The present study was approved by the Ethics Committee of the Tumor Hospital of First People's Hospital of Foshan (Foshan, China). A total of 74 serum samples from patients with NPC and 27 from healthy controls were obtained. The clinical characteristics of the patients with NPC are summarized in Table I (22). All of the participants gave written informed consent. Prior to blood sample collection, none of the patients had received any treatment. Serum was isolated from 10 ml blood by centrifuging at 1,000 × g at room temperature for 5 min, and subsequently at 12,000 × g at 4°C for 5 min. The samples were stored at −80°C prior to usage.

NPC C666-1 cells and the normal nasopharyngeal epithelial cell line NP69 were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) in a 37°C humidified atmosphere containing 5% CO₂.

For transfection, C666-1 cells were cultured to 70% confluence and transfected with 100 nM pcDNA3.1-CDKN2A open reading frame plasmid (Yearthbio, Changsha, China), pcDNA3.1 blank vector (both Promega Corporation, Madison, WI, USA), miR-663 inhibitor (GeneCopoeia, Inc., Rockville, MD, USA), negative control (NC) inhibitor (GeneCopoeia, Inc.), miR-663 mimic (GeneCopoeia, Inc.), or scramble miR mimic (GeneCopoeia, Inc.) using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.), for 48 h according to the manufacturer's protocol. The following links demonstrate the miR-663 mimic and NC sequences, respectively: http://www.fulengen.com/product/search2/?s=miR-663&search_type=1&submit=Submit and http://www.fulengen.com/product/reporter-vector-controls.

Total RNA was extracted from cells using TRIzol Reagent (Thermo Fisher Scientific, Inc.), which was converted to cDNA using an RevertAid Reverse Transcription kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The miR-663 expression levels were examined with qPCR using PrimeScript^® miRNA RT-PCR kit (Takara Biotechnology Co., Ltd., Dalian, China) on an ABI 7500 thermocycler (Thermo Fisher Scientific, Inc.). U6 was used as an internal reference. The mRNA expression was detected by qPCR using the standard SYBR-Green RT-PCR kit (Takara Biotechnology Co., Ltd.). GAPDH was used as an internal reference. The specific primer pairs were as follows: CDKN2A sense, 5′-GATCCAGGTGGGTAGAAGGTC-3′ and antisense, 5′-CCCCTGCAAACTTCGTCCT-3′; GAP DH sense, 5′-ACAACTTTGGTATCGTGGAAGG-3′ and antisense, 5′-GCCATCACGCCACAGTTTC-3′. The reaction conditions were 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 15 sec and an annealing/elongation step at 60°C for 30 sec. The relative expression was analyzed using the 2^−ΔΔCq method (23).

Cells were solubilized in cold radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific, Inc.) to extract the total protein, which was quantified using a bicinchoninic acid assay (Pierce; Thermo Fisher Scientific, Inc.) and 50 µg protein was separated using SDS-PAGE on a 10% gel (Pierce; Thermo Fisher Scientific, Inc.), and transferred onto a polyvinylidene difluoride membrane (Thermo Fisher Scientific, Inc.). The membrane was blocked with 5% non-fat milk in DMEM for 3 h at room temperature, followed by a rabbit anti-CDKN2A monoclonal antibody (1:100; catalog no. ab108349; Abcam, Cambridge, UK) or rabbit anti-GAPDH monoclonal antibody (1:100; catalog no. ab181602; Abcam) at room temperature for 3 h. Following washing with PBST for 15 min, the membrane was incubated with the goat anti-rabbit secondary antibody (1:1,000; catalog no. ab6721; Abcam) at room temperature for 1 h. Chemiluminescent detection was conducted by using an enhanced chemiluminescence kit (Pierce; Thermo Fisher Scientific, Inc.). The protein expression was analyzed using Image-Pro plus software (version 6.0; Media Cybernetics, Inc., Rockville, MD, USA), represented as the density ratio vs. GAPDH.

C666-1 cells (5×10⁴) were cultured in a 96-well plate, each well containing 100 µl fresh serum-free medium with 0.5 g/l MTT (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Following incubation at 37°C for 12, 24, 48 and 72 h, the medium was removed by aspiration and 50 µl dimethyl sulfoxide (Sigma-Aldrich; Merck KGaA) was added to each well. Following incubation at 37°C for a further 10 min, the absorbance at 570 nm of each sample was measured using a plate reader (Tecan Infinite M200; Tecan Group Ltd., Männedorf, Switzerland).

C666-1 cells (1×10⁶) were washed twice in Dulbecco's PBS, resuspended in 70% ethanol and fixed overnight at −20°C. Subsequently, cells were washed twice in PBS with 3% bovine serum albumin (BSA, Sigma-Aldrich; Merck KGaA) and incubated for 30 min at room temperature in propidium iodide (PI) staining buffer containing 3% BSA, 40 µg/ml PI, and 0.2 mg/ml RNase in PBS. DNA content was determined using flow cytometry (FACSCalibur; BD Biosciences, Franklin Lake, NJ, USA) and FACSComp software version 1.0 (BD Biosciences).

Targetscan software (www.targetscan.org) was used to predict the putative target genes of miR-663.

The wild type (WT) or mutant (MT) CDKN2A 3′-UTRs were constructed using PCR and the Quick-Change Site-Directed Mutagenesis kit (Stratagene; Agilent Technologies, Inc., Santa Clara, CA, USA), which was subsequently inserted into the multiple cloning site in the psiCHECK™ vector (Promega Corporation). C666-1 cells were cultured to 70% confluence in a 24-well plate, and co-transfected with 100 ng WT-CDKN2A or MT-CDKN2A vector, plus 50 nM miR-663 mimics or scramble miR using Lipofectamine^® 2000. The dual luciferase reporter assay system (Promega Corporation) was used to determine the luciferase activities 48 h post-transfection. Renilla luciferase activity was normalized to firefly luciferase activity.

Data are expressed as the mean ± standard deviation. Comparisons between groups were analyzed using Student's t test or one-way analysis of variance followed by the Tukey's post-hoc test. The association between serum miR-663 levels and the clinical characteristics of patients with NPC was analyzed using a χ² test. Statistical analysis was performed using SPSS (version 19.0; IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

The serum levels of miR-663 were detected in patients with NPC and healthy controls. RT-qPCR analysis demonstrated that the serum miR-663 levels were significantly increased in patients with NPC compared with healthy controls (Fig. 1).

The association between the serum miR-663 levels and clinical characteristics in patient with NPC was analyzed. Using the mean level of serum miR-663 as a threshold, the patients with NPC recruited for the present study were divided into two groups, a high serum miR-663 expression group and a low serum miR-663 expression group. It was observed that the serum miR-663 levels were significantly associated with grade, clinical stage and lymph node metastasis (Table I). However, no association was observed between the serum miR-663 levels and age, gender, tumor stage, distant metastasis or EBV infection (Table I). Therefore, increased expression of miR-663 may promote the malignant progression of NPC.

In order to further confirm the results detailed above, the expression of miR-663 in NPC C666-1 cells was examined, and a normal nasopharyngeal epithelial cell line, NP69, was used as a control. As demonstrated in Fig. 2A, miR-663 was upregulated in NPC cells compared with NP69 cells.

The regulatory role of miR-663 in NPC cell viability was subsequently investigated. As miR-663 was significantly upregulated in the serum of patients with NPC patients, in addition to NPC cells, C666-1 cells were transfected with a miR-663 inhibitor to knock down its expression. An NC non-specific inhibitor was additionally used. Transfection with the miR-663 inhibitor significantly decreased the miR-663 levels in C666-1 cells, compared with the control group (Fig. 2B). However, transfection with the NC inhibitor did not affect the miR-663 levels in C666-1 cells (Fig. 2B).

An MTT assay was conducted to examine cell proliferation. The results of the present study demonstrated that the knockdown of miR-663 significantly decreased the proliferation of C666-1 cells, compared with the control group (Fig. 2C). Cell cycle progression serves an important role in cell proliferation. As presented in Fig. 2D, inhibition of miR-663 led to a significant arrest at the G1 stage in C666-1 cells. The results of the present study demonstrated that the knockdown of miR-663 decreased the proliferation of C666-1 cells via the induction of cell cycle arrest at the G1 stage.

The present study demonstrated that CDKN2A was a predicted target gene of miR-663 using Targetscan software, and that this association was evolutionarily conserved (Fig. 3A). In order to confirm the targeting of CDKN2A by miR-663, luciferase vectors containing the WT or MT CDKN2A 3′-UTR were generated (Fig. 3B and C). A luciferase reporter assay was performed, and it was observed that the luciferase activity was significantly decreased in C666-1 cells co-transfected with the WT-CDKN2A luciferase reporter vector and miR-663 mimics, compared with the control group (Fig. 3D). However, these effects were reversed by transfection with the MT-CDKN2A luciferase reporter vector. Therefore, the results of the present study indicated that miR-663 may directly bind to the 3′UTR of CDKN2A mRNA.

The effects of miR-663 on CDKN2A expression were further examined. Western blotting data demonstrated that the knockdown of miR-663 significantly increased the protein expression of CDKN2A in C666-1 cells (Fig. 4A). In order to further confirm these results, C666-1 cells were transfected with miR-663 mimic, and the miR-663 levels were significantly increased following transfection (Fig. 4B). Overexpression of miR-663 significantly decreased the CDKN2A protein level in C666-1 cells (Fig. 4C). The results of the present study demonstrated that miR-663 negatively-regulated the expression of CDKN2A via direct binding to the 3′UTR of CDKN2A mRNA in C666-1 cells.

It was hypothesized that CDKN2A may be associated with the miR-663-mediated increase in NPC cell viability and with cell cycle progression. C666-1 cells were transfected with a miR-663 inhibitor, or co-transfected with the miR-663 inhibitor and CDKN2A small interfering (si)RNA. It was observed that the protein expression of CDKN2A was significantly decreased in the miR-663 inhibitor + CDKN2A siRNA group, compared with the miR-663 inhibitor group (Fig. 5A). An MTT assay further demonstrated that the viability of C666-1 cells was significantly decreased in the miR-663 inhibitor + CDKN2A siRNA group, compared with the miR-663 inhibitor group (Fig. 5B), suggesting that inhibition of CDKN2A expression reversed the suppressive effects of miR-663 knockdown on C666-1 cell proliferation. The cell cycle distribution was additionally assessed. As presented in Fig. 5C, a reduced number of C666-1 cells were at the G1 stage in the miR-663 inhibitor + CDKN2A siRNA group compared with the miR-663 inhibitor group, suggesting that the inhibition of CDKN2A reversed the suppressive effect of miR-663 knockdown on cell cycle progression in C666-1 cells. Therefore, CDKN2A is associated with the miR-663-mediated cell cycle progression and proliferation of NPC cells.

The expression of CDKN2A in C666-1 cells was examined, and NP69 cells were used as a control. RT-qPCR analysis and western blotting indicated that the mRNA and protein levels of CDKN2A were significantly decreased in C666-1 cells compared with NP69 cells (Fig. 6A and B). Therefore, the decreased expression of CDKN2A may be caused by the increased expression of miR-663, which promotes NPC cell proliferation.