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), p21 (catalog no. ab109521; 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, 2,000 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-293T 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 3,000 cells/well. After 24 h of incubation, the cells were exposed to cisplatin solution with an appropriate concentration for three 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 (1μg/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.
Bioinformatics analysis
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 two 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.