Introduction
Nasopharyngeal carcinoma (NPC) is a rare malignancy worldwide, while it is particularly common in the Southern China [
1,
2]. More than 70% of patients with NPC are initially diagnosed with a locally advanced cancer because of concealed symptoms and invasiveness [
3]. To date, chemo-radiotherapy has become the main treatment for NPC, owing to its high radio-resistance and chemo-sensitivity [
4]. Although the combination of radiotherapy and chemotherapy has achieved a satisfactory 5 year survival rate (85–90%), 8–10% of patients had recurrence and tumor metastasis [
5,
6]. However, the mechanisms underlying radio-resistance and chemo-resistance of NPC have still remained elusive [
7,
8]. Thus, it is urgent to explore new treatment strategies to reveal these mechanisms for NPC.
In the process of comprehensive analysis of data collected from Gene Expression Omnibus (GEO) database, we found that the mRNA expression level of C2orf40 gene was significantly reduced in NPC cells. In China, C2orf40, also known as esophageal cancer-related gene-4 protein (ECRG4), was preliminarily identified and cloned by comparing the differential gene expression in normal esophageal epithelial cells and in esophageal squamous cell carcinoma (ESCC) cells [
9]. In human, constitutive expression of Ecrg4 in a quiescent state monitors tissue homeostasis. Ecrg4 encodes a non-canonical hydrophobic leader sequence that assists in tethering Ecrg4 to the cell surface [
10]. Upon injury, Ecrg4 is shed immediately from the surface and its gene expression rapidly decreases within 24 h, which is accompanied by the activation of inflammatory cascades [
11]. Besides, naïve T cells highly express Ecrg4, which is down-regulated upon activation [
12]. Although C2orf40 is highly expressed in various normal tissues, its expression is down-regulated in diverse types of cancer, including esophageal cancer [
13], breast cancer [
14], anaplastic thyroid carcinoma [
15], and liver cancer [
16], which may be partially due to the DNA hypermethylation at the promoter region [
17,
18]. Especially, C2orf40 is a tumor suppressor gene and an independent prognostic factor of ESCC [
19]. However, the mechanism of down-regulation of C2orf40 expression and its biological role in NPC have not been fully explored.
In the current study, based on the analysis of three GEO datasets (GSE12452, GSE53819, and GSE12452), we compared the gene expression profile data of normal nasopharyngeal epithelial tissues and NPC tissues, and it was found that the C2orf40 expression in NPC tissues was significantly down-regulated. Survival analysis indicated that decreased expression of C2orf40 was correlated with a poor prognosis of patients with NPC. In vitro and in vivo functional experiments revealed that C2orf40 not only promoted the sensitivity of NPC cells to chemotherapy and radiotherapy by inducing cell cycle arrest at G2/M phase and cell apoptosis, but also inhibited the migration ability of NPC cells.
Materials and methods
Cell culture
The human immortalized nasopharyngeal epithelial cell line (TERT) was cultured in a keratinocyte/serum-free medium supplemented with growth factors (Gibco Inc., Grand Island, NY, USA). Seven human NPC cell lines, including CNE-1, CNE-2, HONE-1, SUNE-1, HNE-1, HK-1, and 5-8F, were grown in a Roswell Park Memorial Institute (RPMI)-1640 medium (Corning Inc., Corning, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand Island, USA) in the presence of 5% CO2 at 37 °C. All NPC cell lines were purchased from the Cell Bank of the Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). All cell lines were routinely tested for mycoplasma contamination and found to be negative.
Data collection
The GEO database was used to obtain the gene expression profiles of human normal nasopharyngeal epithelial tissues and NPC tissues. The gene expression profiles and clinical data of GSE12452 (31 NPC samples and 10 controls), GSE53819 (18 NPC samples and 18 controls), and GSE64634 (12 NPC samples and 4 controls) datasets were downloaded.
Collection of human tissue specimens
In the present study, human NPC tissues (n = 20) and human normal nasopharyngeal epithelial tissues (n = 12) were collected from The Third Xiangya Hospital of Central South University according to the ethical and legal standards of The Third Xiangya Hospital of Central South University. The diagnosis of primary NPC was confirmed by hematoxylin–eosin (H&E) staining by experienced pathologists. Written informed consent was obtained from all patients.
RNA isolation and quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis
Total RNA was extracted from cells or tissues using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Then, 2 μg of each RNA sample was used for cDNA synthesis with the FastKing One Step RT-PCR Kit (Tiangen, Beijing, China). The qRT-PCR was performed in triplicate according to the manufacturer's instructions using the SYBR Green SuperMix system (Bio-Rad Laboratories Inc., Hercules, CA, USA). The gene expression was evaluated for three biological replicates, and the relative changes in gene expression were analyzed by the 2−ΔΔCT method. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. The primer sequences used for qRT-PCR were as follows: human C2orf40: 5′- CCAGCAGTTTCTCTACATGGGC-3′ and 5′-GCAGAGTCTTCATCATAGTGACG-3′; human GAPDH: 5′-GTCTCCTCTGACTTCAACAGCG-3′ and 5′-ACCACCCTGTTGCTGTA GCCAA-3′.
Western blot analysis
Western blot analysis was performed using standard techniques. Briefly, total proteins from cells or tissues were extracted using radio-immunoprecipitation assay (RIPA) lysis buffer (Millipore, Burlington, MA, USA). Protein concentrations were measured with a Pierce BCA Protein kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of total lysate were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were then transferred to polyvinylidene difluoride (PVDF) membranes (IPFL00010; Millipore), blocked with 5% non-fatty milk, and incubated with the appropriate antibodies according to the manufacturer’s instructions. Finally, the blotting was developed using an Enhanced Chemiluminescence (ECL) Detection kit (Merck, Kenilworth, NJ, USA). The band intensity was analyzed by the ImageJ software (National Institutes of Health, Bethesda, MD, USA). The following antibodies were used in the current study: C2orf40 (catalog no. ab224077; Abcam, Cambridge, UK), β-actin(catalog no. ab6276; Abcam), GAPDH (catalog no. ab59164; Abcam), cleaved caspase-3 (catalog no. ab32042; Abcam), caspase-3 (catalog no. ab32351; Abcam), cleaved PARP (catalog no. ab32064; Abcam), PARP (catalog no. ab191217; Abcam), Bax (catalog no. ab32503; Abcam), γ-H2AX (catalog no. ab81299; Abcam), CDK1 (catalog no. ab133327; Abcam), Rb (catalog no. ab181616; Abcam), p-Rb (catalog no. ab184702; Abcam), CCNE1 (catalog no. ab33911; Abcam), CCNB1 (catalog no. ab32053; Abcam), PI3K (catalog no. ab86714; Abcam), AKT (catalog no. ab8805; Abcam), p-AKT (catalog no. ab38449; Abcam), mTOR (catalog no. ab2732; Abcam), p-mTOR (catalog no. ab5536; Abcam), and p-PI3K (catalog no. 17366; Cell Signaling Technology, Inc., Danvers, MA, USA).
Immunohistochemistry (IHC)
To specifically analyze the expression of the C2orf40, IHC was performed on the paraffin-embedded tumor tissue sections. Briefly, after deparaffinization and rehydration, antigen retrieval was undertaken using citrate solution. The primary antibody (dilution, 1:400; Abcam) was added and incubated overnight at 4 ℃, and a secondary antibody was added and incubated for 30 min in a humid chamber, followed by washing with Tris-buffered saline (TBS) buffer. Slides were then incubated for 30 min with EnVision peroxidase reagent (DAKO, Carpentaria, CA, USA). Finally, the slides were stained with 3, 3-diaminobenzidine (DAB) for 5 min, and Mayer’s hematoxylin solution was used for counterstain. The percentage of positively stained NPC cells in three images was assessed using the ImageJ software as previously described [
20].
Construction of plasmids and lentiviral vectors
Herein, pcDNA-3.1 ( +) vectors (Invitrogen) were used for C2orf40 overexpression. Primers used for amplification of C2orf40 were as follows: C2orf40-F: 5′- CTAGCTAGCCCACCGATGGCTGCCTCCCCCGCGCGGCC-3′ and C2orf40-R: 5′-TTAGTAGTCATCGTAGTTGACGCTGATATCCCG-3′. Besides, AgeI (NEB, Beijing, China) and EcoRI (NEB) enzymes were used to clone the C2orf40 into the pCDNA-3.1( +) vector. For transfection of NPC cells, 3 × 105 NPC cells were seeded into 6-well plates and incubated at 37 ℃ for 24 h. Then, 2000 ng pcDNA-3.1( +) vectors containing C2orf40 or empty vectors were transfected with Lipofectamine® 3000 reagent (Invitrogen). Each experiment was repeated at least three times. For construction of NPC cells stably overexpressing C2orf40, the full-length human C2orf40 gene was subcloned into the lentiviral vector pLV (Add-gene). After that, pLV-C2orf40, psPAX2, and pMD2.G were transiently transfected into HEK-293 T cells. The supernatants that contained viruses were subsequently infected with NPC cells for 48 h. Following infection, the stable clones were selected with 0.5 μg ml−1 puromycin (Sigma-Aldrich, St. Louis, MO, USA).
In vitro chemo-resistance assay
Cell sensitivity to the cisplatin was assessed indirectly by the Cell Counting Kit-8 (CCK-8) and colony formation assays. For CCK-8 assay, transfected NPC cells were seeded into a 96-well plate at a density of 3000 cells/well. After 24 h of incubation, the cells were exposed to cisplatin solution with an appropriate concentration for 3 days according to experimental requirements. Then, cells were incubated with 100 µL of a fresh medium containing 10% CCK-8 reagent (DoJinDo Laboratories, Tokyo, Japan) for 1 h at 37 ℃. The absorbance at a wavelength of 450 nm was detected using an automatic spectrometer (PerkinElmer, Waltham, MA, USA).
For colony formation assay, 600 NPC cells were seeded into a 6-well plate after transfection. After a 24 h-incubation, the cisplatin solution was added to the culture medium in accordance with the experimental conditions. After 14 days, cells were fixed with methanol solution and stained with crystal violet.
Cell apoptosis
Apoptosis of HONE-1 and SUNE-1 cells induced by cisplatin (1 μg/ml) was determined using an Annexin V-FITC/PI apoptosis detection kit (Beijing 4A Biotech Co., Ltd., Beijing, China) in accordance to the manufacturer’s protocol. The transfected cells were collected and fixed with 4% paraformaldehyde for 1 h at room temperature, and the cells were treated with 0.5% Triton X-100 for 15 min. The TUNEL reaction mixture was placed in a dark environment at 37 °C for 1 h in a humid atmosphere. DAPI was used to stain nuclei simultaneously. The TUNEL-positive NPC cells were analyzed under a fluorescence microscope.
Gene set enrichment analysis (GSEA) was performed using the Molecular Signatures Database (MSigDB; ver. 6.0). Of the 31 NPC patients from GSE12452 and 18 NPC patients from GSE53819, 12 with the highest C2orf40 expression and 12 with the lowest C2orf40 expression were divided into two groups. A
P-value < 0.05 and a false discovery rate (FDR) < 0.25 were considered significant. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were undertaken using the DAVID (ver. 6.7) website (
https://david.ncifcrf.gov).
Wound healing assay
The migration ability of NPC cells was evaluated by an in vitro wound healing assay. In brief, NPC cells were seeded into 6-well plates at a density of 1 × 106 cells per well in a RIPM-1640 medium supplemented with 10% FBS. After the cells reached a confluence of 80%, the cultured monolayers were mechanically scraped with 200 μl pipette tips and cultured in the RIPM-1640 medium supplemented with 0.5% FBS for 48 h. Images were captured at 0, 12, and 24 h.
Transwell migration and invasion assays
Transwell migration and invasion assays were performed using transwell chambers (Corning Inc.). Briefly, 5 × 104 HONE-1 or SUNE-1 cells were resuspended on a serum-free RIPM-1640 medium with Matrigel and seeded into the upper chamber of the transwell. Then, the transwell chambers were placed on 24-well plates with a RIPM-1640 medium containing 10% FBS. After incubation at 37 ℃ for 24 h, the cells that invaded the lower chamber of the transwell were fixed with methanol, stained with crystal violet, and photographed under a microscope.
Immunofluorescence assay
Immunofluorescence assay was carried out to assess the repair ability of cells after radiation injury. Briefly, 5 × 104 HONE-1 or SUNE-1 cells with or without C2orf40 overexpression were seeded, fixed with 4% paraformaldehyde for 10 min, and blocked with 10% bovine serum albumin (BSA) for 15 min. Pooled anti-γ-H2AX antibodies (dilution, 1:100; Abcam) were reacted with the cells for 1 h at 25 °C with gentle mixing. After washing, the bound antibodies were detected by reactivity with secondary antibodies conjugated with fluorescein isothiocyanate for 1 h in the dark. Images were captured using a fluorescence microscope.
Comet assay
The comet assay was used to assess the oxidative DNA damage in individual cells under radiation exposure. In short, HONE-1 and SUNE-1 cells stably overexpressing C2orf40 or controls were exposed to radiation for the indicated time, and then, the cells were collected (106/ml) and mixed with 0.75% low-melting agarose (Sigma-Aldrich). Afterwards, the cells were spread on a frosted microscopic slide pre-coated with 0.75% normal melting agarose. After solidification of the agarose, cells were lysed with lysis buffer, and the slides were then placed in a gel-electrophoresis apparatus containing electrophoresis buffer (300 mM NaOH and 10 mM Na-EDTA) for 20 min. The electrical field was applied to the same buffer at 4 °C for 20 min to draw the negatively charged DNA towards the anode. After electrophoresis, the slides were rinsed with neutralization buffer and stained with 40 g/mL ETBr (Sigma-Aldrich). The slides were observed under a fluorescence microscope.
An in vivo mouse model of NPC
Tumor sensitivity to chemotherapy and radiotherapy was evaluated using an in vivo mouse model of NPC. Female nude mice (BALB/c nude mice; age, 6–8-week-old) were used. Besides, 2 × 106 HONE-1 cells were mixed with Matrigel and injected subcutaneously into mice. For cisplatin sensitivity assessment, when the tumor volume reached almost 100 mm3, mice were randomly divided into four groups and intraperitoneally injected with phosphate-buffered saline (PBS) or cisplatin (4 mg/kg) every 3 days. Mice were sacrificed on day 30 and tumor volume was calculated using the following equation: (length × width2)/2. For the assessment of sensitivity of tumors to radiation therapy, mice were anesthetized via intraperitoneal injection of 150 μl 4% chloral hydrate. When the tumor volume reached almost 100 mm3, tumor tissues were exposed to 2-Gy γ rays emitted by the Co-60 source every 2 days from day 1 to day 9, and the growth rate of the tumor was recorded as well. Mice were sacrificed on day 21 and volume and weight of tumors were measured.
Statistical analysis
The data were imported into SPSS 23.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA, USA) software for statistical processing, and the results were expressed as mean ± standard deviation (SD) (x̅ ± s). For comparisons, the Wilcoxon signed-rank test, the Pearson chi-square (χ2) test, one-way analysis of variance (ANOVA) with Dunnett's test, or the Student’s t-test was employed as indicated. P-value < 0.05 was considered statistically significant.
Discussion
NPC is a common type of cancer in Africa and Southeast Asia, highly associated with genetic factors, dietary effects, and viral infections [
1,
26]. At present, intensity-modulated radiation therapy (IMRT) combined with platinum-based chemotherapy is still the main treatment choice for patients with NPC [
27‐
29]. However, several factors, including recurrence, distant metastasis, and radiation resistance, may reduce the efficacy of radiotherapy and chemotherapy in the treatment of NPC [
5,
30]. Therefore, a new treatment regimen aimed at improving the sensitivity of radiotherapy and chemotherapy for NPC is expected to improve the overall survival rate and quality of life of patients with NPC. We attempted to investigate the C2orf40 gene because when we reanalyzed the publicly accessible NPC microarray dataset previously stored in the GEO database, it was significantly downregulated in all the three datasets. More importantly, the biological function and mechanism of C2orf40 in NPC have not been reported.
The C2orf40 gene encodes a secretory protein, which is hydrolyzed to produce soluble peptides, and considered to be necessary for C2orf40 to exert its cell type-specific biological activity [
31]. In recent years, a number of scholars have found that C2orf40 is a tumor suppressor gene, which has a variety of functions in the processes of cell proliferation, migration, and invasion. In breast cancer, C2orf40 is mainly silenced by hypermethylation of the promoter. The mRNA level of C2orf40 was significantly correlated with disease-free survival and distant metastasis-free survival. C2orf40 inhibits the proliferation, migration, and invasion of breast cancer cells by down-regulating the expression levels of mitotic genes [
32]. In esophageal cancer, soluble C2orf40 has a dose-dependent inhibitory effect on the growth of esophageal cancer cells in vivo. Additionally, C2orf40 inhibits the growth of tumor cells by reducing telomerase activity and it is therefore expected to be a potential biotherapeutic drug for esophageal cancer. In the present study, the results not only showed that the expression level of C2orf40 in NPC cells was down-regulated, but also revealed that the expression level of C2orf40 was highly correlated with the prognosis of NPC patients. The downregulation of C2orf40 gene in NPC cells could be partially due to its hypermethylated promoter. In addition, as a potential tumor suppressor gene, C2orf40 inhibits the expression levels of cell cycle-related proteins (CCNE1 and CDK1), and blocks the cell cycle in G2/M phase. Furthermore, C2orf40 inhibits the resistance and migration of NPC cells to radiotherapy and chemotherapy by downregulating the expression levels of HRR-related proteins and activation of PI3K/Akt/mTOR signaling pathway.
Epigenetics refers to the change of genetic gene expression without changing DNA sequence. It includes DNA methylation, histone modification, nucleosome remodeling, and RNA-mediated targeting [
33,
34]. Among them, DNA methylation is the most common epigenetic change [
35]. Methylation of CpG island in the promoter or the first exon inhibits gene transcription and leads to inactivation of gene expression [
36,
37]. With the development of sequencing and microarray technologies, we can easily screen the expression and DNA methylation levels of thousands of genes in the human genome simultaneously. DNA methylation is closely associated with the regulation of gene expression, and the high expression levels of oncogenes and low expression levels of tumor suppressor genes are the key factors of tumorigenesis [
38]. Perturbation of DNA methylation is common in various types of cancer, and it has become an important mechanism of tumorigenesis. Aberrant DNA methylation may influence the functions of key genes, especially tumor suppressor genes, thereby participating in the carcinogenesis of NPC. To date, several studies have reported some hypermethylated genes in NPC, and described the overall outline of the interaction network of these aberrantly methylated genes, providing a valuable reference for the study on occurrence and development of NPC at the molecular level [
39‐
41]. Particularly, the promoter hypermethylation of SHISA3 contributes to the downregulation of this gene in NPC. SHISA3 suppresses invasion and metastasis of NPC cells by impeding the TRIM21-mediated ubiquitination and degradation of SGSM1 [
42]. HOPX gene is the most prominent hypermethylated gene in NPC, and restoring the expression level of HOPX inhibits the metastasis of NPC cells and enhances their sensitivity to chemotherapy [
43]. Moreover, numerous scholars demonstrated that the down-regulation of C2orf40 expression in tumor cell lines and tissues is mainly attributed to the hypermethylation of its promoter [
31,
44‐
46]. Consistently, in the present study, through pyrosequencing, it was found that the promoter region of C2orf40 was hypermethylated, which indicated its down-regulated expression level in NPC cells.
In conclusion, This study clarified the biological functions and mechanisms of C2orf40, as a tumor suppressor gene, in NPC, and provided a potential molecular target for improving the sensitivity of NPC cells to radiotherapy and chemotherapy. it may be a promising treatment for NPC to restore the expression level of C2orf40 in NPC cells by epigenetic therapy or the application of recombinant C2orf40-derived peptides.
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