Patients and samples
Twenty normal oral mucosa tissues, forty oral lichen planus (OLP) tissues and 108 cases of OSCC tissues were harvested from patients or volunteers in the Sichuan Provincial People’s Hospital from 2010 to 2015 and stored at -80 °C in the refrigerator for further analysis. Among the 108 patients, 9 patients were progressed from OLP to OSCC. At the same time, we collected peripheral venous blood (5 mL) from these participants, and the supernatant was collected by centrifugation (4 °C, 3 000 × g/min, 20 min). The supernatant was divided into sterile EP tubes and stored at -80 °C in the refrigerator. Inclusion criteria: The diagnosis of OSCC was confirmed by pathological sections and clinical diagnosis; all were first-time cancer patients with complete pathological data; no radiotherapy, chemotherapy and drug treatment; patients and their relatives agreed and signed the informed consent. Exclusion criteria: combination of tumors from other sites; combination of hypertension, diabetes mellitus; patients during pregnancy or lactation. In addition, clinical findings and follow-up data pertaining to these patients were recorded. The study was approved by Ethics Committee of Sichuan Provincial People’s Hospital [Sichuan, P.R. China; approval no. 2015NSF(7)], all patients signed informed consent.
Oral epithelial cells (HOEC) and OSCC cell line Cal27 were purchase from Procell (Wuhan, China), OSCC cell lines (SCC-4, SCC-15, SCC-9, SCC-25, and Tca83) were from ATCC (Manassas, VA). All cells were hatched in DMEM (Gibco) with L-glutamine, sodium pyruvate, 10% fetal bovine serum (FBS, Sigma) at 37 °C and 5% CO2.
Cell treatment and transfection
SCC-15 and SCC-25 cells were first addressed with 0, 0.1, 1, 5, 10, 25, 50 ng/mL IL-6 for 24 h, respectively. SCC-15 and SCC-25 cells were also processed with 25 ng/ml IL-6, 5 μmol/L JAK2 inhibitor (Fedratinib) , and 25 μmol/L STAT3 inhibitor (Protosappanin A) for 24 h . Empty vector (pcDNA4.0), Sox4 overexpression plasmid (pcDNA4.0-Sox4), Sox4 shRNAs (shSox4, 5’-GCGACAAGATCCCTTTCATTC-3’), NLRP3 overexpression plasmid (pcDNA4.0-NLRP3), NLRP3 shRNAs (shNLRP3, 5’-GCTTCATCCACATGACTTTCC-3’), and negative control (NC) shRNAs were gained from HanBio Biotechnology (HanBio, Shanghai, China). SCC-15 and SCC-25 cells (1 × 105 cells/well) in 6-well plates were transfected with shSox4, pcDNA4.0-Sox4, shNLRP3, or pcDNA4.0-NLRP3 for 48 h using lipofectamine 3000 (Invitrogen) in accordance with the specification.
The processed OSCC cells were harvested or the tumors in each group were ground, and total RNAs were isolated applying TRIzol reagent (Invitrogen, MA, USA). Subsequently, cDNAs were synthesized with the RNAs (as template) and PrimeScript™ RT reagent Kit (TaKaRa). Then PCR amplification was then conducted with SYBR Green qPCR Master Mix (DBI Bioscience) after reverse transcription. The data in this experiment were calculated with 2−△△CT method, and GAPDH was the internal control. The primers for different genes are given as followed: IL-6: 5’-ACTCACCTCTTCAGAACGAATTG-3’ (forward) and 5’-CCATCTTTGGAAGGTTCAGGTTG-3’ (reverse); IL-6R: 5’-CCCCTCAGCAATGTTGTTTGT-3’ (forward) and 5’-CTCCGGGACTGCTAACTGG-3’ (reverse); Sox4: 5’-AGCGACAAGATCCCTTTCATTC-3’ (forward) and 5’-CGTTGCCGGACTTCACCTT-3’ (reverse); NLRP3: 5’-GATCTTCGCTGCGATCAACAG-3’ (forward) and 5’-CGTGCATTATCTGAACCCCAC-3’ (reverse); GAPDH: 5’-CTGGGCTACACTGAGCACC-3’ (forward) and 5’-AAGTGGTCGTTGAGGGCAATG-3’ (reverse).
The processed OSCC cells and the ground tumors were increased with the lysates including RIPA (Beyotime, China) and protease inhibitors (Beyotime, China). The extracted proteins were quantified through BCA method, mixed with appropriate loading, and heated at 100℃ for denaturation. Then same amount (50 μg) of proteins were added to 10% SDS-PAGE, separated by electrophoresis at constant pressure, and transferred to PVDF membrane (Millipore). Next, the membrane with protein was sealed with 5% skim milk for 2 h, exposed to primary antibodies, including ASC (Abcam, ab283684, 1:1000), IL-1β (Abcam, ab254360, 1:1000), IL-18 (Abcam, ab207324, 1:1000), Pro-IL-18 (Proteintech, 10,663–1-AP, 1:1000), NLRP3 (Abcam, ab263899, 1:1000), IL-6 (Abcam, ab9324, 1 µg/ml), Sox4 (Abcam, ab70598, 1:500), JAK2 (Abcam, ab108596, 1:1000), pJAK2 (Abcam, ab32101, 1:1000), STAT3 (Abcam, ab68153, 1:1000), pSTAT3 (Abcam, ab267373, 1:1000) at 4℃ overnight, and secondary antibodies (Abcam) for 1 h. Finally, western blotting was developed after processing with ECL kit (Thermo scientific), and the brightness of each strip can be controlled by adjusting the exposure time.
In line with the instruction, IL-1β ELISA kit (Abcam, ab214025 [for human] and ab197742 [for mouse]) and IL-18 ELISA kit (Abcam, ab215539 [for human] and ab216165 [for mouse]) was utilized to test IL-1β and IL-18 activities.
The treated OSCC cells were evenly increased into 96-well plates (100 μL, 1 × 103 cells/well). Then cells were growth in a 37 ℃ incubator and each well was supplemented with 10 μL CCK-8 (Dojindo, Tokyo, Japan) at 0, 24, 48, and 72 h. After additional 2 h of incubation, the OD was monitored by applying a microplate reader (Bio-TekEpoch) at 450 nm.
Colony formation assay
The processed OSCC cells (1 × 103 cells) were routinely hatched in a 6-well plate at 37 ℃ for 14 days. Then the adherent clones were fixed and dyed using 0.1% crystal violet, and clones were counted.
The processed OSCC cells (1 × 105 cells/well) were incubated in 6-well plates until they adhere to the wall. Then cells were processed with 100 μL EdU regent (50 μM, Life Technologies) for 2 h at 37 °C. Then cells were fixed using 4% formaldehyde (30 min), disposed of 50 μL 2 mg/mL glycine and 100 μL 0.5 Triton X-100. The Edu-positive cells were photographed using a fluorescence microscope (Olympus, Tokyo, Japan).
Chromatin immunoprecipitation (ChIP) assay
SCC-15 cells (1 × 106 cells/dish) were uniformly placed in 10 cm cell culture dishes and fixed using 37% formaldehyde at 37 ℃ for 10 min. Then cells were disposed of 0.125 M glycine and SDS Lysis Buffers, and DNA was interrupted into 200–1000 bp through ultrasound. Next, the mixture was addressed with 8 μL NaCl (5 M) at 65 ℃ and cross-linked for 4 h. Subsequently, GenClean Agarose Gel DNA Recovery Kit was applied to extract DNA, and co-precipitation was conducted with the CHIP kit (Millipore). The Sox4 primers for ChIP as followed: forward 5’-GCACCAGAGGCTGATTCT-3’ and reverse 5’-CTGCTTAAAAGCCAAGTG-3’.
Luciferase reporter assay
The downstream target genes for transcription factor STAT3 were identified by JASPAR 2022 (https://jaspar.genereg.net/). We first constructed the wild type (WT) and mutant (Mut) Sox4 promoter plasmids (pGL4.1-Sox4-promoter-WT or pGL4.1-Sox4-promoter-MUT) by referring to the binding sites between STAT3 and Sox4 promoter region. 293 T cells were then co-transfected with the corresponding plasmids and internal plasmids (pTK-RL) containing the Renilla luciferase gene by applying Lipofectamine 3000 (Invitrogen). After 48 h of transfection, luciferase (Firefly/Renilla) activity was measured using the Dual luciferase assay kit (Promega).
For NLRP3, the wild type promoter of NLRP3 was constructed (pGL4.1-NLRP3-promoter-WT). SCC-15 and SCC-25 cells were then co-transfected with pGL4.1-NLRP3-promoter-WT plasmids, different concentration of Sox4 overexpression plasmids (0, 0.1, 0.5, 1, 5, 10, 20, 50 ng) and internal plasmids (pTK-RL) containing the Renilla luciferase gene by applying Lipofectamine 3000 (Invitrogen). After 48 h of transfection, luciferase (Firefly/Renilla) activity was measured using the Dual luciferase assay kit (Promega).
DNA Pull-down assay
The interaction between STAT3 and Sox4 promoter was confirmed by applying a DNA pull-down test kit (Gzscbio, Guangzhou, China). Briefly, cells were lysed after centrifugation. And then we mixed the streptavidin magnetic beads (125 μL) and probes targeting Sox4 promoter (25 μL, 8 μmol/L). And the mixture continued to mix with cell lysis for 12 h on ice. After elution, the conjunct protein was collected and Western blot was utilized to test STAT3 expression.
Tumor xenograft model
BALB/c male nude mice (SPF, 4 weeks, 20 ± 2 g) were obtained from Shanghai slack laboratory. And the experimental mice were fed through separate cages under the conditions of humidity 45%-55%, temperature 22–25 ℃, light for 12 h, adequate standard feed, and purified water. After one week, SCC15 cells were harvested and counted, and the right anterior axilla of each mouse was subcutaneously injected with 2 × 105 cells in 0.2 mL PBS. To evaluate the role of IL-6 on tumor growth, when the tumor volume reached 150 mm3, the tumor xenograft was directly injected with 100 μl PBS (Sigma-Aldrich, P2272, pH = 7.2), 10 mg/kg IL-6 recombinant protein (Fully biologically active, Abcam, ab259381) in 100 μl PBS (Sigma-Aldrich, P2272, pH = 7.2), or 10 mg/kg IL-6 antibody (Abcam, ab259341) in 100 μl PBS (Sigma-Aldrich, P2272, pH = 7.2) once a week, and the mice were divided into control, PBS, IL-6 group and IL-6 antibody groups. To evaluate the role of Sox4 on tumor growth, SCC15 cells were transfected with shSox4 and a Sox4 knockdown stable cell line was constructed. Each mouse was subcutaneously injected with 2 × 105 SCC15 cells with Sox4 knockdown in 0.2 mL PBS. when the tumor volume reached 150 mm3, the tumor xenograft was directly injected with 10 mg/kg IL-6 recombinant protein (Fully biologically active, Abcam, ab259381) in 100 μl PBS (Sigma-Aldrich, P2272, pH = 7.2), and the mice were divided into control, shSox4, IL-6 group and shSox4 + IL-6 groups. Tumor length was tested every 7 days for 21 days. At 21 days, the BALB/c nude mice were dislocated and sacrificed, and the tumors were collected. All animal experiments were done in animal laboratory center as per the study protocol according to the NIH Guide for the Care and Use of Laboratory Animals, approved by the Animal Care and Use Committee of the Sichuan Provincial People’s Hospital.
Normal oral mucosa, OLP and OSCC tissues from human and the tumors from mice were fixed in 10% formaldehyde for 24 h. All tissues were conventionally dehydrated, paraffin embedded, and cut into 4 μm thick slices. Then the slices were conventionally dewaxed, washed, and repaired using sodium citrate (pH = 6.0) for 8 min under high temperature and high pressure. After washing, the slices were added into 3% H2O2 and then heated. Subsequently, the slices were blocked using 10% BSA, placed at 4℃ overnight with anti-IL-6, anti-Sox4, anti-NLRP3, anti-Ki-67, followed by secondary antibody (Abcam) for 1 h. After washing, the slices were then subjected to multiple processing in the later stage, including DAB treatment (10 s), washing, hematoxylin redyeing (30 s), dehydration, neutral gum sealing and natural drying. The results were obtained under a light microscope.
All experiments were conducted in thrice, and all data was displayed with mean ± SD and counted with SPSS 21.0 (SPSS, Inc.). And the statistical charts were made with GraphPad Prism 8.0. And the paired Student’s t test or One-Way ANOVA were utilized for statistical calculations. The curves of Kaplan–Meier and log-rank assessments were employed to evaluate differences in survival outcomes between groups, while associations between IL-6 and Sox4, IL-1β or IL-18 were appraised through Pearson correlation assessments. Chi-square test was adopted to determine the relation between IL-6 or Sox4 expression level and clinical characteristics of OSCC patients. P < 0.05 represented the statistical significance.
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