Human oral epithelial cells (HOECs) were purchased from the Cell Bank of Suzhou University (Suzhou, China). Three OSCC cell lines (HN4, HN30 and HN6) were provided by the Shanghai Key Laboratory of Stomatology (School of Medicine, Ninth People's Hospital, Shanghai Jiaotong University, Shanghai, China). Cells were seeded in Dulbecco's modified Eagle's medium (cat. no. 21013024; Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (cat. no. 35-010-CV; Corning, Inc.) at 37°C in 5% CO₂. XAV939 (100 µM; cat. no. X3004; Sigma-Aldrich; Merck KGaA), a potent small-molecule inhibitor of β-catenin (18), was used to treat HN4 cells for 72 h.

Western blotting was performed as previously described (19). Total protein was analyzed by 10% SDS-PAGE and blotted on a nitrocellulose filter membrane (cat. no. 10401196; Whatman plc; GE Healthcare Life Sciences). The primary antibodies (all used at 1:1,000) were all from Cell Signaling Technology, Inc., and were as follows: PLAC8 (cat. no. 13885), proliferating cell nuclear antigen (PCNA; cat. no. 13110), cyclin D1 (cat. no. 2978), vimentin (cat. no. 5741), c-Myc (cat. no. 5605), E-cadherin (cat. no. 14472), total-β-catenin (cat. no. 9587), glycogen synthase kinase 3β (GSK3β; cat. no. 12456), non-phospho (Active) β-catenin (Ser33/37/Thr41; cat. no. 8814S), phosphorylated-GSK3β (cat. no. 5558), Akt (cat. no. 4685) and phosphorylated-Akt (cat. no. 4060). β-actin (1:2,000; cat. no. 3700) was used as the control standard. Horseradish peroxidase-conjugated rabbit anti-mouse (1:2,000; cat. no. p0161; Dako; Agilent Technologies, Inc.) or goat anti-rabbit (1:5,000; cat. no. sc2357; Santa Cruz Biotechnology, Inc.) were used as secondary antibodies. The blots were probed with primary antibodies overnight at 4°C and then incubated with the secondary antibody for 1 h at room temperature. An electrochemiluminescence western-blotting system (cat. no. 7003; Cell Signaling Technology, Inc.) was used to detect protein bands (Tanon 4600SF; Tanon Science and Techonlogy Co., Ltd.).

Total RNA was extracted from HOEC, HN4, HN30 or HN6 cells using TRIzol^® reagent (cat. no. 15596026; Invitrogen; Thermo Fisher Scientific, Inc.). Complementary DNA was synthesized using the RNA reverse transcriptase kit (cat. no. RR036A; Takara Bio, Inc.) according to the manufacturer's protocol. The temperature protocol was as follows: 37°C for 15 min and 85°C for 5 sec. Quantitative PCR was performed using SYBR™ Premix Ex Taq™ II (cat. no. RR820Q; Takara Bio, Inc.) in an ABI 7300 system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: 94°C for 5 min; 40 cycles at 94°C for 30 sec, 58°C for 45 sec and 70°C for 50 sec. The expression of mRNA was assessed by evaluating the threshold cycle (CT) values, and GAPDH was used as the internal reference gene. Relative expression was calculated using the 2^−ΔΔCq method (20). The following primers were used: GAPDH forward, 5′-AGGTCGGAGTCAACGGATTTGGT-3′ and reverse, 5′-GTGCAGGAGGCATTGCTGATGAT-3′; and PLAC8 forward, 5′-CTGTCTGTGTGGAACAAGC-3′ and reverse, 5′-GAGGACAGCAAAGAGTTGCC-3′.

Transfection experiments were performed using Lipofectamine^® 2000 (cat. no. 11668030; Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Briefly, HN4 and HN30 cells were transfected with PLAC8 plasmid (Sangon Biotech, Co., Ltd.) and control vector (cat. no. 631070; Takara Bio Inc.). HN6 cells were transfected with PLAC8 small interfering (si)RNA and negative control (Hippobiotec, Inc., http://www.hippobiotec.com). The OSCC cell lines were seeded into 6-well plates (2.5×10⁵ cells/well) and treated with 10 ug plasmid or 20 µM siRNA after 24 h of culture. Cells were harvested after 72 h of culture for western blot analysis. Cells were regularly passaged for the Cell Counting Kit-8 (CCK-8) assay. Targeted sequences of PLAC8 siRNAs were: siRNA#1 sense, 5′-CUUUGCCAAAUCAAGAGAGAUdTdT-3′ and antisense, 5′-AUCUCUCUUGAUUUGGCAAAGdTdT-3′; siRNA#2 sense, 5′-GCUGAUAUGAAUGAAUGCUGUdTdT-3′ and antisense, 5′-ACAGCAUUCAUUCAUAUCAGCdTdT-3′; and negative control siRNA (siNC) sense, 5′-UUCUCCGAACGUGUCACGUTT-3′. Altered expression of PLAC8 was verified by western blotting.

Cells were seeded at 5×10³ cells/well in a 96-well plate for 1–5 days. Cell proliferation was measured using CCK-8 (CK04; Dojindo Molecular Technologies, Inc.) according to the manufacturer's protocol. The absorbance was measured at a wavelength of 450 nm using a spectrophotometer (Omega Bio-Tek, Inc.).

Invasion of OSCC cells was measured using Matrigel^®-coated Transwell chambers (cat. no. 354480; Corning Inc.). Briefly, Matrigel-coated Transwell chambers were rehydrated at 37°C in 5% CO₂ for 2 h. Subsequently, 2.5×10⁴ cells were seeded in the Matrigel-coated Transwell chambers and culture medium without serum was placed in the lower compartment and incubated for 24 h at 37°C in 5% CO₂. Non-invasive cells in the upper membrane were removed using a cotton swab, whilst the invasive cells were stained at room temperature for 2 min using the Differential Quik Stain kit (cat. no. B4132-1A; Allegiance Chemicals LLC), and manually counted in five pre-determined fields under a light microscope (magnifications, ×10 and ×40; BX51, Olympus Corporation).

Immunofluorescence staining was performed as previously described (21). Briefly, adherent cells grown on coverslips were washed thrice with PBS and incubated in PBS containing 0.2–0.3% Triton X-100. Subsequently, E-cadherin (cat. no. 14472) and vimentin (cat. no. 5741) antibodies (both Cell Signaling Technology, Inc.) were diluted to 1:100 and incubated overnight at 4°C with cells. Next, Alexa Fluor 555 anti-mouse IgG (H+L) (cat. no. 4409) and Alexa Fluor 488 anti-rabbit IgG (H+L) (cat. no. 4412) (both Cell Signaling Technology, Inc.) were incubated for 1 h at room temperature using a 1:200 dilution. Subsequently, DAPI (cat. no. H-1200; Vector Laboratories, Inc.) was used for nuclear staining for 1 min at room temperature. Images were captured using a fluorescence microscope (BX51; Olympus Corporation; magnification, ×200).

Statistical analyses were conducted using GraphPad Prism 5.0 (GraphPad Software, Inc.). Paired Student's t-test and one-way ANOVA followed by Tukey's post hoc test were used for comparisons of two or multiple groups, respectively. All experiments were performed in triplicate and data are presented as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

To assess whether PLAC8 is expressed in OSCC, three OSCC cell lines and primary HOECs were selected to measure PLAC8 expression. Relative expression levels of the PLAC8 protein were decreased in HN4, HN30 and HN6 cells compared with in HOECs, with statistically significant differences in HN4 and HN30 cells (P<0.001; Fig. 1A). Additionally, PLAC8 mRNA expression was significantly downregulated in HN4 and HN30 cells compared with that in HOECs (P<0.05); however, HN6 cells exhibited no significant difference in PLAC8 mRNA expression compared with HOECs (Fig. 1B). Overall, the present findings suggested that OSCC cells had a low PLAC8 expression.

In order to understand the function of PLAC8 in OSCC, HN4 and HN30 cells were selected for the overexpression of PLAC8, since PLAC8 expression was lower in these two cell lines compared with that in HN6 cells (Fig. 1). Western blot analysis was performed to confirm that PLAC8 was significantly increased following transfection with PLAC8 plasmid compared with non-transfected cells and cells transfected with empty vector (Fig. 2A). PLAC8 overexpression significantly suppressed cells proliferation from day 3 according to the CCK-8 assay, compared with non-transfected HN4 and HN30 cells (P<0.05; Fig. 2B. In additional experiments, PLAC8 overexpression significantly decreased the expression levels of PCNA, c-Myc and cyclin D1 in HN4 and HN30 cells (P<0.001; Fig. 2C and D, respectively). The present results suggested that PLAC8 overexpression suppressed the proliferation of OSCC cells.

Two siRNAs were used to silence PLAC8 expression for further evaluation of the function of PLAC8 in OSCC. Expression levels of the PLAC8 protein were significantly decreased by transfection with siRNA#1 (si-#1) and siRNA#2 (si-#2) compared with those transfected with the negative control, therefore, si-#1 was randomly selected for further experimentation (P<0.001; Fig. 3A). The proliferation of HN6 cells was significantly increased from day 2 after silencing of PLAC8 expression (P<0.05; Fig. 3B). Western blotting revealed that protein levels of PCNA, c-Myc and cyclin D1 were significantly increased following PLAC8-knockdown (P<0.001; Fig. 3C). The present findings further indicated that PLAC8 repressed the proliferation of OSCC cells.

Transwell assays were performed to assess the role of PLAC8 on the invasive ability of OSCC cells. The invasive ability of HN6 cells was significantly increased after PLAC8 expression was silenced (P<0.001; Fig. 4A and B), which suggested that PLAC8 inhibited the invasion of OSCC cells.

To ascertain if EMT is involved in how PLAC8 influences the invasion of cells, western blotting was used to detect EMT-associated markers. E-cadherin expression was significantly increased, whereas vimentin expression was significantly decreased following PLAC8-overexpression in HN4 and HN30 cells (P<0.001; Fig. 4C). Knockdown of PLAC8 expression resulted in a significant downregulation of E-cadherin expression and significant upregulation of vimentin expression in HN6 cells (P<0.001; Fig. 4D). Immunofluorescence staining indicated higher EMT activity in HN6 cells in the silenced-PLAC8 group compared with in the control group (Fig. 4E and F). Overall, the present data suggested that PLAC8 may inhibit the invasive ability of OSCC cells via EMT suppression.

Overexpression or knockdown of PLAC8 affected c-Myc and cyclin D1 expression (Figs. 2D and 3D), which are target genes of the Wnt signaling pathway. β-catenin is a pivotal player of Wnt signaling and promotes EMT in cancer (22). Therefore, the role of PLAC8 in regulating β-catenin expression was analyzed. PLAC8-overexpression significantly decreased the protein levels of non-phospho β-catenin (Ser33/37/Thr41) compared with HN4 cells transfected with empty vector, whereas PLAC8 silencing performed after overexpression of PLAC8 reverted non-phospho β-catenin (Ser33/37/Thr41) expression in HN4 cells (P<0.001). Furthermore, PLAC8-overexpression slightly increased the protein levels of total β-catenin compared with HN4 cells transfected with empty vector, whereas PLAC8 silencing performed after overexpression of PLAC8 significantly decreased total β-catenin expression in HN4 cells (P<0.05; Fig. 5A).

Whether inhibition of β-catenin expression affected PLAC8 function was also evaluated. Expression levels of non-phospho β-catenin (Ser33/37/Thr41) were significantly decreased when XAV939 (a β-catenin inhibitor) was used to treat HN4 cells in which PLAC8 expression had been knocked down (Fig. 5B). Furthermore, protein levels of c-Myc, cyclin D1 and vimentin were significantly downregulated, while those of E-cadherin were significantly upregulated after XAV939 treatment (P<0.001; Fig. 5B). The present findings indicated that PLAC8 may inhibit the proliferation and EMT pathway of OSCC cells by inactivating the Wnt/β-catenin pathway.

Akt phosphorylation of GSK3β may lead to stabilization of β-catenin and accumulation of nuclear β-catenin (22). Numerous studies have suggested that Wnt signaling cross-talks with the PI3K/Akt signaling pathway (17,23). Therefore, the present study investigated whether PLAC8 could also contribute to the PI3K/Akt/GSK3β signaling pathway in OSCC. PLAC8 overexpression significantly decreased the ratio of phosphorylated/total protein of GSK3β and Akt (P<0.001), while silencing overexpressed-PLAC8 slightly increased the ratio of phosphorylated/total protein of GSK3β and Akt in HN4 cells (P<0.005; Fig. 5C). XAV939 treatment resulted in a significant decrease in the protein levels of phosphorylated-GSK3β and phosphorylated-Akt in PLAC8-silenced HN4 cells, which suggested that β-catenin contributed to the inhibitory effect of PLAC8 in the PI3K/Akt signaling pathway (Fig. 5D). Overall, these results suggested that PLAC8 may regulate the PI3K/Akt and Wnt/β-catenin signaling pathways via suppression of β-catenin expression in OSCC cell lines.