Human nasopharyngeal epithelial cell line (NP69, as a control) and NPC cell lines (C666-1, 5-8F and SUNE-1) were purchased from the American Type Culture Collection (Manassas, MA, USA), and cultured in 5% CO₂ at 37°C. Dulbecco's modified Eagle's medium (DMEM) was used as basic culture medium. 10% fetal bovine serum (FBS), 100 U/ml streptomycin and 100 µg/ml penicillin were then added in to the medium. DMEM, FBS, streptomycin and penicillin were purchased from Biological Industries (Kibbutz Beit Haemek, Israel). Lithium chloride (LiCl; 20 mM; incubation time 24 h) was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany) and was used to activate the Wnt signaling pathway as previously described (31).

The well-differentiated SUNE-1 cells were evenly plated in 6-well plates at the initial concentration of 6×10⁴ cells/well. After being cultured for 12 h, the cells reached 60–70% confluence. The cells were cultured in DMEM without FBS. A mixture of 50 pmol KRT6A-siRNA (Shanghai GeneChem Co., Ltd., Shanghai, China), with the sequence of sense 5′-CCAGCAGGAAGAGCUAUA-3′, and antisense 3′-GGUCGUCCUUCUCGAUAUU, and transfection reagent Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) (2:1), or 50 pmol scrambled siRNA (as control; Shanghai GeneChem Co., Ltd.), with the sequence of sense: 5′-UUCUCCGAACGUGUCACGUTT-3′, and antisense: 5′-ACGUGACACGUUCGGAGAA-3′ in mixture with Lipofectamine^® 3000 was respectively added to the cells, and cultured for 8 h at 37°C. The transfected cells were accordingly named the siKRT6A group and the mock group. Cells with non-treatment were named the Cotl group and treated as the control. The cells were then cultured in DMEM with FBS for another 48 h before further measurement of activity.

The effect of KRT6A silencing on cell viability of NPC SUNE-1 cells was measured using Cell Counting Kit-8 (CCK-8; Beyotime Institute of Biotechnology, Haimen, China) assay. To be more specific, SUNE-1 cells transfected with KRT6A-siRNA or scrambled-siRNA, or untreated SUNE-1 cells were respectively seeded in 96-well plates at an initial density of 2×10³ cells/well and incubated at 37°C for 12, 24, and 48 h. Next, 20 µl CCK-8 reagent was added to each well. Then the plate was incubated at 37°C for 3 h. Finally, optical density values were read at 450 nm using a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). The ratios of cell viabilities to Cotl group at 0 h were compared.

The effect of KRT6A silencing on cell metastasis ability of NPC SUNE-1 cells was measured using wound healing assay. To be more specific, SUNE-1 cells transfected with KRT6A-siRNA or scrambled-siRNA, or SUNE-1 cells without transfection were respectively seeded in 6-well plates at an initial density of 6×10⁴ cells/well. After reaching 80% confluence, a wound was created using pipette tip scratching. After being cultured for 24 h, the wound closure size images in various fields were captured and measured by light microscope.

Cell invasion abilities were measured using Transwell plates with 8-µm pore filter (Corning, Inc., Corning, NY, USA) coated with Matrigel (BD Biosciences; Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Cells (6×10⁴ cells/well) suspended in serum-free medium were seeded in the upper-chambers, while medium with 20% FBS was added to the lower-chambers in order to induce cell invasion. After being cultured at 37°C for 24 h, the cells were stained with crystal violet (Beyotime Institute of Biotechnology) for 15 min at room temperature. Finally, cells images were captured and invaded cell numbers were counted under a light microscope.

The mRNA expression levels of KRT6A in NP69 nasopharyngeal epithelial cells and different NPC cell lines (C666-1, 5-8F and SUNE-1) were determined. After conducting KRT6A silencing, levels of KRT6A, E-cadherin, tissue inhibitor of metalloproteinase-2 (TIMP-2), MMP-2, MMP-9, β-catenin, lymphoid enhancer binding factor 1 (LEF-1) and T-cell specific factor 4 (TCF-4) were determined in the Cotl, mock and siKRT6A cell groups. Primers used are listed in Table I and GAPDH was treated as an internal control. The process was performed as follows: Total RNA was first extracted using the TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and then reverse transcribed to cDNA using Transcriptor Universal cDNA Master (Roche Diagnostics, Indianapolis, IN, USA), with the protocol of 70°C for 5 min and 42°C for 60 min. Then, the PCR amplification process was conducted using LightCycler^® Multiplex Masters (Roche Diagnostics) with LightCycler^® 480II System (Roche Diagnostics) under the conditions as follows: Pre-denaturation at 95°C for 2 min, 36 cycles (denaturation at 95°C for 25 sec, annealing at 60°C for 25 sec, extension at 72°C for 30 sec) and a final extension at 72°C for 10 min. The changes were calculated by the 2^−ΔΔCq method (32).

The protein levels of KRT6A were detected in NP69 nasopharyngeal epithelial cells and in the different NPC cell lines (C666-1, 5-8F and SUNE-1). After conducting KRT6A silencing, levels of KRT6A, E-cadherin, TIMP-2, MMP-2, MMP-9, β-catenin, LEF-1 and TCF-4 were measured in Cotl, mock and siKRT6A cell groups. The cells were lysed on ice using radio immunoprecipitation assay lysis buffer (Wuhan Boster Biological Technology, Ltd., Wuhan, China) for 30 min and centrifuged at 1×10⁴ × g at 4°C for 20 min. Next, the proteins in supernatant were quantified using BCA protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). A total of 20 µg protein was first loaded to each well that contained 12% SDS-PAGE and then separated and transferred onto polyvinylidene fluoride membranes (Thermo Fisher Scientific, Inc.). Next, the membranes were blocked in 5% non-fat dry milk for 1 h at room temperature and incubated with specific primary antibodies overnight at 4°C. Finally, the membranes were incubated with secondary antibodies conjugated with horseradish peroxidase: Anti-rabbit IgG, HRP-linked Antibody (Cell Signaling Technology, Inc., Danvers, MA, USA; cat. no. 7074, 1:5,000) for 1 h at room temperature. GAPDH was treated as a loading control. The proteins were detected by enhanced chemiluminescence detection reagents (Pierce; Thermo Fisher Scientific, Inc.) and analyzed by Bio-Rad ChemiDoc XRS densitometry with Image Lab™ Software version 4.1 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The primary antibodies used were as follows: Rabbit anti-KRT6A (Abcam, Cambridge, UK; cat. no. ab18586; 1:200), E-cadherin (Abcam; cat. no. ab15148; 1:500), TIMP-2 (Abcam; cat. no. ab180630; 1:1,000), MMP-2 (Abcam; cat. no. ab92536; 1:1,000), MMP-9 (Abcam; cat. no. ab38898; 1:1,000), β-catenin (Abcam; cat. no. ab16051; 1:5,000), LEF-1 (Abcam; cat. no. ab22884; 1:500), TCF-4 (Abcam; cat. no. ab185736; 1:1,000) and GAPDH (Abcam; cat. no. ab9485; 1:2,000).

SPSS software (version 20.0; IBM Corps, Armonk, NY, USA) was used for data analysis. Three replications were conducted in each assay. Data were expressed with the mean ± standard deviation. The data were analyzed by one-way analysis of variance and Dunnett t's test. P<0.05 was considered to indicate a statistically significant difference.

The expression levels of KRT6A in the nasopharyngeal epithelial cell line (NP69, as control) and NPC cell lines (C666-1, 5-8F and SUNE-1) were measured. The results of RT-qPCR and western blotting demonstrated that the mRNA and protein levels of KRT6A were significantly upregulated in all detected NPC cells, among which the value was the highest in the SUNE-1 cells (P<0.01; Fig. 1). Therefore, SUNE-1 cells were selected for the following gene modification experiments.

KRT6A-siRNA was transfected into NPC SUNE-1 cells using the liposome method. RT-qPCR and western blot assays analysis demonstrated that the mRNA and protein levels of KRT6A were significantly decreased in the siKRT6A group, compared with those of the Cotl and mock groups (P<0.01; Fig. 2A-C). CCK-8 assay results demonstrated that cell viabilities in the siKRT6A group was inhibited compared with the two groups, Cotl and mock (P<0.01; Fig. 2D).

Cell metastasis and invasive abilities of SUNE-1 NPC cells prior to and following performing KRT6A silencing were measured by scratch wound healing and Transwell assays, respectively following 24 h of cell growth. The wound distance siKRT6A group were significantly increased compared with the Cotl and mock groups, indicating that KRT6A silencing inhibited cell metastasis of SUNE-1 NPC cells (P<0.01; Fig. 3A and B). In addition, the invasion rates of SUNE-1 NPC cells significantly decreased in the siKRT6A group, suggesting that KRT6A silencing inhibited cell invasion of SUNE-1 NPC cells (P<0.01; Fig. 3C and D).

EMT is the major mechanism of cell metastasis and invasion, Therefore, the epithelial marker E-cadherin and matrix degrading enzyme-associated factors were measured. RT-qPCR demonstrated though the mRNA and protein expression levels of E-cadherin and TIMP-2 were significantly promoted in the siKRT6A group (P<0.01), the mRNA and protein expression levels of MMP-2 and MMP-9 were significantly reduced compared with those in both the Cotl and mock groups (P<0.01; Fig. 4).

As the Wnt/β-catenin pathway strongly affects tumorigenesis, the effect of KRT6A silencing on the Wnt/β-catenin pathway was investigated using RT-qPCR and western blot assays. The results demonstrated that both the mRNA and protein levels of β-catenin, LEF-1 and TCF-4 were significantly reduced in the siKRT6A group, compared with those in both the Cotl and mock groups (P<0.01; Fig. 5).

LiCl was used to activate the Wnt/β-catenin pathway and determined the association between the Wnt/β-catenin pathway and si-KRT6A. It was observed that decreased expression of MMP2/9 caused by si-KRT6A was then rescued by LiCl. By contrast, the expression of TIMP2 was significantly decreased in LiCl+si-KRT6A group compared with the si-KRT6A group (P<0.05; Fig. 6). This suggested that the effect of si-KRT6A on NPC cells may be largely dependent on the inactivation of the Wnt/β-catenin pathway.