The study was approved by the Medical Ethics Committee of the Affiliated Hospital of Inner Mongolia Medical University (Hohhot, China). In total, 19 human mucoepidermoid carcinoma specimens of the submaxillary salivary gland and 18 control submaxillary salivary gland specimens were obtained by surgical resection prior to the administration of radiotherapy or chemotherapy for the SGMN patients or pleomorphic adenoma patients. Informed consent and agreement were provided by all the patients. Clinicopathological data of the patients were recorded prospectively, including the age at diagnosis, tumor size, axillary lymph node metastasis and histological grade. All fresh tumor specimens were immediately frozen in liquid nitrogen and stored at −70°C following resection. An epidermoid carcinoma cell line originating from the human submaxillary salivary gland (A-253 cell line) was purchased from the American Type Culture Collection (Rockville, MD, USA). The cells were cultured at 37°C in a humidified atmosphere of 5% CO₂ in McCoy's 5a Medium Modified (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% fetal bovine serum (Gibco Life Technologies, Rockville, MD, USA).

To generate an A-253 cell line that overexpressed PTTG, the wild-type PTTG coding sequence was amplified and cloned into a pcDNA3.1 (+) vector (Invitrogen Life Technologies, Carlsbad, CA, USA). Subsequently, the PTTG-2A-EGFP-pcDNA3.1 (+) or control CAT-pcDNA3.1 (+) vectors were transfected into the A-253 cells using Lipofectamine 2000 (Invitrogen Life Technologies). The PTTG- and EGFP-positive cell clones were selected under the pressure of 1.5 mg/ml G418 (Sigma-Aldrich), and maintained in medium containing 1 mg/ml G418.

SGMN tissue slides were successively deparaffinized by heating at 55°C for 30 min, and rehydrated serially in 100, 90 and 70% ethanol, and phosphate-buffered saline (PBS). Subsequently, antigen retrieval was performed by heating for 20 min at 98°C in 10 mM sodium citrate (pH 6.0), after which the endogenous peroxidase activity was blocked by incubation with 0.3% hydrogen peroxide for 20 min. Rabbit polyclonal antibodies targeting human PTTG (1:100; PA5-14240) and β-actin (1:300; PA1-183; Thermo Fisher Scientific, Waltham, MA, USA) were utilized to stain for PTTG and β-actin expression. The samples were incubated with the primary antibodies against PTTG and β-actin for 1 h at room temperature, followed by incubation with a goat anti-rabbit/mouse IgG horseradish peroxidase (HRP)-conjugated secondary antibody (1:1,000; ab6721; Abcam, Cambridge, UK) at room temperature for 1 h. Subsequently, the HRP substrate, 3,3′-diaminobenzidene (Abcam), was added for 1–5 min and counterstained with Mayer's hematoxylin, after which the samples were dehydrated and sealed with cover slips.

Total cellular mRNA was isolated from the tumor specimens or from the A-253 cells using TRIzol reagent (Invitrogen Life Technologies), according to the manufacturer's instructions. RT-qPCR was performed to quantify the mRNA expression levels of PTTG using a SYBR PrimeScript RT-qPCR kit (Takara Bio, Inc., Tokyo, Japan) and a LightCycler 2.0 (Roche Diagnostics Gmbh, Mannheim, Germany), where β-actin was used as an internal control. The primer sequences for PTTG were as follows: 5′-ACC TTT GCT TCT CCC ACC TT-3′ (sense) and 5′-CAAATA CAC ACA AAC TCT GAA GCA-3′ (antisense). The primer sequences for β-actin were as follows: 5′-TTC TAC AAT GAG CTG CGT GTG-3′ (sense) and 5′-GGG GTG TTG AAG GTC TCA AA-3′ (antisense). All the primers were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). PTTG expression was normalized against β-actin, and was expressed as the fold change over the control, as calculated according to the ∆∆Ct method (19).

PTTG protein expression levels in the tumor specimens were analyzed by western blot analysis. The tumor specimens were homogenized prior to protein isolation. The homogenized specimens or cultured cells were collected and lysed with a lysis reagent (Pierce Biotechnology, Inc., Rockford, IL, USA), according to the manufacturer's instructions, which was supplemented with a protease inhibitor cocktail (Thermo Fisher Scientific). Protein samples were separated by 12% SDS-PAGE and transferred to a nitrocellulose membrane (EMD Millipore, Bedford, MA, USA). The protein expression levels of PTTG and β-actin were quantified via successive incubation at 37°C for 1 h with anti-PTTG (1:300; PA5-14240) or anti-β-actin (1:1,00; PA1-183) polyclonal rabbit primary antibodies, followed by a monoclonal peroxidase-conjugated secondary antibody against rabbit IgG (1:1,000; A1949; Sigma-Aldrich). An electrochemiluminescence detection system (GE Healthcare Life Sciences, Uppsala, Sweden) was used for visualization of the membranes, according to the manufacturer's instructions.

A cell count assay was performed as previously described (20). Briefly, 1×10³ or 1×10⁴ A-253 PTTG (+) and A-253 PTTG (-) cells/ml were seeded in six-well plates and incubated at 37°C for 12, 24 or 48 h, or for 1, 2 or 4 days, after which the cells were trypsinized. The number of viable cells was counted in a hemocytometer (Tainuokeji, Taian, China) with the use of trypan blue staining (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The rate of cell migration was determined by a scratch assay. A-253 PTTG (+) or A-253 PTTG (-) cells were cultivated to 90% confluence on six-well plates. Subsequently, cell scrapers (Corning, Inc., Corning, NY, USA) were utilized to scratch the confluent cells. After 48 h, the cells that had migrated across the baseline were observed and counted using a BX60 microscope (Olympus Optical Co., Ltd., Tokyo, Japan). All the experiments were repeated in triplicate.

Statistical analysis was performed using GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). All data are expressed as the mean ± standard error of the mean. The difference in the PTTG-positivity rate between the two groups was evaluated using the χ² test. Comparisons between the expression of PTTG at an mRNA or protein level, or the cell number in the two groups, were analyzed using the Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

To identify a possible oncogenic role of PTTG in SGMNs, the PTTG expression levels in the SGMN specimens were determined. Firstly, as shown in Fig. 1A and B, there was a higher rate of PTTG-positive cells in the SGMN tissues when compared with the control submaxillary salivary gland tissues (14/19 vs. 7/18, P=0.0327), with a cutoff of 2 positive cells per field. To further reveal the difference in the PTTG expression levels between the tumor and non-tumor groups, the mRNA expression levels of PTTG in all the specimens from the two groups were analyzed. The results demonstrated that the mRNA expression level of PTTG in the SGMN specimens was 1.772±0.187 (n=19), as compared with the average level of 1.032±0.096 (n=18) in the control group, which was a statistically significant difference (P=0.0014; Fig. 2A). In addition, the protein expression levels of PTTG were further analyzed using western blot analysis in 16 SGMN specimens and 14 control specimens that were of sufficient sample volume to facilitate the assay. As shown in Fig. 2B, the protein expression level of PTTG in the SGMN specimens was also significantly higher when compared with the control specimens (P<0.05, paired-samples t-test). Therefore, the overexpression of PTTG in SGMN specimens was confirmed.

To identify the oncogenic role of PTTG in SGMN cells, a PTTG and EGFP coexpressing A-253 cell line was constructed. As shown in Fig. 3A, the cDNA of PTTG and EGFP were linked by a 2A peptide coding sequence, which guaranteed the transcription of PTTG and EGFP in one mRNA molecule, but enabled the translation of two separate proteins (21,22). EGFP-positive A-253 cells, which were transfected with the recombinant PTTG-2A-EGFP-pcDAN3.1 (+) plasmid, were selected under G418 pressure. Following serial passages with 1.5 mg/ml G418, >85% of the A-253 cells were determined to be EGFP-positive (Fig. 3B). Subsequently, the overexpression of PTTG was determined in the EGFP-positive A-253 cells. Stabilized PTTG overexpression at the mRNA level in the A-253 PTTG (+) cells was determined using a RT-qPCR method with the PTTG specific primers. Significantly higher expression levels of PTTG mRNA were observed in the A-253 PTTG (+) cells at the passages of 1, 3 or 5 (P<0.01, vs. control A-253 cells, using the t-test; Fig. 3C). In addition, PTTG overexpression at a protein level was stabilized in the A-253 PTTG (+) cells (P<0.01, vs. control A-253 cells, as determined with the t-test; Fig. 3D). Therefore, the A-253 PTTG (+) cells were shown to stably express PTTG.

The influence of PTTG overexpression on the growth of SGMN cells was subsequently evaluated. The growth of the A-253 PTTG (+) or A-253 PTTG (-) cells in vitro was assessed using a cell count assay, in which cell counting was conducted at 12, 24 and 48 h after cell inoculation. As shown in Fig. 4A, when inoculated at a density of 10⁴ cells/ml, the A-253 PTTG (+) cells grew more efficiently compared with the A-253 PTTG (-) cells at 48 h after cell inoculation, and the difference was statistically significant (4.51±0.38×10⁴/ml for A-253 PTTG (+) cells and 3.733±0.32×10⁴/ml for A-253 PTTG (-) cells at 48 h after inoculation; P<0.05). In addition, the difference in growth between the two cell lines was reconfirmed following inoculation at 10³ cells/ml, where the growth of the A-253 PTTG (+) cells was also significantly more efficient compared with the A-253 PTTG (-) cells (Fig. 4B; P<0.05 for 24 h; P<0.01 for 48 h). Thus, the promotive role of PTTG in the growth of SGMN cells was confirmed. Cell migration contributes to the extent of tumor metastasis; thus, the migration of the A-253 PTTG (+) and A-253 PTTG (-) cells were further assessed using a scratch assay. As shown in Fig. 4B and C, at 48 h after the initiation of the scratch assay, a significantly increased number of A-253 PTTG (+) cells had migrated across the baseline when compared with the A-253 PTTG (-) cells (92.67±7.51 vs. 41±4.62, P<0.01). Therefore, the results indicated that overexpression of PTTG stimulated the growth and migration of SGMN cells.