Human oral squamous cell carcinoma cell lines (FaDu, YD-8, YD-10B and YD-15) were purchased from the Korea Cell Line Bank in July, 2015. Immortalized normal oral keratinocytes (iNOK) were provided by Dr E.C. Kim (Wonkwang University, Korea) in March, 2010 (14). Cell lines were subjected to identity authentication using a short tandem repeat profiling method. Last tested in May 20, 2021. FaDu cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.); YD-8, YD-10B, and YD-15 in RPMI 1640 (Gibco; Thermo Fisher Scientific, Inc.); and iNOK cells in defined keratinocyte serum-free medium (K-SFM, Gibco; Thermo Fisher Scientific, Inc.). All media were supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were maintained at 37°C in 5% CO₂. The DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine was obtained from MilliporeSigma.

The SMART database was used to investigate CpG-methylated gene expression in tumors and adjacent normal tissues. The β value was used in the DNA methylation analysis. The bioinformatics analysis utilized publicly available datasets derived from the SMART database (https://smart.embl.de/smart/change_mode.cgi). The distributions of the methylation expression levels were displayed in box plots. Plots based on Kaplan-Meier analyses were generated to compare the overall survival rates between the high- and low-expression groups (*P<0.05, **P<0.01, and ***P<0.001).

Genomic DNA was purified using the Wizard Genomic DNA Purification Kit (Promega Corporation) and eluted in 100 µl of ddH₂O from 2×10⁶ cells. Bisulfite treatment was performed using the EpiJET bisulfite conversion kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. For denaturation and DNA bisulfite conversion, a PCR tube was placed in a thermal cycler under Protocol A conditions: 98°C for 10 min and 60°C for 150 min. In the initial step, MSP was conducted in a reaction volume of 10 µl. This reaction volume consisted of 2 µl of bisulfite DNA serving as the template, 300 nmol/l of both forward and reverse primers, 7 µl of deionized water, and AccuPower^® ProFi Taq PCR PreMix (Bioneer Corporation). PCR was performed under the following conditions: Denaturation at 94°C for 5 min; 35 cycles of 90°C for 30 sec, annealing at 50°C for 30 sec, and extension at 72°C for 45 sec; and a final extension at 72°C for 5 min. Subsequently, the MSP products were visualized via 1.5% agarose gel electrophoresis and HiQ Blue Mango Dye (Bio-D Co. Ltd.) staining. The specific sequences of the primers used for PCR amplification are listed in Table I.

The pcDNA4-Myc-HisA vector served as the backbone for constructing pcDNA4-ITGA4-Myc-HisA using an In-Fusion^® Cloning Kit (Takara Bio USA, Inc.). Briefly, the pcDNA4-Myc-HisA vector was linearized with KpnI and XhoI restriction enzymes and purified. The ITGA4 coding sequence was PCR-amplified from the pITGA4-GFP plasmid (Addgene, Inc.) using GoTaq^® Long PCR Master Mix (Promega Corporation) and the designed primers. The vector and the insert were both purified from agarose gels using a DNA-spin™ Plasmid DNA Purification Kit (Intron Biotechnology, Inc.) according to the manufacturer's instructions. The In-Fusion cloning reaction was then established according to the recommended protocol. The In-Fusion mixture was transformed into DH5α competent cells, and the resulting plasmids were isolated and confirmed by DNA sequencing.

The cells were lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with a protease inhibitor cocktail (1 µg/ml) and 1X Xpert phosphatase inhibitor cocktail (GenDEPOT). The protein concentration was determined using a Bradford assay. Protein lysates (30 µg) were separated on 10% SDS-PAGE gels and then transferred to PVDF membranes (MilliporeSigma). The membranes were then blocked with 5% skimmed milk for 2 h at room temperature before incubation with primary antibodies (1:1,000 dilution) against ITGA4 (cat. no. Sc-365569), Pro-Caspase 7 (cat. no. Sc-28295), Pro-Caspase 9 (cat. no. Sc-8355), Slug (cat. no. Sc-166476), Vimentin (cat. no. Sc-6260), N-cadherin (cat. no. Sc-8424) and β-actin (cat. no. Sc-47778), all purchased from Santa Cruz Biotechnology, Inc. The membranes were washed three times with TBST (Tris-buffered saline with 0.1% Tween 20) and incubated with an anti-rabbit IgG conjugated with horseradish peroxidase (W4018) or an anti-mouse IgG conjugated with horseradish peroxidase (cat. no. W4028; 1:2,500 dilution) from Promega Corporation for 1 h at room temperature. Protein signals were visualized using Clarity Western ECL Substrate (Bio-Rad Laboratories, Inc.) and detected via an Amersham ImageQuant 800 western blot imaging system (Cytiva).

TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to extract total RNA from the samples. Total RNA was extracted from approximately 1×10⁶ cells per sample. The RNA quality and concentration were determined via a Nanodrop Denovix Ds-11 Series (DeNovix Inc.), where the aim was to obtain an OD 230/260 greater than 1.8 before downstream experiments. For reverse transcription quantitative polymerase chain reaction, both cDNA synthesis and qPCR were conducted within a single reaction tube, Reverse transcription and quantitative PCR were performed using the GoTaq^® Probe 1-Step RT-qPCR System (Promega Corporation) according to the manufacturer's instructions, with reverse transcription at 37°C for 15 min, initial denaturation at 95°C for 2 min, followed by 40 cycles of denaturation at 95°C for 15 sec and annealing/extension at 60°C for 1 min. The primers used for RT-qPCR are listed in Table I. Relative quantification was performed according to the 2^−ΔΔCq method, as previously described (15). All experiments were performed in triplicate and data are presented as the mean ± standard deviation.

All oral cancer cell lines were transiently transfected with the ITGA4 expression plasmid or the pcDNA4 empty vector using the Neon Electroporation System (Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. Electroporation was performed under the following conditions: 1,100 V, one pulse, and a pulse width of 30 msec. For the FaDu cell line, transfection was carried out in DMEM, whereas RPMI-1640 medium without antibiotics was used for the YD cell lines. After electroporation, cells were incubated for 24 h, transferred to fresh complete culture medium, and incubated for an additional 24 h before being subjected to subsequent analyses and experiments.

An MTT assay was used to evaluate cell proliferation. Cells were seeded at a density of 2×10⁵ cells/well in a 12-well plate 12 h prior to transfection with pcDNA4-ITGA4. Then, the cells were rinsed with PBS, and the media were replaced with fresh media containing a 0.5 mg/ml MTT solution. The cells were incubated for an additional 3 h for formazan crystal formation. Next, the insoluble formazan was dissolved in an acidified isopropanol solution containing 40 mM HCl. Finally, the absorbance of the dissolved solution was evaluated at 540 nm using a DTX 880 multimode detector (Beckman Coulter, Inc.).

Transwell chamber assays were performed to investigate the effects of ITGA4 on the migration and invasiveness of oral cancer cell lines. FaDu and YD-15 cells (2.5×10⁶/ml) were transfected with either the ITGA4 plasmid or the empty vector control for 24 h. Each chamber was prepared by adding 200 µl of serum-free medium, followed by the addition of a suspension of 2.5×10⁵ control and transfected cells in another 200 µl of serum-free medium. Then, 600 µl of culture medium was added to the lower compartment. The chambers were placed in the wells containing culture medium and incubated at 37°C for an additional 24 h. Following this incubation, the chambers were washed twice with PBS to remove nonadherent cells. The cells were subsequently fixed, washed, and stained with crystal violet at room temperature for 15 min. Nonadherent cells were removed, and the remaining stained cells were visualized under an inverted microscope (Olympus IX2-SLP; Olympus Corporation) at 40× magnification. Finally, the stain was dissolved in 10% acetic acid, and the absorbance of the solution was measured to quantify the number of migrated cells.

FaDu, YD-15 and YD-8 oral cancer cell lines were seeded in a 6-well plate and transfected with either the ITGA4 plasmid or the empty vector control for 24 h in culture medium without antibiotics and containing fetal bovine serum (FBS). The next day, the medium was replaced with fresh complete culture medium, and the monolayer of adherent cells was scratched with a pipette tip to generate an approximately 2 mm wide scratch. Wound closure was monitored by capturing images under an Olympus IX2-SLP inverted microscope (Olympus) at 40× magnification. The wound-healing assay was evaluated qualitatively based on representative images.

A Guava Muse^® Cell Analyzer (Luminex Corporation) was used to examine the cell cycle distribution and cell apoptosis according to the manufacturer's instructions. Briefly, cells were seeded in a 6-well plate and transfected with either an empty vector control or an ITGA4 plasmid. The cells were harvested and diluted to a concentration of 1×10⁶ cells/ml and then fixed in 70% cold EtOH overnight at −20°C. For cell cycle analysis, cells were treated with Muse^® Cell Cycle Reagent (Luminex Corporation) at room temperature for 30 min and analyzed via the Guava Muse^® Cell Analyzer (Luminex Corporation). For the apoptosis analysis, a cell suspension (10⁶ cells/ml) was incubated with 100 µl of Muse Annexin V and Dead Cell Reagent (Luminex Corporation). The cells were then incubated for 20 min at room temperature in the dark before analysis with a Guava Muse Cell Analyzer (Luminex Corporation). Data acquisition and analysis were performed using Muse Cell Analyzer Software (Luminex Corporation). The apoptotic rate was calculated as the percentage of early and late apoptotic cells combined, as defined by Annexin V-positive staining.

The cells were seeded in a 6-well plate and transfected with either the ITGA4 plasmid or the empty vector. The cells were subsequently harvested and seeded in 6-well plates at a density of 2,000 cells per well. The plates were incubated for 14 days for colony formation. Next, the colonies were fixed in 10% formalin at room temperature for 20 min and stained with 0.05% crystal violet at room temperature for 30 min. Subsequently, the cell colonies were imaged and counted under an IX2-SLP inverted microscope (Olympus Corporation).

For the protein network analysis, cells were transfected with/without the pcDNA4-ITGA4 plasmid for 48 h. The proteins were identified by proteomic analysis. Total proteins were extracted by filter-aided sample preparation digestion, which was performed according to the protein concentration measured in a BCA assay according to the manufacturer's instructions. The proteomic profile was determined via LC-MS/MS analysis according to the manufacturer's instructions. Briefly, a proteomic analysis was conducted on protein samples (150 µg) from each group using UPLC Exactive Equipment (Agilent 1290; Agilent Technologies, Inc.). LC-MS/MS data were analyzed using Proteome Discoverer. Raw MS data were analyzed using Proteome Discoverer software and searched against the UniProt Homo sapiens database. Carbamidomethylation of cysteine was set as a fixed modification, while oxidation of methionine and acetylation were considered variable modifications. The proteomics data have been uploaded to the PRIDE database and are now fully accessible via the provided accession number (project accession: PXD057268, ID: IlgbsEDjjjFO).

Predesigned siRNAs targeting human SNX5 (ID: 27131, NM_001282454.2) were purchased from Bioneer Corporation and transfected into cultured oral cancer cells using Lipofectamine^® RNAiMAX reagent (Invitrogen; Thermo Fisher Scientific, Inc.). siRNA (1 µl) and 6 µl of Lipofectamine^® RNAiMAX were diluted in 100 µl of OptiMEM (Invitrogen; Thermo Fisher Scientific, Inc.) separately; the mixture was mixed together and incubated at room temperature for 5 min. The final complexes were added to the cell culture medium. Cells were cultured with the siRNA complex for 48 h prior to downstream analysis. The specific primer pairs were as follows: siSNX5 sense (5′-GUGAAGGGUCUAUGACCAA-3′) and anti-sense (5′-UUGGUCAUAGACCCUUCA-3′); siRNA control sense (5′-GCAGCGAGAGAATGAATTA-3′) and anti-sense (5′-CAGTCGCGTTTGCGACTGG-3′).

For the CAM assay, fertilized chicken eggs (1 day) were sterilized with 70% ethanol and incubated at 37°C for 3 days. After 3 days incubation, a window approximately 2 cm in diameter was carefully generated in the eggshell using nippers to form an air pocket without damaging the CAM. The CAM was then visualized by meticulously separating a section of the eggshell membrane. This exposed area was then injected with control cells (1×10⁷ cells) or ITGA4-transfected cells. After placement in a 60-mm culture plate sealed with parafilm, the fertilized eggs were incubated at 37°C in suitable humidity for 5 days additionally. After 5 days incubation, blood vessel formation within the injected area was visually assessed and documented with images. Angiogenesis was quantified by counting the number of blood vessel branching points. At the end of the CAM assay, chicken embryos were humanely euthanized by rapid cooling on ice, and death was confirmed prior to tissue collection, in accordance with established ethical guidelines for embryos (16).

Data were obtained from three independent replicates in each experiment. The results are presented as the means ± standard deviations for variables with a normal distribution. Statistical analysis was performed via two-way ANOVA or one-way ANOVA followed by Tukey's post hoc test for multigroup comparisons. P<0.05 was considered to indicate a statistically significant difference.

According to the SMART database, several genes, including ITGA4 and zinc finger protein 82 (ZFP82), are significantly hypermethylated in head and neck squamous cell carcinoma (HNSSC) tissues compared with normal tissues (Table SI). The present study further examined the correlation between these hypermethylated genes and OSCC patient survival using Kaplan-Meier analysis. Low expression of ITGA4 and ZFP82 was significantly associated with reduced survival in OSCC patients (P≤0.05), while high expression correlated with prolonged survival, indicating an inverse relationship between gene hypermethylation and prognosis (Fig. S1A-D). MSP was performed to assess ITGA4 and ZFP82 methylation in OSCC cell lines (FaDu, YD-8, YD-10B and YD-15). The two genes were highly methylated in cancer cells. Notably, ZFP82 was also methylated in normal iNOK cells, whereas ITGA4 was completely unmethylated in iNOK cells (Fig. 1A). Additional candidate genes were likewise hypermethylated in OSCC cells (Fig. S2). The present study next examined whether methylation affected gene expression. ITGA4 and ZFP82 mRNA levels were significantly lower in OSCC cells than in iNOK cells (Fig. 1B-D), supporting the association between hypermethylation and transcriptional repression. To assess methylation-dependent suppression, we treated OSCC cells with 10 µM 5-aza-2′-deoxycytidine (5-aza), a DNA demethylating agent. After 48 h, RT-qPCR revealed significant upregulation of ITGA4 and ZFP82 in treated cells compared with controls (Fig. 1E and F).

The present study overexpressed ITGA4 in FaDu and YD-15 cells to evaluate its role in oral cancer progression. The pcDNA4-ITGA4 plasmid and empty vector were transfected via electroporation. As shown in Fig. 2A and B, ITGA4 expression significantly increased at both mRNA and protein levels following transfection. Cell proliferation was significantly reduced in ITGA4-overexpressing FaDu and YD-15 cells at 48 and 72 h (Fig. 2C). To investigate the underlying mechanism, cell cycle distribution and apoptosis were analyzed using flow cytometry. ITGA4 overexpression increased the G₁-phase population and decreased the S/G2-phase population, indicating G₁-phase arrest (Fig. 2D). Moreover, apoptotic cell percentages increased in both cell lines: viability dropped from ~90% in controls to 77% in FaDu and 59% in YD-15 cells (Fig. 2E). Western blotting confirmed increased cleavage of procaspase-7 and −9 in ITGA4-overexpressing FaDu and YD-15 cells (Fig. 2F). Supporting these results, MTT and apoptosis assays in YD-8 and YD-10B cells showed ~43 and 46% proliferation inhibition at 48 h, respectively (Fig. S3A). Similarly, apoptotic cells (late apoptosis) increased to 38% in YD-8 and 35% in YD-10B cells, respectively, compared with controls (Fig. S3B).

Next, the effect of ITGA4 on cell migration was detected. For this experiment, a wound healing assay and a Transwell migration assay were performed. ITGA4-transfected cells were incubated for 24 h to create a monolayer prior to generating a scratch ~2 mm wide, after which a wound healing assay was performed to evaluate cell motility at 24 and 48 h. ITGA4 overexpression reduced the wound closure rate of both FaDu and YD-15 cells (Fig. 3A), while ITGA4 overexpression inhibited wound closure in YD-8 cells (Fig. S4). Next, the effects of ITGA4 overexpression on the migration of FaDu and YD-15 cells was observed via Transwell migration assays. Cell migration was markedly reduced in ITGA4-overexpressing cells compared with control cells (Fig. 3B and C). These results indicated that ITGA4 inhibited the migration and invasiveness of oral cancer cells. EMT is a process by which epithelial cells lose their cell polarity and cell adhesion ability and gain migratory and invasive properties to become mesenchymal cells. EMT is broadly recognized for its involvement in cell migration and invasion. Therefore, the expression of mesenchymal markers in ITGA4-overexpressing cells was detected. Notably, the present study showed that ITGA4 overexpression inhibited the expression of mesenchymal proteins (slug, vimentin and N-cadherin) in FaDu and YD-15 cells (Fig. 3D and E). The long-term proliferative activity of ITGA4 was further evaluated via colony formation assays, which suggested that the colony-forming ability of FaDu and YD-15 cells was markedly reduced at 14 days after transfection with ITGA4 (Fig. 3F and G). Compared with control cells, FaDu and YD-15 cells transfected with ITGA4 presented colony formation rates of 0 and 11.4%, respectively. The ability of ITGA4 to affect long-term colony formation may be partially due to both the inhibition of cell proliferation and/or the induction of cell death.

Proteins differentially expressed after ITGA4 overexpression in two oral cancer cell lines, FaDu and YD-15, were identified via LC-MS/MS analysis to examine the protein network through which ITGA4 may exert its anticancer effects on oral cancer cells. The present study identified 2,813 proteins, of which 2,597 could be quantified. The heatmap and volcano plots of the proteomic data revealed the differentially expressed proteins in both FaDu and YD-15 cells following ITGA4 overexpression. A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed to determine the pathways associated with the differentially expressed proteins. The KEGG-based enrichment analysis revealed that the differentially expressed proteins were mostly related to cell migration, cell differentiation, apoptosis, the extracellular matrix and secretion. Proteins that were upregulated >2.0-fold or downregulated <2.0-fold were considered significantly differentially expressed. According to this standard, compared with those in control cells, the levels of 68 and 295 proteins were increased and the levels of 117 and 173 proteins were decreased in FaDu and YD-15 cells, respectively, as a result of ITGA4 expression. The Venn diagram also revealed that 34 proteins were significantly upregulated, whereas 16 proteins were downregulated in both FaDu and YD-15 cells (Fig. S5). The top 50 upregulated and downregulated proteins according to the ITGA4 expression/control ratio are listed in Tables SII and SIII. The proteomic results were confirmed by quantifying 16 proteins downregulated by ITGA4 in both cell lines (data not shown). Many of these proteins have oncogenic potential. Among them, SNX5 was consistently downregulated in both ITGA4-overexpressing lines and selected for further study. Using The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus datasets, it was found that SNX5 expression was significantly upregulated in OSCC tissues compared with normal tissues (Fig. 4A), and high SNX5 levels were significantly associated with poor survival in OSCC patients (P=0.042; Fig. 4B). SNX5 mRNA and protein expression in oral cancer cells were analyzed via RT-qPCR and western blotting. The two were elevated in cancer cells compared with normal iNOK cells (Fig. 4C and D). Western blotting showed that SNX5 expression was markedly decreased in FaDu and YD-15 cells overexpressing ITGA4 (Fig. 4E). To validate this, SNX5 was knocked down using siRNA in FaDu and YD-15 cells. RT-qPCR and western blotting confirmed effective silencing of SNX5 mRNA and protein (Fig. 4F and G). MTT assays showed significantly reduced proliferation in SNX5-knockdown cells (Fig. 4H). Flow cytometry revealed a marked increase in apoptosis: late apoptotic cell percentages rose to 57.95% (FaDu) and 54.75% (YD-15) vs. controls (Fig. 4I). Western blotting showed cleavage of procaspase-9 and PARP-1 after SNX5 knockdown (Fig. 4J), consistent with results from ITGA4 overexpression. The present study then assessed the effect of SNX5 depletion on migration using scratch and Transwell assays. Knockdown significantly impaired migration in FaDu and YD-15 cells (Fig. 4K and L). Finally, colony formation was significantly reduced after 14 days of SNX5 knockdown, with FaDu and YD-15 cells showing >68 and 71% reductions, respectively, compared with controls (Fig. 4M).

Angiogenesis and invasion are crucial processes in tissue expansion, which, when enhanced, lead to the formation of malignant tumors. After ITGA4-overexpressing cells were implanted in fertilized eggs using a microsyringe, they were allowed to grow for 5 days to further clarify the role of the ITGA4/SNX5 axis in OSCC angiogenesis and invasion in vivo. The findings revealed a significant reduction of ~70% in angiogenesis surrounding ITGA4-overexpressing tumors compared with the control groups for both FaDu and YD-15 cells (Fig. 5A and B). Histology of the tumor tissues revealed that the number of invading tumor cells was markedly increased in the control group. Conversely, fertilized eggs bearing ITGA4-overexpressing tumors had lower numbers of invading tumor cells than the control eggs (Fig. 5C). SNX5 siRNA-transfected FaDu and YD-15 cells were seeded on chicken embryo CAMs and incubated for 3 days. The results revealed that the number of blood vessels on the surface of the tumors in the siSNX5-transfected group was greater than that in the control group (Fig. 5D), and the microvessel density was markedly suppressed by siSNX5. Fig. 5E summarized the main findings of the results.