibits the proliferation of NPC cells

The effect of FTO on the malignant behavior of nasopharyngeal carcinoma cells was first studied using the gain-of-function method. We successfully constructed a 5-8F cell line overexpressing FTO, which could be verified by qPCR and western blotting (Figure 3E and Figure 3F). The experiment of the effect of FTO on the proliferation ability of nasopharyngeal carcinoma cells was done by Soft agar clone proliferation assay, concluded that forced expression of FTO inhibits the proliferation of 5-8F cells (Figure 4A and Figure 4B).

FTO inhibited the invasion and migration of NPC cells

We selected the 5-8F cell line for overexpression and compared it to the normal control, we verified this by western blotting. The effect of FTO on the migratory ability of nasopharyngeal carcinoma cells was demonstrated by cell invasion and migration assays. Forced expression of FTO inhibited the migration of 5-8F cells (Figure 4C and Figure 4D). Taken together, these results suggest that FTO inhibits the migration and invasion of nasopharyngeal carcinoma cells.

FTO inhibits tumor cell proliferation in vivo

In order to explore the in vivo proliferation effects of FTO in nasopharyngeal carcinoma cells, we established a mouse model. We injected equal amounts of FTO-overexpressing nasopharyngeal carcinoma cells and the control group into the axilla of mice to observe the changes in their tumorigenic ability and proliferation rate. We found that the tumorigenicity of NPC cells was good, with 100% tumor formation, and the growth rate of NPC cells overexpressing FTO after tumor formation was lower than that of the control group (Figure 5A and Figure 5B). After the mice were sacrificed, the lung tissues of the mice were taken out for observation, and no nodules or metastases were found under the naked eye or microscope.

Gene Set Enrichment Analysis revealed the potential downstream pathways of FTO in NPC

To better understand the mechanism by which FTO regulates the biological behavior of NPC, we conducted Gene Set Enrichment Analysis (GSEA) to explore the potential downstream pathways of FTO. This result shows that FTO expression had a significant correlation with KEGG gene sets closely related to cancer development, including focal adhesion [21], TGF-β signaling pathway [22,23], and WNT signa [24] ling pathway [25,26]. Yang X, et al. found that in pancreatic cancer, m6A demethylase FTO modifies Praja ring finger ubiquitin ligase 2 (PJA2), reduces mRNA decay, inhibits WNT signaling, and ultimately inhibits pancreatic cancer cell proliferation, invasion, and metastasis. The top 20 significantly correlated pathways were shown in Figure 6A. Given the possibility that the same gene exists in different pathways, we then established a network to show the interactive sets of these pathways (Figure 6B). Our results suggested that FTO may play a role in the occurrence and development of NPC through these signaling pathways. Our results suggested that the function of FTO in nasopharyngeal carcinoma may be related to these signaling pathways.

Discussion

Nasopharyngeal carcinoma is one of the most common head and neck cancers in its high incidence areas, including southern China, Southeast Asia, the Arctic, and the Middle East/North Africa, effective treatments and new treatments for nasopharyngeal cancer are so urgent [27]. The m6A regulators (readers, writers, and erasers) are a research hotspot, and their role is reflected in a variety of cancers. Including human breast cancer [9], kidney cancer [7], pancreatic cancer [10], and so on. However, there are few reports on the expression pathways and functions of m6A regulators in nasopharyngeal carcinoma. In addition, its pathogenesis is still unclear. In our current research, we first performed a differential analysis of FTO expression alone. Survival analysis showed that high FTO expression was associated with slower disease progression and better prognosis. However, the difference was not statistically significant, possibly due to the small sample size and the incidence of terminal events. Clinical correlation analysis showed that the FTO expression of clinical-stage IV patients is higher than those of clinical stage II or III, implying that high FTO expression is associated with higher disease progression. Immune signature of FTO against NPC immunity, indicating that FTO promoted immune cell infiltration and activation in the nasopharyngeal carcinoma microenvironment. Next, we did molecular biology experiments. We found that the expression of FTO was reduced in nasopharyngeal carcinoma tissues. In functional studies, FTO can inhibit the migration and invasion of nasopharyngeal carcinoma cells. Then we used a mouse model to mimic the tumorigenic environment in vivo in animal experiments and found that high expression of FTO inhibited the proliferation of tumor cells in vivo. Finally, to better understand the mechanism by which FTO regulates the biological behavior of NPC, we conducted Gene Set Enrichment Analysis (GSEA) to explore the potential downstream pathways of FTO. This result shows that FTO expression had a significant correlation with KEGG gene sets closely related to cancer development, including focal adhesion, TGF-β signaling pathway, and WNT signaling pathway. Our data demonstrate the significant correlation of FTO expression with the KEGG genome closely related to cancer development, and the protective role of FTO during NPC progression, and suggest that FTO may be a promising target for future NPC therapy.

Conclusion

In this work, we evaluated FTO, one of the writers, was downregulated in NPC. Functional studies showed that FTO was underexpressed in nasopharyngeal carcinoma and promoted the invasion and migration of NPC cells. However, overexpression of FTO reversed this effect and inhibited the migration and invasion of nasopharyngeal carcinoma cells. In addition, GSVA enrichment analysis found that FTO also had a positive effect on the infiltration and activation of immune cells in the NPC microenvironment. Our data demonstrate the significant correlation of FTO expression with the KEGG genome closely related to cancer development, and the protective role of FTO during NPC progression. Collectively, our study demonstrates the positive effect of FTO in NPC and suggests that FTO may be a future therapeutic target for NPC.

Acknowledgments

The authors would like to thank Jiandong Huang's group at Shenzhen Institute of Advanced Technology, China, for their technical guidance on molecular biology and epigenetics. Thanks to the central laboratory provided by the University of Hong Kong Shenzhen Hospital. We also acknowledge the Gene Expression Omnibus (GEO) for providing their platforms and contributors for uploading their meaningful datasets.

Funding

This work was supported by High-Level Hospital Program, Health Commission of Guangdong Province, China (No. HKUSZH201901033; No. HKUSZH201901039).

Availability of Data and Materials

The data is valid, and if necessary, you can contact the corresponding author by email or other means to obtain it.

Authors' Contributions

Pingan Wu made significant contributions to the conception and design of the study. Zhencheng Liao, Fan Ye performed the experiments and analyzed the data. Jun Wang is in charge of immunohistochemistry related matters. Siyi Yang, Zhaoqun Lu, Guan Huang and Honglei Zhu participated in the collection and arrangement of samples and data. Zhencheng Liao participated in the drafting and revision of the manuscript. Pingan Wu critically revises research for important knowledge content. All participating authors have read and the final manuscript approved.

Ethics Approval and Consent to Participate

This study was approved and performed under the supervision of the Ethics Committee of the University of Hong Kong Shenzhen Hospital. All patients were aware of the whereabouts and purpose of the specimens and signed informed consent. All experiments in this article were performed by relevant guidelines and regulations, and animal experiments were performed by ARRIVE guidelines (https://arrive guidelines.org).

Consent for Publication

Not applicable.

Competing Interests

There is no conflict of interest between the authors.

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