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Article Open Access

Hypermethylation‑induced silencing of ITGA4 promotes oral squamous cell carcinoma progression through SNX5 upregulation

  • Authors:
    • Nguyen Ngoc Thuy Tien
    • Han-Cheol Choe
    • Sang-Gun Ahn
  • View Affiliations / Copyright

    Affiliations: Department of Pathology, School of Dentistry, Chosun University, Gwangju 61452, Republic of Korea, Department of Dental Materials, School of Dentistry, Chosun University, Gwangju 61452, Republic of Korea
    Copyright: © Tien et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 119
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    Published online on: April 24, 2026
       https://doi.org/10.3892/or.2026.9124
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Abstract

Epigenetic modifications, especially DNA methylation, play an increasingly important role in oral cancer. However, their specific contributions to the progression of oral squamous cell carcinoma (OSCC) remain unclear. The present study used the Shiny Methylation Analysis Resource Tool (SMART) database (https://smart.embl.de/smart/change_mode.cgi) to identify methylation‑driven genes associated with OSCC. Among the identified candidates, integrin subunit α4 (ITGA4) exhibited significantly elevated methylation levels in head and neck cancers. A methylation‑specific PCR assay showed that ITGA4 is highly methylated in OSCC cells compared with normal immortalized human normal oral keratinocyte (iNOK) cells. Additionally, the mRNA expression levels of ITGA4 were significantly lower in OSCC cell lines compared with normal iNOK cells. ITGA4 overexpression markedly inhibited the cell proliferation, migratory ability and capacity of colony formation and induced apoptosis in FaDu and YD‑15 cells. In proteomic analysis, ITGA4 suppressed the expression of Sorting Nexin 5 (SNX5), a protein linked to cancer progression. siRNA‑mediated knockdown of SNX5 importantly inhibited cell proliferation, migration, and colony formation in FaDu and YD‑15 cells. Moreover, in a chick chorioallantoic membrane xenograft model, overexpression of ITGA4 or small interfering SNX5 significantly inhibited OSCC tumor growth and angiogenesis in vivo. Collectively, these findings demonstrated that ITGA4 acts as a tumor suppressor in OSCC by downregulating SNX5 and suggested that ITGA4 may serve as a valuable prognostic biomarker and potential therapeutic target for OSCC.
View Figures

Figure 1

Methylation levels of ITGA4 and ZFP82
in different types of human cancers compared with normal tissues in
the SMART database. (A) Representative results of the MSP.
Verification of ITGA4 and ZFP82 gene promoter methylation in oral
cancer cell lines (FaDu, YD-8, YD-10B and YD-15) and iNOKs via MSP.
PCR products of unmethylated (U) and methylated (M) ITGA4 and ZFP82
from sodium bisulfite-treated genomic DNA from cell lines were
visualized by ethidium bromide staining. (B-D) The expression of
ITGA4 and ZFP82 was determined by RT-PCR and RT-qPCR. ITGA4 and
ZFP82 mRNA expression. The quantities of the ITGA4 and ZFP82 mRNAs
were determined in each sample using RT-qPCR. The data are
presented as the mean ± standard deviation., n=3. Statistical
significance of the differences between oral cancer cells (FaDu,
YD-8, YD-10B, and YD-15) and iNOKs: *P<0.05, **P<0.01 and
***P<0.001 (E and F) Effect of 5-aza-2′-deoxycytidine (5-aza)
treatment on oral cancer cells. RT-qPCR analysis of ITGA4 and ZFP82
expression in oral cancer cells treated with or without 10 µM 5-Aza
for 48 h. The columns represent the mean ± standard deviation of
triplicate qPCR experiments. **P<0.01 and ***P<0.001. ITGA4,
integrin subunit α 4; ZFP82, zinc finger protein 82; SMART, Shiny
Methylation Analysis Resource Tool; iNOKs, immortalized human
normal oral keratinocytes; MSP, methylation-specific PCR; RT-PCR,
reverse transcription PCR; RT-qPCR, reverse
transcription-quantitative PCR.

Figure 2

ITGA4 inhibits cell proliferation and
induces cell cycle arrest and death in oral cancer cells. FaDu and
YD-15 cells were transfected with the vector or the pcDNA4-ITGA4
plasmid for 48 h. (A) RT-qPCR analysis of ITGA4 mRNA levels in FaDu
and YD-15 cells transfected with the pcDNA4-ITGA4 plasmid. β-Actin
served as an internal control for RT-qPCR and western blot
analyses. *P<0.05 vs. the control. (B) Western blot analysis of
ITGA4 expression levels in ITGA4-overexpressing FaDu and YD-15
cells. (C) Cell proliferation was examined by MTT assays at the
indicated time points after transfection with the pcDNA4-ITGA4
plasmid. The data are shown as the mean ± standard deviation of
three separate experiments. **P<0.01 and ***P<0.001 vs. the
control. (D) FaDu and YD-15 cells were transfected with
pcDNA4-ITGA4 for 48 h, and then the cell cycle distribution was
assessed by flow cytometry analysis after staining with PI. (E)
Apoptotic cells were evaluated by dual staining with Annexin V/PI
and counted via flow cytometry. The graph shows the quantification
of the relative numbers of apoptotic cells among the
ITGA4-overexpressing cells. (F) Procaspase-9 and procaspase-7
expression in FaDu and YD-15 cells 48 h after ITGA4 transfection,
as measured by western blotting. ITGA4, integrin subunit α 4;
RT-qPCR, reverse transcription-quantitative PCR.

Figure 3

ITGA4 inhibits the migration and
invasiveness of oral cancer cells. FaDu and YD-15 cells were
transfected with the ITGA4 plasmid or the control empty vector. (A)
Wound healing assays were used to assess the effect of ITGA4
overexpression on cell migration at the indicated times. Original
magnification, ×10. (B and C) Transwell migration assays. The graph
summarizes the data from three independent experiments. The bars
indicate the mean ± standard deviation (n=3). *P<0.05 and
**P<0.01. Effects of ITGA4 on the expression of EMT markers. (D)
FaDu and (E) YD-15 cells were transfected with the ITGA4 plasmid or
the control empty vector for 48 h. The expression levels of
mesenchymal markers in the indicated cells were analyzed by western
blotting. (F) Colony formation assay in ITGA4-overexpressing cells.
Vector- or ITGA4-transfected cells were plated in 12-well plates
and cultured for 14 days. The colonies were stained with crystal
violet for quantification. (G) Results of the quantification of
Fig. 3F. The data are presented as
the mean ± standard deviation. from three independent experiments.
**P<0.01 and ***P<0.001. ITGA4, integrin subunit α 4.

Figure 4

SNX5 is upregulated and associated
with a poor prognosis of OSCC patients. (A) The expression of SNX5
in HNSCC tissues (red) was compared with that in corresponding
noncancerous normal tissues (blue) in TCGA datasets (n=520). (B)
Kaplan-Meier analysis of the overall survival of OSCC patients
stratified by the SNX5 expression level. The black plot indicates
the survival of patients in the low-risk group, and the red plot
represents the survival of patients in the high-risk group. The
survival curves were statistically significant (P<0.05). The
expression of SNX5 mRNA in oral cancer cells was compared with that
in corresponding noncancerous iNOK cells using (C) RT-qPCR and (D)
western blotting. *P<0.05 vs. control iNOK cells. (E) ITGA4
reduced SNX5 expression in FaDu and YD-15 cells. The cells were
transfected with the ITGA4 plasmid or the control empty vector for
48 h. The expression levels of SNX5 in the indicated cells were
analyzed by western blotting. SNX5 protein expression in
siSNX5-induced FaDu and YD-15 cells was confirmed by (F) RT-qPCR
analysis and (G) western blotting. Actin served as a loading
control. (H) SNX5 knockout attenuated cell growth and induced cell
death in FaDu and YD-15 cells. The growth of control or
SNX5-knockout FaDu and YD-15 cells was detected by an MTT assay.
The bars indicate the mean ± standard deviation from three
independent experiments. *P<0.05 and **P<0.01. (I) Apoptotic
cells were evaluated by dual staining with Annexin V/PI and counted
using flow cytometry. The percentage represents the relative number
of apoptotic siSNX5-treated cells. (J) Caspase-9 and PARP
expression/activation in FaDu and YD-15 cells 48 h after the
transfection with siSNX5, as measured by western blotting. (K) SNX5
silencing inhibits oral cancer cell migration. FaDu and YD-15 cells
were transfected with 100 nM siRNA for 48 h. Cell migration was
determined via a wound healing assay at the indicated times after
siRNA transfection. (L) Transwell migration assay in
siSNX5-overexpressing cells. siSNX5-transfected cells were plated
on soft agar and incubated for 48 h. (M) A clonogenic assay was
performed, and the colonies were stained with crystal violet for
quantification. Images of 12-well plates with colonies were
captured on day 14, and a bar graph was generated by calculating
the percentages of colonies from each cell line relative to those
of the controls. The data are representative of three independent
experiments. **P<0.01, ***P<0.001. SNX5, Sorting Nexin 5;
OSCC, oral squamous cell carcinoma; HNSCC, head and neck squamous
cell carcinoma; TCGA, The Cancer Genome Atlas; iNOK, immortalized
human normal oral keratinocyte; RT-qPCR, reverse
transcription-quantitative PCR; si, short interfering.

Figure 5

ITGA4 inhibits angiogenesis. A CAM
assay was used to analyze the effects of ITGA4 and siSNX5 on
angiogenesis in vivo. (A) Fertilized eggs were implanted
with ITGA4- or siSNX5-transfected FaDu and YD-15 cells
(1×107/ml) and maintained in a humidified incubator at
37°C for an additional 5 days (n=5). (B) Graphs showing the results
of the quantitative evaluation of angiogenesis. The values are
presented as the means ± S.D *P<0.05 and **P<0.01. (C)
H&E staining of tumors (blue arrow head). (D) Representative
images of oral cancer cell xenografts from the CAM assay.
Fertilized eggs were inoculated with siSNX5-transfected FaDu and
YD-15 cells and incubated for an additional 5 days (n=5). The
graphs show the quantitative evaluation of angiogenesis.
*P<0.05. (E) Images of summarized the main findings. ITGA4,
integrin subunit α 4; CAM, chick chorioallantoic membrane; si,
short interfering; SNX5, Sorting Nexin 5; OSCC, oral squamous cell
carcinoma.
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Copy and paste a formatted citation
Spandidos Publications style
Tien NN, Choe H and Ahn S: Hypermethylation‑induced silencing of ITGA4 promotes oral squamous cell carcinoma progression through SNX5 upregulation. Oncol Rep 55: 119, 2026.
APA
Tien, N.N., Choe, H., & Ahn, S. (2026). Hypermethylation‑induced silencing of ITGA4 promotes oral squamous cell carcinoma progression through SNX5 upregulation. Oncology Reports, 55, 119. https://doi.org/10.3892/or.2026.9124
MLA
Tien, N. N., Choe, H., Ahn, S."Hypermethylation‑induced silencing of ITGA4 promotes oral squamous cell carcinoma progression through SNX5 upregulation". Oncology Reports 55.6 (2026): 119.
Chicago
Tien, N. N., Choe, H., Ahn, S."Hypermethylation‑induced silencing of ITGA4 promotes oral squamous cell carcinoma progression through SNX5 upregulation". Oncology Reports 55, no. 6 (2026): 119. https://doi.org/10.3892/or.2026.9124
Copy and paste a formatted citation
x
Spandidos Publications style
Tien NN, Choe H and Ahn S: Hypermethylation‑induced silencing of ITGA4 promotes oral squamous cell carcinoma progression through SNX5 upregulation. Oncol Rep 55: 119, 2026.
APA
Tien, N.N., Choe, H., & Ahn, S. (2026). Hypermethylation‑induced silencing of ITGA4 promotes oral squamous cell carcinoma progression through SNX5 upregulation. Oncology Reports, 55, 119. https://doi.org/10.3892/or.2026.9124
MLA
Tien, N. N., Choe, H., Ahn, S."Hypermethylation‑induced silencing of ITGA4 promotes oral squamous cell carcinoma progression through SNX5 upregulation". Oncology Reports 55.6 (2026): 119.
Chicago
Tien, N. N., Choe, H., Ahn, S."Hypermethylation‑induced silencing of ITGA4 promotes oral squamous cell carcinoma progression through SNX5 upregulation". Oncology Reports 55, no. 6 (2026): 119. https://doi.org/10.3892/or.2026.9124
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