Paired primary TSCC samples and adjacent histological normal tissues were collected from 20 TSCC patients, consisting of 14 male and 6 female patients (age, 62.36±5.74 years), at the Department of Oral and Maxillofacial Surgery, Guanghua School of Stomatology, Sun Yat-sen University (Guangzhou, China), between September 2006 and December 2011. Tumor tissues and adjacent normal tissues that were at least 1.5 cm distal to the tumor margins were snap-frozen in liquid nitrogen and then stored at −80°C until use. All samples were evaluated by two pathologists according to the World Health Organization guidelines and pathological tumor-node-metastasis Union for International Cancer Control pathological staging criteria (20), and the samples were examined for the expression level of survivin, miR-335 and miR-182. Patients that received chemotherapy or radiotherapy prior to surgery were excluded from the present study. Informed consent was obtained from all patients, and the study was approved by the Human Research Ethics Committee of Sun Yat-Sen University.

The human TSCC SCC-15 cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The SCC-15 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin (Gibco; Thermo Fisher Scientific, Inc.) and 100 µg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a 5% CO₂ atmosphere.

The SCC-15 cells were transfected with miR-335 mimics, miR-182 mimics or the negative control (scramble control sequence) and incubated for 48 h at room temperature. Subsequently, 30 µl 0.5% MTT (Sigma-Aldrich, St. Louis, MO, USA) solution was added to each well and the wells were incubated for another 4 h. The medium was then replaced with 150 µl dimethyl sulfoxide (DMSO; Sigma-Aldrich) in the microplate, and then agitated on a rotary platform for 10 min. The survival rate was determined by measuring the optical density (OD) values of the mimic-transfected cells relative to the OD of the control at a 492-nm wavelength using a Jenway spectrophotometer 7300 (67 series VU Visible Scanning spectrophotometer; Bibby Scientific Ltd., Stone, Staffordshire, UK).

Total RNA was obtained using TRIzol One-Step RNA Isolation kit (Invitrogen; Thermo Fisher Scientific, Inc.). Complementary DNA specific for miR-335, miR-182, survivin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which acted as an internal control, was synthesized from total RNA using gene-specific primers with the TaqMan MicroRNA assay, according to the manufacturer's protocol (Applied Biosystems; Thermo Fisher Scientific, Inc.). Relative quantification of target miRNA expression was measured using the 2^−ΔΔCq method (21). Each sample was examined in triplicate and the raw data were expressed as the relative quantity of target miRNA, normalized with respect to GAPDH.

The SCC-15 cells were cultured in DMEM supplemented with 10% FBS at 37°C in an atmosphere of 5% CO₂ to 70–80% confluency. Using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), the cells were then transfected with the WT-Sur-UTR DNA plasmid, which consisted of a firefly luciferase reporter vector containing the wild-type survivin 3′-UTR, or the mutant Sur-UTR DNA plasmids (Mut1 and Mut2), which consisted of a firefly luciferase reporter vector containing the mutant survivin 3′-UTR, (Fig. 1) and the control pRL-TK vector, which contained Renilla luciferase (Promega Corporation, Madison, WI, USA). The firefly and Renilla luciferase activities were measured consecutively using Dual Glo Luciferase assays (Promega Corporation) 48 h subsequent to transfection.

Lysates were resolved on 12% sodium dodecyl sulfate-polyacrylamide gels and the bands were transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk at room temperature for 1 h followed by incubation with a rabbit anti-human survivin antibody (catalog no., ab2050; Abcam, Cambridge, UK) under room temperature for 2 h and subsequently incubated for 2 h with horseradish peroxidase-conjugated anti-rabbit secondary antibody at room temperature for 1 h. Chemical fluorescence was detected using an Enhanced Chemiluminescence Western Blotting Detection Reagent kit (GE Healthcare, Little Chalfont, Buckinghamshire, UK), according to the manufacturer's protocol. The target bands underwent densitometric analysis with Quantity One^® 1-D analysis software version 170-9600 (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and were normalized against β-actin. A total of 3 independent experiments were performed.

The SCC-15 cells were digested with trypsin-ethylenediaminetetraacetic acid solution (Beyotime Institute of Biotechnology, Haimen, China), washed with phosphate-buffered saline (PBS; Invitrogen; Thermo Fisher Scientific, Inc.), and fixed in 70% ethanol 48 h subsequent to transfection. Cell cycle analyses were conducted using propidium iodide staining. FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) was used to determine G0/G1, S and G2/M fractions. For the apoptosis analysis, an Annexin V-FLUOS Staining kit purchased from Roche (Mannheim, Germany) was used 48 h subsequent to transfection, according to the manufacturer's protocol. The results were analyzed using CellQuest Pro software (BD Biosciences).

For the cell invasion assay, 8-µm pore polycarbonate membrane filters (BD Biosciences) pre-coated with 50 µl Matrigel (1.25 mg/ml; BD Biosciences) were used. The cells transfected with each miR were collected and seeded at a density of 1×10⁵ cells/well with 1% FBS-supplemented DMEM in the cell culture chambers, with the bottom chambers filled with 0.6 ml medium. The chamber was incubated at 37°C for 48 h prior to the removal of the Matrigel. The invaded cells were fixed, stained and calculated. The invasiveness of cells was evaluated by the following equation: Invasion index (%) = invaded cell number / total cell number × 100.

The data were expressed as the mean ± standard deviation. The differences were assessed for significance using Student's t-test, χ² test or one-way analysis of variance followed by Fisher's least significant difference test, as appropriate. The statistical analysis was conducted by SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

To evaluate the effect of miR-335 and miR-182 on the proliferation and invasion of TSCC cells, the SCC-15 cells were transfected with miR-335 and miR-182 mimics. As shown in Fig. 1A and B, the levels of miR-335 and miR-182 were increased >30-fold at 24 h subsequent to transfection compared with the control. The MTT assay results showed that exogenous overexpression of miR-335 and miR-182 individually and synergistically suppressed the proliferation of SCC-15 cells compared with the control (Fig. 1C). In addition, the migratory ability of the cells with or without overexpression of miR-335 and miR-182 was examined, and the introduction of either miR-335 or miR-182 was not found to affect the migratory ability of the cells (data not shown). The results indicated that miR-335 and miR-182 each played inhibitory roles in the regulation of cell proliferation.

To investigate the mechanism underlying the effect of miR-335 and miR-182 on cell proliferation, cell cycle distribution analysis was performed in TSCC cells using flow cytometry. The data revealed that overexpression of miR-335 and/or miR-182 arrested the growth of TSCC cells in the G1 phase, with a decrease in the proportion of cells in the G2/M and S phases (Fig. 2). The result showed that compared with the control group, the percentage of TSCC cells in the G1 phase was increased between 61.35 and 75.14% in cells transfected with miR-335 alone (P<0.01), 61.35 and 72.11% in cells transfected with miR-182 alone (P<0.01) and 61.35 and 88.32% in cells transfected with miR-335 combined with miR-182 (P<0.01). The percentage of cells in the S phase decreased between 29.26 and 16.24% in cells transfected with miR-335 alone (P<0.01), 29.26 and 19.50% in cells transfected with miR-182 alone (P<0.01) and 29.26 and 4.23% in cells transfected with miR-335 combined with miR-182 (P<0.01). The population of cells in the G2 phase decreased between 9.39 and 8.63% in cells transfected with miR-335 alone (P<0.01), between 9.39 and 8.39% in cells transfected with miR-182 alone (P<0.01) and between 9.39 and 7.45% in cells transfected with miR-335 and miR-182 (P<0.01)(Fig. 2A-D). These data demonstrated that overexpression of miR-335 and miR-182 suppressed the proliferation of TSCC cells by arresting cell growth in the G1 phase.

The miRNA databases miRanda (22) and TargetScan (23) were searched for potential target genes of miR-335 and miR-182, in order to identify the mediators of the inhibitory effect of miR-335 and miR-182 on cell proliferation. By screening the candidate genes that were identified based on their functions combined with the information about the miRNA obtained from the DIANA lab-based microRNA pathway analysis tool (diana.imis.athena-innovation.gr/DianaTools/index.php), all predicted genes were categorized functionally to identify candidate genes that are functionally associated with the regulation of the cell cycle. Finally, the present study focused on survivin, a key cell cycle regulator and a putative shared target of miR-335 and miR-182 (Fig. 1E). Therefore, it was hypothesized that miR-335 and miR-182 may participate in the regulation of the cell cycle by targeting survivin. To test the hypothesis, the present study measured and compared the expression level of survivin in the TSCC cells transfected with miR-335 and miR-182 mimics using western blot analysis. The effects of these mimics on survivin knockdown were comparable with the survivin level in cells co-transfected with the two miRs, which demonstrated an additional decrease (Fig. 1D). Furthermore, a luciferase assay was conducted to confirm that survivin is a direct shared target of miR-335 and miR-182. Wild-type or mutant survivin 3′-UTR was cloned downstream of a firefly luciferase gene (Fig. 1F), and the constructs were transfected into miR-335 or miR-182-overexpressing cells or the negative control. In addition, overexpression of miR-335 significantly decreased the relative luciferase activity of the wild-type and Mut1 survivin 3′-UTR, but had minimal effects on the relative luciferase activity of the Mut2 survivin 3′-UTR (Fig. 1G). Overexpression of miR-182 suppressed the luciferase activity of wild-type and Mut2 (Fig. 1H), but the luciferase activity of Mut1 was almost unaltered, suggesting that miR-335 and miR-182 suppress the expression of survivin by binding directly to the corresponding ‘seed sequence’ in the 3′-UTR of survivin.

The levels of miR-335, miR-182 and survivin were measured and compared in cancerous tissues (n=20) and adjacent normal tissues (n=20). As shown in Fig. 2E, it was found that the relative expression level of miR-335 and miR-182 in cancerous tissue was significantly downregulated to ~20 and 10%, respectively, compared with normal tissues. The expression level of survivin mRNA was measured by RT-qPCR, and it was found that the mRNA level of survivin in the cancerous tissue was significantly increased compared with normal tissues (Fig. 2F). The survivin protein level was also measured using western blotting, and the relative density of survivin in the cancerous tissue was almost 5-fold higher compared with the normal control (Fig. 2G and H).