1. Clinical samples
OSCC tissue and serum samples from 67 patients who underwent surgical resection were collected at Chungnam National University Hospital (CNUH) (Daejeon, Republic of Korea) between 2007 and 2023. The clinical information of patients with OSCC is summarized in Table S1. All the OSCC specimens were confirmed through histopathological examination. This study was conducted with signed informed consent under the approval and guidance of the Ethics Review Board of CNUH (CNUH-2022-11-004-001).
2. Cell culture and transfection
The normal human cell line hFB and the head and neck cancer cell lines YD8, HEP2, SCC15, SCC25, SNU1041, SNU1076, and FADU were obtained from the Korean Cell Line Bank (Seoul, South Korea). The YD8, SNU1041, and SNU1076 lines YD8 were cultured in RPMI-1640 (Welgene, Gyeongsan, Korea). The HEP-2 cells were cultured in EMEM (ATCC, Manassas, VA, USA), while the SCC15 and SCC25 cell lines were maintained in DMEM/F12 (Welgene). FADU cells were cultured in high-glucose Dulbecco Modified Eagle Medium (DMEM; Gibco, Grand Island, NY, USA). For all cell lines, the medium was supplemented with 10% fetal bovine serum (FBS) and 5% penicillin-streptomycin (Gibco). Cells were grown in a cell culture incubator at 37°C with 5% CO2 under humidified conditions.
SCC25 and YD8 cells were transfected using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) with miR-92b-3p mimic, inhibitor (Bioneer, Daejeon, Korea), ATXN1-overexpressed vector, CPEB3-overexpressed vector (Origene, Rockville, MD, USA) and negative controls. Cells were collected 24 or 48 hours later for subsequent analysis.
3. Bioinformatics
All genomic data for OSCC were acquired from The Cancer Genome Atlas (TCGA) through its portal (https://tcga-data.nci.nih.gov) and the UCSC Xena cancer browser (https://genome-cancer.ucsc.edu). In addition, the GSE45238 and GSE168227 datasets were acquired from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/). Gene set enrichment analysis (GSEA) was conducted using GSEA software (version 4.1.0). The co-expression network was reconstructed using WGCNA. In brief, the gene expression matrices were transformed into matrixes with similarity scores for mRNA pairs. These were then converted to adjacency matrices using Pearson correlation coefficients. Next, the represented scale-free gene co-expression topological technique was used to examine the minimal possible value, confirming that the adjacency matrix satisfied the criteria for scale-free topology. A topological overlap matrix (TOM) was built by defining a metric of node dissimilarity. We then used dynamic hierarchical tree clustering to identify network modules. Finally, we chose the most relevant module for further analysis using Gene Ontology (GO) annotation and the Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) enrichment analysis. Using a threshold value for each dataset, modules that exhibited high similarity scores were combined.
4. Cell proliferation assay
In total, 6 × 103 SCC25 or YD8 cells per well were cultured at 37°C after being seeded in 96-well plates. Cell viability was subsequently assessed using the WST-1 assay (Roche Diagnostics, Indianapolis, IN, USA). The conversion of WST-1 to formazan was quantified at 450 nm using an enzyme-linked immunosorbent assay reader. The results are shown as percentages relative to the control group.
5. Colony formation assay
Transfected cells were seeded in 6-well plates at a density of 500 cells/well in 2 mL of medium. With medium replacement at 3-day intervals, the cells grew for 14 days until visible colonies formed. Fixation with 4% formaldehyde was then performed, followed by crystal violet staining, and the cells were subsequently visualized and counted under a dissection microscope.
6. Cell migration and invasion assay
For the invasion assay, transwell chamber membranes (24-well; Costar, Cambridge, MA, USA) were coated with Matrigel for 6 hours, while no Matrigel was used for the migration assay. The lower chamber contained 750 µL of medium with 10% FBS, while 2 × 105 cells in serum-free medium were seeded onto the upper chamber. After 48 hours of incubation, a cotton swab was used to remove cells that adhered to the membrane’s upper surface. Crystal violet staining was applied to invasive or migratory cells adherent to the lower surface, and the cells were counted in four representative fields under light microscopy at ×40 magnification.
7. Dual-luciferase reporter assay
The wild type (WT) fragment of ATXN1 or CPEB3 3'-UTR, containing the miR-92b-3p binding sequence, was cloned into the pmirGLO vector (Promega, Madison, WI, USA) to construct ATXN1-WT or CPEB3-WT. Subsequently, the target-binding sequence between ATXN1 or CPEB3 and miR-92b-3p was mutated to create ATXN1-MUT or CPEB3-MUT. ATXN1-WT (CPEB3-WT) or ATXN1-MUT (CPEB3-MUT) and miR-92b-3p mimic or miR-NC were then co-transfected into SCC25 and YD8 cells using Lipofectamine 3000. After 48 hours, the luciferase activity was assessed using the dual-luciferase reporter gene detection system (Promega) according to the provided protocols.
8. RNA isolation and reverse transcription-polymerase chain reaction
Total RNA was extracted from tissues and cells using TRIzol (Invitrogen, Carlsbad, CA, USA), and complementary DNA (cDNA) was synthesized with 2 µg of total RNA and TOPscriptTMRT DryMIX (Enzynomics Inc., Daejeon, Korea) according to the instructions provided by the manufacturer. Amplification was carried out using SYBR Green qPCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA), and the primer sequences are listed in Table S2.
Circulating miRNA was isolated from 200-µL serum samples to purify RNA using miRNeasy serum/plasma kits (Qiagen, Hilden, Germany) according to the protocol provided by the manufacturer. Total miRNA was used as the cDNA synthesis template with the miScript II RT Kit/miRCURY LNA RT kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. For miRNA analysis, qRT-PCR was conducted using the miScript SYBR Green PCR kit/miRCURY LNA SYBR Green PCR kit (Qiagen, Hilden, Germany) along with the protocols provided by the manufacturer, utilizing the universal primer and miRNA-specific forward primers in the 7500 system. The miRNA-specific primers were acquired from the miScript primer assays, the miRCURY LNA miRNA PCR assay (Qiagen), and Bioneer (Daejeon, Korea).
9. Western blotting analysis
All samples were lysed in a buffer that contained 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate, 1.0% Nonidet-P40, a protease inhibitor, 50 mM Tris (pH 8.0), and 150 mM NaCl (Roche Applied Science, Vienna, Austria, pH 7.4). Tissue samples were minced using scissors, and the total protein was subsequently extracted. Electrophoresis was carried out as previously described. All the primary antibodies used for western blotting are listed in Table S3. Incubation with the appropriate horseradish peroxidase-conjugated secondary antibodies (1:5000; Santa Cruz Biotechnology) was followed by the visualization of immunoreactive bands using enhanced chemiluminescence detection (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
10. Animal experiments
Six-week-old BALB/c nude mice were acquired from Orient Bio (Seongnam, South Korea). The mice were kept under specific pathogen-free conditions, at 24°C with a 12-hour day/night cycle. All experiments adhered to the relevant guidelines and regulations of the animal care unit at Chungnam National University. The animal protocols for these experiments received approval from the Ethics Committee of Animal Experimentation of Chungnam National University (No. CNUH-2022-IA0100-00). Twenty mice were randomly assigned to four groups for the first and second animal experiments. Negative-control SCC25 cells and stable miR-92b-3p–overexpressing SCC25 cells were prepared, and 5 × 106 cells in 200 µL of a suspension mixed with Matrigel were administered through a subcutaneous injection into the mice’s lower left flank. A caliper was used to measure tumor dimensions, and tumor volumes were calculated with the following formula: tumor volume = length × width2 × 0.52, where length is the largest tumor diameter and width corresponds to the diameter along the axis perpendicular to the length. The tumors were collected and utilized for histological analyses. For the second experiment, when the size of the tumor reached 62.5 mm3, each mouse was intraperitoneally administered cisplatin at a dose of 5mg/kg body weight twice a week. All animal experiments were conducted at least twice, yielding similar results.
11. Histological and immunohistochemical analysis
For immunohistochemistry, tissue sections were deparaffinized in xylene, hydrated in graded alcohol solutions, and heated at 100°C for 15 minutes in Antigen Retrieval Citra Solution (pH 6.0) to remove antigens. For single immunostaining, endogenous peroxidase activity was blocked by incubating the sections in a 1% hydrogen peroxide solution (Sigma-Aldrich, St. Louis, MO, USA) in phosphate-buffered saline (PBS) with 0.3% Triton X-100 for 30 minutes at room temperature. The sections were then incubated with the specified antibodies overnight at 4°C, followed by incubation with the corresponding horseradish peroxidase-conjugated secondary antibody. Finally, 3,3′-diaminobenzidine (DAB; Dako, Agilent, Santa Clara, CA, USA) was utilized to detect the labeled antibodies, and the nuclei were stained with hematoxylin. The samples were rinsed with PBS, mounted, and analyzed using an automatic digital slide scanner (Pannoramic MIDI; 3DHISTECH, Hungary).
12. TUNEL staining
TUNEL staining was used to measure cell apoptosis. Briefly, after dewaxing and dehydration, paraffin sections were stained with a TUNEL assay kit (ab206386; Abcam, Cambridge, MA, USA) according to the instructions from the manufacturer. The photomicrographs were taken using an optical microscope.
13. Statistical analysis
All in vitro and in vivo experiments were conducted in triplicate, and statistical significance was assessed using the Student t-test. Data are presented as mean ± standard deviation. To evaluate the association between miR-92b-3p and target gene expression in the TCGA dataset, we employed Spearman correlation analyses. A p-value of < 0.05 was considered to indicate statistical significance. Statistical analyses were carried out using SPSS version 26.0 (IBM Corp, Armonk, NY, USA).