Patients and specimen collection

A total of 30 OSCC specimens used for immunohistochemical (IHC) and quantitative real-time PCR analysis were collected from the Hospital of Stomatology of China Medical University (Shenyang, China) between 2021 and 2022. The inclusion criteria were patients who had confirmed primary OSCC without other malignancies and were undergoing tumorectomy. The pateints’ clinical data is shown in Supplementary Table 1. The tumor grading and TNM classification were completed by two independent pathologists. The use of OSCC samples for this study was approved by the Affiliated Stomatological Hospital of China Medical University Institutional Review Board (K2021010) and all patients gave informed consent.

Immunohistochemical staining

Formalin-fixed paraffin-embedded (FFPE) tumor tissues were serially sectioned into 4 μm sections. Tissue paraffin sections were de-waxed, rehydrated, blocked, and incubated with the primary antibodies, listed in Supplementary Table 2, overnight at 4 °C. Subsequently, each section was incubated with polyperoxidase-anti-rabbit/mouse IgG (ZSGB-BIO, China) at room temperature for 30 min, stained with diaminobenzidine, and counterstained with haematoxylin.

Cell and bacteria culture

The OSCC cell lines HSC-4 and SCC-9 were originally derived from a patient with tongue cancer and respectively obtained from the Japanese Collection of Research Bioresources Cell Bank (JCRB; Shinjuku, Japan) and American Type Culture Collection (ATCC, Manassas, USA). HSC-4 cells were cultured in DMEM/high glucose (Gibco, USA) supplemented with 10% fetal bovine serum (FBS). SCC-9 cells were cultured and passaged in DMEM/Ham’s F-12 medium with 10% FBS and 400 ng/mL hydrocortisone (Sigma-Aldrich, USA). P. gingivalis ATCC 33277 (American Type Culture Collection) were cultured on brain heart infusion (Difco Laboratories, MI, USA) agar plates at 37 °C in an anaerobic chamber (80% N2, 10% H2, and 10% CO2). For all the experiments in our study, cells were challenged with P. gingivalis at a multiplicity of infection (MOI) of 10 for 4 weeks. Subsequently, long-term infection cells lysed with sterile distilled water for 40 min were collected, inoculated on agar plates with appropriate dilution and cultured anaerobically for 7 days.

Cell viability assay

HSC-4 and SCC-9 cells were seeded into 96-well plates at a density of 3 000 cells per well overnight. Subsequently, cells were treated with P. gingivalis for 24, 48, and 72 h. 10 μL CCK-8 reagent (SparkJade, China) was added to each well. After 2 h of incubation at 37 °C in the dark, the plates were determined at 450 nm by a microplate reader (Tecan, Mechelen, Belgium).

As for drug resistance assay, cells were exposed to different concentrations of cisplatin (0, 2.5, 5, 10, 20, and 40 μmol/L) for 48 h.

kFluor488 Click-iT EdU assay

Cell proliferation was measured using EdU detection kit (5-Ethynyl-2’-deoxyuridine) (KeyGENBioTECH, China) according to the producer’s instructions. OSCC cells were seeded at 5 × 104 per well on 24-well plates. Subsequently, 10 μmol/L EdU labeling media was added to culture cells for another 2 h and then the cells were stained with 1× Click-iT EdU staining mix for 30 min in the dark. These cells were subsequently counterstained with Hoechst33342 for 20 min and imaged by a fluorescence microscope (200×) (Nikon DS-Ri2).

Cell migration and invasion assay

For wound-healing assay, a wound was created and incubated in serum-free medium. Cell motility was determined by measuring the average scratch width change. For transwell invasion assay, Matrigel (BD Biosciences, USA) was used to precoated the upper chamber of transwell (pore size, 8 μm; Corning, NY, USA) for 1 h at 37 °C and the cells were seeded into the upper chambers (1 × 105 cells per well) in 200 μL serum-free medium. After incubation for 48 h, cells were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet dye. Then, the chambers were washed and photographed under the microscope (Nikon DS-Ri2).

Colony formation assay

1 000 cells were seeded in the six-well plate and incubated for 12 days. The cells were fixed with 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet for 15 min. Then, excess stain was washed with water and number of colonies more than 50 cells was counted.

Sphere formation assay

2 000 cells were plated in the ultra-low attachment 6-well plate (Corning, USA). Cells were grown in a serum-free DMEM or DMEM/F12 medium supplemented with 2% B27 supplement (ThermoFisher Scientific, USA), 20 ng/mL human EGF (Novoprotein, China), and 20 ng/mL human bFGF (Novoprotein, China). The medium was changed every 2–3 days. After incubation for 12 days, spheres larger than 100 μm in diameter were counted under Nikon DS-Ri2 microscope.

Quantitative real-time PCR

Total RNA was extracted from OSCC tissues or OSCC cells using TRIzol reagent (ThermoFisher Scientific, Waltham, MA, USA) and synthesized cDNA was prepared with a PrimeScriptTM RT Reagent Kit (Takara Bio Inc., Dalian, China). Quantitative real-time PCR (qRT-PCR) analysis were performed with a 7500 Real-time PCR system using SYBR™ Green Master Mix (Takara Bio Inc., Dalian, China). The primers used in this study are listed in Supplementary Table 3. Fold changes were analyzed using the comparative 2−ΔΔCt determination method.

Western blot analysis

The protein concentrations were quantified by BCA protein assay kit (Beyotime Biotechnology, China). Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology, China) was used to separate nuclear and cytoplasmic proteinaccording to the manufacturer’s instructions. The primary antibodies used in this study were listed in Supplementary Table 2. Secondary antibodies include HRP-linked anti-mouse IgG or anti-rabbit IgG (Absin, China). Protein bands were visualized using Tanon 5200 (Tanon, China) and signals were quantified using ImageJ software.

ALDH1 assay

The ALDEFLUORTM kit (STEMCELL, Canada) was applied to determine the ratio of ALDH enzymatic activity. 5 × 105 cells were re-suspended in the ALDEFLUOR assay buffer containing 1 μmol/L ALDH1 substrate BAAA, and then 500 µL of the cell suspension was immediately transferred into the control tube and incubated with 5 µL of ALDH inhibitor diethylaminobenzaldehyde (DEAB) for calculating background. After incubated at 37 °C for 45 min in the dark, cells were washed twice and the samples were detected using a flow cytometry (FACS, BD, USA).

Nile red staining

The cells were washed twice with PBS and fixed in 4% paraformaldehyde for 20 min. Then the cells were incubated in Nile red staining solution (1 mg/L, Solarbio, China) in the dark for 20 min at 37 °C. Finally, the immunostained samples were counterstained with DAPI (Beyotime Biotech. Co.). Images were captured an Olympus FV-1000 confocal microscope (Tokyo, Japan).

Transcriptome sequencing (RNA-Seq) analysis

Total RNA from HSC-4 cells and long-term P. gingivalis-infected HSC-4 cells was isolated using TRIzol (Takara, Japan). After detected by NanoDrop, the samples were applied by ILLumina Hiseqxten/NovaSeq6000 for transcriptomic high-throughput sequencing (Magorbio Ltd, Shanghai, China). The RNA-seq analysis was performed on the Majorbio Cloud Platform (www.majorbio.com). Differential expression analysis was performed using DESeq2 packages. The criteria including |Log2FC| ≥1.3, P < 0.05, and FDR < 0.05 were applied to select the significantly differential mRNAs. Gene Ontology (GO) enrichment and KEGG analysis were completed to identify the biological functions of the differential mRNAs and the pathways they participate in.

Lentivirus and RNA interference

Lentivirus containing SCD1 short hairpin (sh) RNA, lentivirus containing KLF5 short hairpin (sh) RNA, and empty vector lentiviral particles were synthesised by Genechem (Shanghai, China). The shRNA sequences are listed in Supplementary Table 4. Puromycin (2 μg/mL, MCE, Shanghai, China) was used to selecte the stably transfected cells. Specific siRNA against NOD1 and siNC were designed and synthesised by KELBiotech (Shanghai, China) and listed in Supplementary Table 5. Lipofectamin®3000 reagent (L3000008, Invitrogen) was used to transfect cells with siRNA according to the manufacturer’s instructions.

Transmission electron microscopy

After different treatment, HSC-4 cells were collected and fixed in 2.5% glutaraldehyde for 2 h at 4 °C. Then, the samples were cut into ultra-thin slices and the ultrastructure was acquired using a transmission electron microscope (H7650, Hitachi, Tokyo, Japan).

Immunofluorescent staining

Tissue paraffin sections were de-waxed, rehydrated, and blocked. Then, the sections were incubated with hybridization buffer containing P. gingivalis oligonucleotide probe (5′-GGTTTTCACCATCAGTCATCAACA-3′) in a dark humid chamber at 48 °C for 2 h and then incubated with the ALDH1 overnight at 4 °C. For OSCC cells, the samples were fixed with 4% PFA and treated with 0.2% TritonX-100. Subsequently, primary antibodies were incubated overnight at 4 °C. The samples were immunostained with Alexa Fluor 594-conjugated secondary antibodies (Proteintech, China) for 2 h and counterstained with DAPI (Beyotime Biotech. Co.). Images were captured with a laser-scanning confocal fluorescence microscope (OLYMPUS FV3000, Japan).

Mouse xenografts

The experiment was approved by the Ethics Committee of the School and Hospital of Stomatology at China Medical University. Six-week-old male BALB/c nude mice were purchased from Si Pei Fu (Beijing, China). The mice were maintained in a specific-pathogen free facility with a 12 h:12 h light:dark cycle at 21–23 °C. Two hundred microliters of diluted Martrigel containing 3 × 106 tumor cells were injected subcutaneously into the mice. The size of the tumor was measured every 5 days and tumor volumes were calculated using the digital caliper (width2 × length)/2. After 35 days, mice were euthanized and the tumor tissues were removed and analyzed.

ChIP-qPCR assay

The BeyoChIP™Assay Kit (Beyotime Biotechnology) was performed according to manufacturer’s instructions. In brief, P. gingivalis-infected HSC-4 cells were cross-linked with 1% formaldehyde for 10 min. Glycine was used to terminate the cross-linking reaction. Then, the cells were collected, lysed and sheared into 200–1 000 base pair fragments on ice, which were immunoprecipitated with anti-KLF5 antibody (Proteintech) or rabbit IgG (Proteintech) overnight at 4 °C. The fold-enrichment of KLF5 binding on specific region of the SCD1 promoter was detected by qRT-PCR. The primer sequences were listed in Supplementary Table 6.

Dual luciferase reporter assay

293 T cells, obtained from ATCCC, were seeded in 24-well plate and transfected with corresponding plasmids (pcDNA3.1, pGL3-basic, KLF5-pcDNA3.1, pGL3-basic-SCD, PRL-TK) with Lipofectamin® 3000. After culture for 48 h, the relative luciferase activity was determined with a Dual-Luciferase Reporter Assay System (11402ES60, Yeasen Biotechnology, China) according to the manufacturer’s manuals.

Statistical analysis

Statistical analyses were performed by GraphPad Prism 9.0. One-way ANOVA analysis was used for comparison among multiple groups (Tukey’s multiple comparisons test was used for multiple comparison afterwards), and independent sample t test was used for comparison between two groups. Pearson correlation analysis was applied for correlation analysis. All experiments were repeated three times. P-value < 0.05 was considered statistically significant.