Cell culture
Human OSCC cell lines (CAL27, HN30, HN6) and 293T were obtained from the School of Stomatology at Fujian Medical University. High-glucose Dulbecco's modified Eagle medium (DMEM, Hyclone, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) was used for cell culture in an incubator at 37℃ in a humidified atmosphere with 5% CO2.
Construction of stable 5FU-resistant OSCC cells
Parental CAL27 and HN30 cell lines were treated with increasing concentrations of 5FU (MedChemExpress (MCE), China). Over an approximate 12-month period, the 5FU-resistant cells (5FU-R) were successfully stabilized, resulting in the CAL27/5FU and HN30/5FU strains, which were then consistently nurtured in a medium supplemented with 10 μM 5FU.
CCK8 cell viability assay
To determine the half-maximal inhibitory concentration (IC50) values of 5FU or MSAB, cells were seeded in 96-well plates in triplicate for overnight incubation and later exposed to specified drug concentrations for 48 hours. To investigate the reversal of drug resistance, MSAB (MCE, China) was introduced at 0.2 μg/ml, alongside a range of 5FU concentrations. A combination chemotherapy model across a constant-ratio concentration gradient was used to determine the in vitro interaction between 5FU and MSAB at various dosages. After drug exposure, the Cell Counting Kit-8 (CCK8, Dojindo, Japan) reagent was added, and absorbance readings were captured at 450 nm on a SpectroMax iD3 microplate reader (Molecular Devices, USA).
Dose-response curves were constructed using GraphPad Prism software (version 8.0) to determine the IC50 values. The resistance index (RI) values were deduced by comparing the IC50 values of the resistant cells to those of their parental counterparts. The combination index (CI) values were determined as antagonism (CI>1), additive effect (CI=1), or synergism (CI<1) by employing the Chou-Talalay method with CompuSyn software (version 1.0) [30, 31].
Cell proliferation assay
Cells were seeded in 96-well plates and cultured for 3-5 days in the presence of the indicated drugs. Subsequently, cell viability was measured every 24 hours using a CCK8 assay. Growth curves were constructed using GraphPad Prism 8.0.
Colony formation assay
The cells were seeded in 12-well plates and incubated overnight. These cells then prospered in the presence or absence of 5FU for 2 weeks. The colonies were stained with 0.5% crystal violet solution (Beyotime, China) for photographic documentation. Colony occupation was quantified using Fiji (ImageJ) software.
Flow cytometry analysis of cell apoptosis
Cells were seeded in 6-well plates overnight before 5FU exposure. Following treatment, cells were washed and suspended in binding buffer and stained with YF®488 Annexin V and propidium iodide (PI) (UElandy, China) for 15 minutes. Data acquisition was carried out on an Accuri™ C6 Flow Cytometer (BD Biosciences, USA), and subsequent analysis was executed using FlowJo software.
Morphological observation
The cells were seeded in 12-well plates and treated with or without 5FU. Consequent cellular morphological shifts were observed under bright field using an inverted fluorescence microscope (ZEISS, German) at 100× magnification.
Wound-healing assay
Cells were seeded into 12-well plates. Scratches were created across the monolayered cells. Wound closure in serum-free medium with 5FU was monitored and photographed (100× magnification) over the next 1-3 days, and the healing percentages were quantified using ImageJ.
Transwell migration and invasion assay
The migration assay employed non-coated transwell® inserts with 8 µm pore size polycarbonate membranes (Corning, USA) positioned in a 24-well plate. For the invasion assay, Transwell inserts were pre-coated with Matrigel (Corning, USA) to form matrix barriers. Cell suspensions in serum-free culture medium containing 5FU were introduced into the upper chamber. The lower chamber was supplemented with 10% FBS. Following incubation at 37℃ for 48-72 hours, the cells were stained using crystal violet. The lingering cells on the upper surface were removed. Stained cells were photographed (100× magnification) and quantified.
Immunofluorescent staining and confocal microscopy
Cells grown to 30-50% confluence on coverslips in 12-well plates were fixed with 4% paraformaldehyde solution (Biosharp, China), followed by permeabilization using 0.5% Triton X-100 (Biofroxx, German). Ensuing steps included blocking in 3% bovine serum albumin (BSA, Biofroxx, German) and overnight incubation at 4℃ with primary antibodies against β-catenin (1:200, BD Biosciences, USA). The samples were then incubated with goat anti-mouse secondary antibody conjugated to Alexa Fluor® 488 (Cell Signaling Technology (CST), USA). In another experiment, the fixed and permeabilized cells encountered YF®594-Phalloidin (UElandy, China). The nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, Beyotime, China). The coverslips were mounted on glass slides using a protective antifade mounting medium. Samples were observed using confocal microscope (Olympus, Japan) at 400× or 630× magnification, particularly emphasizing the nuclear translocation of β-catenin, or marking the subcellular actin.
RNA sequencing analysis (RNA-seq)
Total RNA was isolated from CAL27 and CAL27/5FU cells using the TRIzol reagent (Invitrogen, USA). RNA sequencing assay (RNA-seq) was performed by Novogene Company. The dataset was available from the Gene Expression Omnibus (GEO) repository of Series GSE248792.
Total RNA isolation and Real-time quantitative PCR (RT-qPCR)
Total RNA was isolated from cells using NucleoZol (Macherey Nagel, German). Extracted RNA was reverse-transcribed into cDNA using PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara Bio, Japan), and RT-PCR was performed using the SYBR Green Kit (Bio-Rad, USA). Amplification was performed on a Real-time PCR system (Applied Biosystems, USA). The primers (Table 1) were purchased from SunYa Company. The relative mRNA expression was normalized to the GAPDH expression using the 2-ΔΔCt method.
Table 1 Primer Sequences for Target Genes in Real-time qPCR
Gene name (Homo sapiens)
|
Forward primer (5ʹ→3ʹ)
|
Reverse primer (5ʹ→3ʹ)
|
GAPDH
|
GGTGTGAACCATGAGAAGTATGA
|
GAGTCCTTCCACGATACCAAAG
|
WNT3
|
TACTCGCTCTTCAAGCCACC
|
CTTCTCCGTCCTCGTGTTGT
|
WNT5A
|
GCCAGTATCAATTCCGACATCG
|
TCACCGCGTATGTGAAGGC
|
WNT7A
|
CGCAAGCATCATCTGTAACAAG
|
TGAGCCTTCTCCTATGACGAT
|
WNT7B
|
GCTTTGGCGTCCTGTACGTG
|
AACTGGTACTGGCACTCGTTG
|
WNT10B
|
TTGTGCAGTCGGGCTCTAAG
|
GATGTGCAGACCCTGAAGCG
|
WNT11
|
GACCTCAAGACCCGATACCTG
|
TAGACGAGTTCCGAGTCCTTC
|
WNT16
|
AGTATGGCATGTGGTTCAGCA
|
GCGGCAGTCTACTGACATCAA
|
AXIN2
|
CCTAAAGGTCGTGTGTGGCT
|
ACAGTTTCCGTGGACCTCAC
|
CCND1
|
CAGACCTTCGTTGCCCTCTG
|
CAGTCCGGGTCACACTTGAT
|
CCND2
|
GCTGTCTCTGATCCGCAAGC
|
CTCAGTCAGGGCATCACAAGT
|
CDH1
|
GACCACCTTAGAGGTCAGCG
|
TAAGGGCTCTTTGACCACCG
|
CDH2
|
TGGGAATCCGACGAATGGATG
|
GAGCCACTGCCTTCATAGTCA
|
VIM
|
CAGGACTCGGTGGACTTCTC
|
TAGTTGGCGAAGCGGTCATT
|
Protein extraction and western blot
Total protein was extracted from the samples using RIPA lysis buffer (Beyotime, China) containing protease inhibitors, PMSF and cocktail (Beyotime, China). Nuclear and cytosolic proteins were extracted using the Nuclear Protein Extraction Kit (Beyotime, China). Protein concentrations were quantified using BCA Protein Assay Kit (Beyotime, China). Equal amounts of the samples were separated by SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 3% (w/v) BSA and incubated with primary antibodies (detailed in Table 2) overnight at 4℃. The subsequent step involved incubation with goat anti-rabbit or anti-mouse HRP-conjugated secondary antibodies (Boster, China). Visualization of protein bands was achieved with Pierce™ Enhanced Chemiluminescence (ECL) Western Blotting Substrate (Beyotime, China) on ChemiDoc™ XRS+ Imaging System (Bio-Rad, USA).
Table 2 Information on Primary Antibodies Used
Primary antibody
|
Source
|
Dilution
|
WNT3
|
CST
|
1:1000
|
HSP90
|
CST
|
1:1000
|
N-Cad
|
CST
|
1:1000
|
VIM
|
CST
|
1:1000
|
GAPDH
|
CST
|
1:1000
|
β-catenin
|
CST
|
1:1000
|
LaminA/C
|
CST
|
1:1000
|
Flag
|
CST
|
1:1000
|
Caspase-3
|
CST
|
1:1000
|
c-Caspase-3
|
CST
|
1:1000
|
PARP
|
CST
|
1:1000
|
c-PARP
|
CST
|
1:1000
|
α-tubulin
|
CST
|
1:1000
|
Small interfering RNA (siRNA) transfection
The sequences of siRNAs (GenePharma, China) targeting WNT3 (siWNT3) and the negative control (siNC) are detailed in Table 3. Resistant cells were seeded in 6-well plates and transiently transfected with siRNAs using the Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, USA). Forty-eight to seventy-two hours after transfection, both RNA and proteins were harvested for subsequent assays. Cells for subsequent functional assays were processed on the day after transfection.
Table 3 siRNA Sequences Targeting WNT3
Sequences
|
Sense (5ʹ→3ʹ)
|
Antisense (5ʹ→3ʹ)
|
siWNT3-646
|
GCCAUGAACAAGCACAACATT
|
UGUUGUGCUUGUUCAUGGCTT
|
siWNT3-1120
|
CAGGAGUGUAUUCGCAUCUTT
|
AGAUGCGAAUACACUCCUGTT
|
siNC
|
UUCUCCGAACGUGUCACGUTT
|
ACGUGACACGUUCGGAGAATT
|
Plasmid transfection
The overexpression plasmid WNT3-flag (WNT3) and its corresponding control (vector) were obtained from Dahong Biotechnology. After seeding OSCC cells in 6-well plates, they were transiently transfected with plasmids using the Lipofectamine 2000 reagent (Invitrogen, USA) and the medium was replaced with fresh medium. Post-transfection protein analysis verified the overexpression efficiency, and the cells were subsequently used for additional experiments.
Dual-luciferase reporter assay
293T cells at 80-90% confluence were co-transfected with the WNT3 plasmid or its control vector with Top-flash (Addgene, USA) and Renilla plasmids (Promega, USA) using the Lipofectamine 2000. After 48 hours, cell lysates were harvested. Luciferase activity was tested using the Dual Luciferase Assay System (Yeason, China), with firefly luciferase activity normalized to that of Renilla luciferase.
Statistical analysis
All data are presented as mean ± standard deviation, as determined using GraphPad Prism 8.0. Statistical analyses were performed using Student’s t-test, with p-values <0.05 as statistically significant (ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001).