A total of 50 TSCC and 10 cancer-adjacent normal tongue tissues were collected at the Stomatology Hospital of Southern Medical University (China) between January 2013 and December 2015. Patients did not receive any treatment and had no history of other malignancies prior to tumor surgery. Recorded clinicopathological parameters included sex, age, pTNM status, histology grade and clinical stage. All experimental procedures were approved by the Ethics Committee of Southern Medical University. All patients provided written informed consent.

All tissue samples were examined for TMEM40 expression by immunohistochemistry (IHC) using hematoxylin-eosin (H&E) stain and a phase-contrast microscope (Nikon Eclipse Ti-S; Nikon Corp., Tokyo, Japan). Firstly, a tissue microarray (TMA) was constructed according to a standard method. Then, the TMA block was cut into 5-µm sections for IHC analysis. It was processed based on standard procedures (9). Tissues were dehydrated and incubated with 5% normal goat serum for blocking. Subsequently, tissues were incubated with TMEM40 primary antibody (mouse monoclonal; dilution 1:200; cat. no. sc-393601; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at 4°C overnight followed by incubation with goat anti-rabbit secondary antibody [dilution 1:5,000; cat. no. 70-GAR007; Hangzhou MultiSciences (Lianke) Biotech Co., Ltd., Hangzhou, China]. Sections were visualized using a DAB Horseradish Peroxidase (HRP) Color Development kit (Beyotime Institute of Biotechnology, Haimen, China) and counterstained with hematoxylin. Two independent investigators determined the staining score by multiplying the staining intensity and the percentage of positive cells. A receiver operating characteristic (ROC) curve analysis, based on clinicopathological parameters, was used to obtain the optimal cut-off score. If the TMEM40 score was below the cut-off score, tissue was defined as TMEM40 negative; otherwise it was TMEM40 positive.

Human tongue cell lines CAL27 and SCC9 were purchased from the China Center for Typical Culture Collection (CCTCC; Wuhan, China). Cells were cultured in Dulbecco's modified Eagle's medium/nutrient mixture F-12 (DMEM/F12; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; HyClone Laboratories; GE Healthcare Life Sciences, Logan, UT, USA) and 100 U/ml penicillin/streptomycin and cells were maintained at 37°C in a humidified atmosphere with 5% CO₂.

Plasmid DNA was transfected to evaluate TMEM40 overexpression. The TMEM40 sequence and negative control were synthesized and cloned into a pEZ-M98 vector (Guangzhou Fulengen Co., Ltd., Guangzhou, China). According to the manufacturer's protocol, the pEZ-M98-TMEM40 plasmid or empty pEZ-M98 vector were used to transfect CAL27 and SCC9 cells using Lipofectamine 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and cultured in 6-well plates. siRNAs were used to knockdown TMEM40 expression. siRNAs targeting TMEM40 and a negative control (NC) were purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). The specific siRNA sequences were as follows: TMEM40-siRNA-1, 5′-GGATGAGCTTCAACTCTAT-3′; TMEM40-siRNA-2, 5′-GTGGAGGCCTCTCAGTTAA-3′; NC, 5′-UUCUCCGAACGUGUCACGUTT-3′. Total RNAs were isolated 48 h after transfection to detect TMEM40 mRNA expression levels.

To quantify TMEM40 mRNA expression, total RNAs were isolated from the cells 48 h after transfection. Total RNAs were extracted using RNAiso Plus (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's protocol and dissolved into nuclease-free water. A PrimeScript^® RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China) was used for RT of mRNA to cDNA. Then, qPCR was performed to detect TMEM40 expression levels using the SYBR^® Premix Ex TaqTM II kit (Takara Biotechnology Co., Ltd.) and an ABI 7500 real-time PCR amplifier (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermal cycling conditions were as follows: 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 34 sec. The following primers were used: TMEM40 forward, 5′-CAGAGCAACCGGAAAACATCG-3′ and reverse, 5′-CTGGGCTACACTGAGCACC-3′; GAPDH forward, 5′-AGAAGGCTGGGGCTCATTTG-3′ and reverse, 5′-AAGTGGTCGTTGAGGGCAATG-3′. Each reaction was analyzed in triplicate. The comparative Ct (2^−ΔΔCq) (10) method was used to calculate the relative levels of TMEM40.

To detect TMEM40 protein expression, total proteins were extracted from cells 72 h after transfection. Radioimmunoprecipitation assay buffer containing 1 mM PMSF (Beyotime Institute of Biotechnology) was used. Protein concentrations were determined using a BCA Protein Assay kit (Beyotime Institute of Biotechnology). Protein samples were mixed with loading buffer [4:1 (v/v); Beyotime Institute of Biotechnology] and were boiled for 10 min. Samples were separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA) with a wet-transfer machine (Trans-blot SD; Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membrane was blocked with 5% non-fat milk at room temperature for 2 h and incubated with TMEM40 primary antibody (mouse monoclonal; 1:500; Santa Cruz Biotechnology, Inc.) and β-actin primary antibody (mouse monoclonal; 1:2,000; ProteinTech Group, Inc., Chicago, IL, USA) at 4°C overnight. The membrane was washed with 1X TBST (3X) and incubated with HRP-conjugated secondary antibody (goat anti-mouse IgG; 1:5,000; NeoBioscience, Shenzhen, China) at room temperature for 2 h, followed by washing with 1X TBST (3×). Immunoreactive bands were visualized with the SmartChemi610 (Beijing Sage Creation Science Co., Ltd., Beijing, China) using the Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore).

Cell Counting Kit-8 (CCK-8; Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) was used to assess cell viability. A total of 1,000-2,000 transfected cells were seeded in 96-well plates and incubated for 3 days. At the indicated time-point, 10 µl of the CCK-8 stock solution was added to each well together with 100 µl medium and incubated at 37°C for 2 h. The absorbance value (optical density) was measured at 450 nm using a multifunctional microplate reader (Tecan Infinite M200; Tecan Group Ltd., Mannedorf, Switzerland). All experiments were performed in triplicate and five duplicate wells were screened.

A total of 2 ml complete medium containing 2,000 transfected cells was added to 6-well plates and incubated at 37°C with 5% CO₂ for 10 days. After discarding the supernatant, cells were washed with phosphate-buffered saline (PBS) (3X), fixed using 500 µl of methanol at room temperature for 20 min and stained with 0.1% crystal violet (Nanjing KeyGen Biotech Co., Ltd.) for 20 min. The colony formation efficacy was determined. Assays were performed in triplicate.

Flow cytometry was used to evaluate the cell apoptosis ratio of the transfected cells. Harvested cells were double-labeled with Annexin V-FITC and PI apoptosis detection kits (Nanjing KeyGen Biotech Co., Ltd.) and a BD FACSVerse flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) was used for the detection of apoptosis. For cell cycle measurements, transfected cells were harvested, digested with 0.05% trypsin and fixed with 70% ethanol at −20°C overnight. Cells were stained with 0.1 mg/ml propidium iodide (PI; Sigma-Aldrich; Merck KGaA, Darmstadt, USA) for 30 min. A FACSVerse flow cytometer was used to measure the intensities of the fluorescence signals. A minimum of 20,000 cells was observed for each sample. Percentages of cells in the G₀/G₁, S and G₂/M phases were evaluated using FlowJo software (Tree Star, Inc., Ashland, OR, USA). All experiments were performed in triplicate.

A wound-healing assay was performed to assess cell migration. When cells reached a density of 80–90% after transfection, a 10-µl pipette tip was used to inflict a straight wound. PBS was used to rinse the cell debris from the dishes and serum free medium was added for cell culturing. Scrape lines were based on the border of the majority of migratory cells and photographed using a phase-contrast microscope (Nikon Eclipse Ti-S; Nikon Corp.) at different time-points (0, 12, 24 and 48 h). The scratch area and wound-healing percentage were calculated using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The experiment was repeated in triplicate and we selected the simultaneous groups.

Cell invasion assays were performed using Transwell chambers (8-µm pore size; Corning Inc., Corning, NY, USA) in 24-well plates. Transwell chambers were coated with 30 µl Matrigel (Corning Inc.) and incubated for 60 min at 37°C. Transfected CAL27 and SCC9 cells were counted and 1×10⁵ cells/chamber were seeded in serum-free DMEM/F12 medium. The lower well was filled with 800 µl complete medium. After 36 h of culturing at 37°C in a 5% CO₂ incubator, the cells in the upper layer were removed with a swab. The cells on the bottom of the Transwell chamber were fixed with methanol and stained with 0.1% crystal violet (Nanjing KeyGen Biotech Co., Ltd.). The results were photographed at five randomized visual fields and experiments were repeated three times.

All statistical analyses were completed using the SPSS 20.0 software. Data are presented as the mean ± SD. ROC was adopted to select an optimal cut-off score for TMEM40 positive. A t-test was used to evaluate differences in expression between two groups. One-way ANOVA followed by the Tukey's post hoc test was used to analyze the CCK-8 assay data. A Chi-square test was used to assess the association between the IHC staining score and the clinicopathological parameters of TSCC. Differences were considered statistically significant at P<0.05.

To investigate the protein expression level of TMEM40 in TSCC tissues, IHC was performed in 50 TSCC and 10 cancer-adjacent normal tongue tissue sections, which were formalin-fixed and paraffin-embedded. Four representative samples of different levels of TMEM40 IHC staining are shown in Fig. 1. As shown in Table I, TMEM40 was observed in 44.0% (22/50) of the TSCC specimens and 0.00% (0/10) of the normal tongue tissues. The results indicated that TMEM40 expression was significantly increased in tumor tissues compared with the normal tongue tissues (χ²=2.756; P=0.008; Table I).

The results of the association analysis between clinicopathological characteristics and TMEM40 protein expression levels in 50 patients with TSCC are summarized in Table II. The results indicated that high TMEM40 expression levels were correlated with pTNM status (χ²=4.884; P=0.027), histological grade (χ²=17.949; P<0.001) and clinical stage (χ²=10.189; P=0.006). However, there was no association between TMEM40 expression levels and sex or age.

TSCC cell lines CAL27 and SCC9 were cultured and transfected with pEZ-M98-TMEM40 plasmid or empty pEZ-M98 vector, or siRNA or negative control. The results revealed that 40% of the transfected pEZ-M98-TMEM40 plasmid or empty pEZ-M98 vector cells died with the transfection experiment while no cells died with transfection of siRNA or the negative control. The expression level of TMEM40 was analyzed by RT-qPCR and amplification efficiency by qPCR was estimated at 98–100% for all samples. The results revealed that the relative mRNA expression levels of TMEM40 in plasmid-transfected cells were significantly upregulated and in siRNA-transfected cells were significantly downregulated (Fig. 2A; P<0.05; P<0.01). As shown in Fig. 2A, the RT-qPCR results revealed that both TMEM40 siRNAs (TMEM40-siRNA-1 and TMEM40-siRNA-2) efficiently silenced TMEM40 expression in CAL27 and SCC9 cells. TMEM40-siRNA-1, which exhibited a more pronounced inhibitory effect, was selected for the following experiments. Western blot analysis was performed to detect the protein expression levels of TMEM40, which revealed consistent results (Fig. 2B).

To explore the influence of TMEM40 on the growth of TSCC cells, CCK-8 assays were applied to determine the cell viability of CAL27 and SCC9 cells. The pEZ-M98-TMEM40 plasmid-transfected cells increased significantly in cell viability compared with the negative control group (Fig. 3A). In contrast, TMEM40-siRNA suppressed the growth of CAL27 and SCC9 cells (Fig. 3A). The results indicated that TMEM40 induced cell proliferation in TSCC cells.

The role of TMEM40 in the regulation of colony formation in TSCC cells was investigated. CAL27 and SCC9 cells transfected with pEZ-M98-TMEM40 plasmid indicated a significant higher colony-forming efficacy compared with the control group, which was transfected with empty pEZ-M98-TMEM40 plasmid. Low expression of TMEM40 suppressed colony formation abilities of cells transfected with TMEM40-siRNA (Fig. 3B and C; P<0.05; P<0.01; P<0.001). The data demonstrated that TMEM40 promoted colony formation.

The influence of TMEM40 on TSCC apoptosis and cell cycle was assessed using flow cytometric assays. CAL27 and SCC9 cells were transfected with pEZ-M98-TMEM40 plasmid or empty pEZ-M98 vector and the early and late cell apoptosis ratios were observed (Fig. 4A). We calculated the total apoptosis and the results revealed that apoptosis in TMEM40-overexpressing cells was inhibited, while apoptosis in TMEM40-siRNA cells was increased (Fig. 4C; P<0.01; P<0.001). These results indicated that TMEM40 inhibited cell apoptosis in TSCC. In addition, cell cycle analysis was performed to investigate the influence of TMEM40 on TSCC cell growth (Fig. 4B). The results revealed that overexpression of TMEM40 decreased the population of the CAL27 and SCC9 cells in the G₀/G₁ phase and increased S-phase cells while silencing of TMEM40 increased the population of the G₀/G₁ phase and decreased the S phase (Fig. 4D; P<0.01; P<0.001). With the G₂/M phase a consistent conclusion was not able to be reached. The results indicated that high expression of TMEM40 promoted proliferation and low expression of TMEM40 induced cell cycle arrest at the G₀/G₁ phase.

Cell migration was detected in TSCC cells using wound-healing assays. We performed a scratch wound-healing assay in TMEM40-overexpressing and silenced CAL27 and SCC9 cells. As expected, cell migration activities of TSCC cells transfected with pEZ-M98-TMEM40 were significantly increased compared with the control cells transfected with empty pEZ-M98 vector. Silencing of TMEM40 significantly inhibited cell mobility compared with the control cells (Fig. 5A and B; P<0.01; P<0.001). Transwell assays were processed to determine further effects of TMEM40 on TSCC cells. The results revealed that high expression of TMEM40 significantly increased cell invasion in CAL27 and SCC9 compared with the control cells transfected with empty pEZ-M98 vector. In contrast, the inhibition of TMEM40 revealed a marked suppressive effect on cell invasion in CAL27 and SCC9 compared with the negative control (Fig. 6A and B; P<0.01; P<0.001). These findings indicated that TMEM40 promoted cell migration and invasion of TSCC.

To explore the changes on a molecular level of TMEM40 regulation of TSCC cell apoptosis and invasion, we detected alterations of tumor suppressor p53, apoptosis-associated protein Bax and invasion-associated protein MMP-9 by RT-qPCR. Knockdown of TMEM40 increased the expression levels of p53, activated Bax and decreased the levels of MMP-9 in CAL27 and SCC9 cells. These molecular changes indicated that TMEM40 knockdown enhanced apoptosis and reduced the invasion of TSCC cells involved with p53, Bax and MMP-9 (Fig. 6C; P<0.05; P<0.01; P<0.001).