The present study was approved by the Ethics Committee of First Affiliated Hospital of Xinjiang Medical University (Urumqi, China). Serum samples from 85 NPC patients (age range, 32-66 years; male, 48; female, 37) and 30 healthy controls (age range, 29-61 years; male, 18; female, 12) were collected at the Department of Otolaryngology, First Affiliated Hospital of Xinjiang Medical University.

Differentiation grade, lymph node metastasis, T stage, advanced clinical stage and Epstein-Barr virus (EBV) infection were recorded (21). All patients were treated by radiotherapy and serum samples were collected at 1 day prior to and 1 day following radiotherapy. Blood samples were collected (10 ml) and serum was obtained by centrifugation (1,500 x g; 5 min; 4˚C). All NPC patients and healthy controls recruited for the present study did not receive any therapy prior to blood collection. The serum samples were stored at -70˚C prior to use. The clinical characteristics of NPC patients are presented in Table I. Written informed consent was obtained from all patients. Patient follow-ups were conducted and no patients were lost during the 60 months follow-up period.

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

Total RNA was extracted from tissue and cell samples using a QIAamp RNA Blood kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer's protocol. RNA was then reverse transcribed into cDNA using a miRNA Reverse Transcription Kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. qPCR was then performed to detect the miR-93 expression on an ABI 7300 Plus Thermocycler (Applied Biosystems; Thermo Fisher Scientific, Inc.) using a miRNA Q-PCR Detection kit (GeneCopoeia, Inc., Rockville, MD, USA), according to the manufacturer's protocol. U6 gene was used as an internal reference. The PCR conditions were 95˚C for 5 min, followed by 45 cycles of 95˚C for 30 sec and 60˚C for 30 sec. Each sample was examined in triplicate. The relative expression was analyzed by the 2^-ΔΔCq method (22). Primers for miR-93 and U6 were purchased from Guangzhou Fulengen Co., Ltd. (Guangzhou, China) with the following sequences: miR-93, forward, 5'-AGGCCCAAAGTGCTGTTCGT-3' and reverse, 5'-GTGCAGGGTCCGAGGT-3'; and U6, forward, 5'-CTCGCTTCGGCAGCACA-3' and reverse, 5'-AACGCTTCACGAATTTGCGT-3'.

For transfection, C666-1 cells were cultured as described to 70% confluence and transfected with 100 nM miR-93 inhibitor (cat. no., HmiR-AN0836-SN-10), negative control (NC) inhibitor (cat. no., CmiR-AN0001-SN), miR-93 mimic (cat. no., HmiR0110-MR04) or scramble miR mimic (miR-NC; cat. no., CmiR0001-MR04; all Guangzhou FulenGen Co., Ltd., Guangzhou, China), or co-transfected with miR-93 mimic and 200 ng pcDNA3.1-programmed cell death 4 (PDCD4) open reading frame plasmid (www.amspring.com; Amspring, Changsha, China), using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Following experiments were conducted following 48 h.

Cells (5x10⁴) were seeded in 96-well plates and 100 µl of fresh serum-free medium containing 0.5 g/l MTT (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) were added to each well. Following incubation at 37˚C for 0, 24, 48 and 72 h, medium was removed, 50 µl of dimethyl sulfoxide (Sigma-Aldrich; Merck KGaA) was added and samples were incubated at 37˚C for 10 min. The absorbance at 570 nm was recorded.

Bioinformatics analysis software, including TargetScanHuman 7.2 (www.targetscan.org), miRanda 1.0 (www.microrna.org) and PicTar 1.0 (pictar.mdc-berlin.de) were used to predict the putative target genes of miR-93 according to the manufacturers' protocols. Amspring provided the wild type (WT)-PDCD4 and mutant type (MT)-PDCD4 reporter plasmid. The WT or MT of PDCD4 3'-UTR were constructed by PCR and a Quick-Change Site-Directed Mutagenesis kit (Agilent Technologies, Inc., Santa Clara, CA, USA), which was then inserted into the multiple cloning sites in the psiCHECK™2 vector (Promega Corporation, Madison, WI, USA). C666-1 cells were cultured as described to 70% confluence in a 24-well plate, and co-transfected with 100 ng WT-PDCD4 or MT-PDCD4 vector, and 50 nM miR-93 mimics or scramble miR (NC) using Lipofectamine 2000. The xDual-Luciferase^® Reporter Assay system (Promega Corporation) was used to determine the luciferase activities at 48 h following transfection. Renilla luciferase activity was normalized to firefly luciferase activity.

Cells were solubilized in cold radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific, Inc.) to extract protein. The protein concentration was determined using a bicinchoninic acid Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Protein (50 µg) was separated by 10% SDS-PAGE (Pierce; Thermo Fisher Scientific, Inc.), and transferred onto a polyvinylidene difluoride membrane (Thermo Fisher Scientific, Inc.). Following incubation with PBS containing 5% non-fat milk at 4˚C overnight, the membrane was incubated with rabbit anti-human PDCD4 primary antibody (1:50; ab80590; Abcam, Cambridge, MA, USA) or rabbit anti-human GAPDH primary antibody (1:100; ab9485; Abcam), respectively, at room temperature for 3 h. Following washing with PBS-Tween-20 for 15 min, the membrane was incubated with the horseradish peroxidase-conjugated mouse anti-rabbit secondary antibody (1:10,000; ab99697; Abcam) at room temperature for 1 h. Chemiluminescent detection was conducted using an Novex™ ECL Chemiluminescent Substrate Reagent kit (Pierce; Thermo Fisher Scientific, Inc). The protein expression was analyzed using Image-Pro Plus software 6.0 (Media Cybernetics, Inc., Rockville, MD, USA), represented as the density ratio vs. GAPDH.

Data in the present study are expressed as the mean ± standard deviation. The differential expression of serum miR-93 between NPC patients and healthy controls was analyzed using an independent sample t-test, whereas the differential expression of serum miR-93 between pre-treated and post-treated NPC patients was analyzed using a paired Student's t-test. The clinical association between serum miR-93 expression and clinical variables was analyzed using a χ² test. Kaplan-Meier method was used to analyze the association between serum miR-93 expression and survival time. Univariate and multivariate analysis were used to identify the independent predicators for the prognosis of NPC patients. The statistical association of data between groups was analyzed by Student's t-test or one-way analysis of variance. SPSS 19.0 software (IBM Corp., Armonk, NY, USA) was used to perform the statistical analysis. P<0.05 was considered to indicate a statistically significant difference.

miR-93 has been reported to be significantly upregulated in NPC tissues and cell lines (19,20); however, whether the serum expression of miR-93 is also upregulated in NPC still remains unclear. To explore the clinical significance of serum miR-93 expression in NPC, RT-qPCR was used to detect the serum miR-93 expression in 85 NPC patients and 30 healthy controls. As indicated in Fig. 1, the serum miR-93 expression was significantly increased in NPC patients compared with healthy controls.

Radiotherapy is a conventional treatment strategy for NPC, and all of the patients recruited for the present study received radiotherapy. Serum samples were collected from the NPC patients following treatment, and the serum miR-93 levels were compared between the NPC patients prior to and following treatment. The data demonstrated that following receiving the standard chemo-radiotherapy, the serum miR-93 expression was significantly decreased (Fig. 2).

Subsequently, the clinical association between serum miR-93 expression and clinical characteristics in NPC patients was analyzed. The mean expression (3.88±1.75) of serum miR-93 was defined as a cut-off point and NPC patients were divided into two groups, high serum miR-93 group (n=48) and low serum miR-93 group (n=47). A χ² test demonstrated that high serum miR-93 expression was significantly associated with a poor differentiation grade, lymph node metastasis and advanced clinical stage, but not associated with age, gender, T stage, and EBV infection (Table I). These findings suggest that increased serum levels of miR-93 are significantly associated with the malignant progression of NPC.

The clinical association between serum miR-93 expression and survival time of NPC patients was further evaluated using the Kaplan-Meier method. The data indicated that those NPC patients with higher serum miR-93 levels exhibited a shorter overall survival time, when compared with those with lower serum miR-93 expression (Fig. 3). Accordingly, the increased serum miR-93 expression was associated with worse prognosis of NPC patients.

The potential factors that may predicate the prognosis were further analyzed using univariate and multivariate analyses. As indicated in Table II, several clinical factors including grade, lymph node metastasis, clinical stage, and serum miR-93 expression were significantly associated with the survival time. Furthermore, multivariate analysis indicated that serum miR-93 expression level, grade, lymph node metastasis, and clinical stage were independent factors for predicating the prognosis of NPC patients (Table III).

These findings suggest that miR-93 may serve a promoting role in NPC progression. Subsequently, the expression levels of miR-93 in NPC cell line C666-1 were evaluated, and the normal nasopharyngeal epithelial cell line NP69 was used as a control. As presented in Fig. 4A, qPCR data indicated that miR-93 was also significantly upregulated in C666-1 cells compared with NP69 cells. C666-1 cells were then transfected with miR-93 inhibitor or NC inhibitor as a control. As indicated in Fig. 4B, transfection with miR-93 inhibitor caused a significant reduction in miR-93 levels, when compared with the NC inhibitor group. MTT assay data further demonstrated that knockdown of miR-93 significantly decreased the proliferation of C666-1 cells, when compared with the NC inhibitor group (Fig. 4C). To further support these data, C666-1 cells were transfected with miR-93 mimic or miR-NC as the control group. Following transfection, the miR-93 levels were significantly increased in the miR-93 group compared with the miR-NC group (Fig. 4D). Furthermore, overexpression of miR-93 significantly promoted C666-1 cell proliferation (Fig. 4E). These findings suggest that miR-93 serves a promoting role in NPC growth.

The potential target of miR-93 in NPC was then evaluated. Bioinformatics analysis indicated that PDCD4 was a target gene of miR-93 (Fig. 5A). To confirm this targeting association, luciferase vectors containing WT or MT PDCD4 3'-UTR were generated (Fig. 5A and B). Luciferase reporter assay data indicated that the luciferase activity was significantly reduced in C666-1 cells co-transfected with the WT-PDCD4 luciferase reporter vector and miR-93 mimics, when compared with the control group (Fig. 5C). However, these effects were eliminated by transfection with MT-PDCD4 luciferase reporter vector. Therefore, miR-93 was demonstrated to directly bind to the 3'UTR of PDCD4 mRNA. The effects of miR-93 on PDCD4 protein expression were further determined. Western blotting data demonstrated that knockdown of miR-93 significantly increased the protein level of PDCD4 in C666-1 cells (Fig. 5D). Conversely, overexpression of miR-93 significantly reduced the PDCD4 protein expression in C666-1 cells (Fig. 5E). Accordingly, miR-93 negatively regulates the protein expression of PDCD4 via directly binding to the 3'UTR of its mRNA in C666-1 cells.

It was then speculated that the promoting effects of miR-93 on NPC cell proliferation may be through negatively regulating PDCD4 expression. To clarify this speculation, C666-1 cells were co-transfected with miR-93 mimic and pcDNA3.1-PDCD4 expression plasmid. Transfection with miR-93 mimic was used as the control group. Following transfection, the protein expression levels of PDCD4 were examined. As demonstrated in Fig. 6A, the protein levels of PDCD4 were significantly higher in the miR-93+PDCD4 group when compared with those in the miR-93 group. MTT assay data further indicated that the proliferation of C666-1 cells was significantly reduced in the miR-93+PDCD4 group when compared with the miR-93 group (Fig. 6B). These findings suggest that miR-93 promotes NPC cell proliferation, at least partly, through directly targeting PDCD4.