Tissue samples and clinical specimens

This study collected oral mucosa tissues from 15 healthy individuals and 17 OSF patients who underwent oral outpatient surgery at Xiangya Stomatological Hospital, Central South University, Hunan, China. OSF tissue samples were derived from the patient’s first visit with no history of treatment and had definitely pathological diagnosis by two experienced pathologists. Written informed consent was obtained from all patients and the study was approved by the Protection of Human Subjects Committee of Xiangya Stomatological Hospital, Central South University (No. 20220102).

Cell culture and treatment

Primary buccal oral mucosa fibroblast was obtained from healthy individuals who did not have areca quid chewing habits, which were isolated and cultivated by tissue adherence method. The primary fibroblast, HaCat cell lines and HEK293T cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Grand Island, NY, USA) containing 10% bovine calf serum (Sigma-Aldrich, MO, USA) under at 37 °C with 5% CO2. Plasmid transfection using Lipo8000™ transfection reagent was carried out following the manufacturer’s instructions (Beyotime, Shanghai, China). After reaching 70%–80% confluence, HaCat cells were washed with PBS and then grown in DMEM without FBS and exposed to Arecoline (Sigma-Aldrich, MO, USA) for 24 h. In a co-culture model, PBS or Arecoline-treated HaCat cells were plated on 0.4-μm pore, hanging cell culture chambers (Millipore, MA, USA), and fibroblast cells were seeded on 12- or 6-well plates.

Exosome isolation and characterization

Exosomes were isolated from the exosomes-free culture medium of PBS or Arecoline-treated HaCat cells by differential centrifugation. The culture medium was first centrifuged at 300 × g for 100 min and then at 2 000 × g for 20 min at 4 °C. The supernatant was then through a 0.22-μm filter (Millipore, MA, USA) to remove cell debris and other larger vesicles. Finally, the supernatant was centrifuged at 100 000 × g for 90 min. The collected exosomes were resuspended in PBS and subjected to the next experiments. Meanwhile, the isolated exosomes from the culture medium and blood serum were collected by exosome isolation reagent (Ecotop Scientific, Guangzhou, China). The size distribution and concentration of exosomes were analyzed by nanoparticle tracking analysis (NTA) (ZetaView, Particle Metrix, Germany). The expression of the exosomal markers CD9, CD63 and CD81 (exosome panel, ab275018, Abcam) was assessed by Immunoblotting analysis.

DiI staining for exosomes

Exosomes derived from epithelial cells were labeled with DiI cell membrane red fluorescent probe (Beyotime, Shanghai, China) according to the manufacturer’s protocol with minor modifications. Briefly, exosomes were suspended in PBS and mixed with diluted DiI (10 μmol/L, for labeling of cell membranes) and incubated at room temperature. After 30 min, the labeled exosomes were finally washed with PBS, and they were resuspended for uptake experiments.

RNA extraction and real-time PCR

Total RNA from cultured cells and tissues of mice was isolated by use of Trizol reagent (Invitrogen, CA, USA) according to the manufacturer’s recommendations. Extraction of miRNA from exosomes was accomplished using a commercial RNAeasy small RNA isolation Kit (Beyotime, Shanghai, China). The purified RNA was eluted with RNase-free water and stored at –80 °C until analysis. MiRNA qRT-PCR synthesis and primer sets kits (Sangon Biotech, Shanghai, China) specific for miR-17-5p and U6 snRNA were used to measure the levels of miRNAs. The U6 snRNA and GAPDH were used as endogenous controls. Real-time PCR was performed by use of SYBR Green (Vazyme, Jiangsu, China) with the Applied Biosystems QuantStudio 5 (Thermo Fisher, MA, USA). To analyze the qRT-PCR results for experiments, the 2−ΔΔCt method was used. The PCR primers are shown in Supplementary Table 1.

Immunoprecipitation, Immunoblotting and Immunostaining

Cell or tissue samples were lysed on ice by RIPA buffer mixed with protease and phosphatase inhibitor cocktails (Beyotime, Shanghai, China). The proteins were then quantitatively analyzed by a BCA Protein Assay Kit (23225, Thermo Fisher, MA, USA). The proteins were then separated by 8%–10% SDS-PAGE gel and transferred to PVDF membranes. Membranes were then incubated overnight at 4 °C with a primary antibody for COL1A1 (1:2 000, 67288, Proteintech), α-SMA (1:2 000, 14395, Proteintech), Smad7 (1:2 000, 25840, Proteintech), TGFBR1 (1:2 000, A0708, ABclonal), WWP1 (1:1 000, sc-390897, Santa Cruz Biotechnology), Fibronectin (1:2 000, 66042, Proteintech), Phospho-Smad2 (Ser465/Ser467) (1:2 000, #18338, Cell Signaling Technology), GAPDH (1:2 000, 5174 S, Cell Signaling Technology). The membranes were then incubated with a 1:5 000 dilution of horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse antibodies (Cell Signaling Technology) for 1 h at room temperature and images were visualized using a chemiluminescent imaging system (BIO-RAD, USA).

The interaction between proteins was verified by immunoprecipitation. In brief, cell lysates were incubated with indicated antibodies and Protein A/G Agarose (Beyotime, Shanghai, China) at 4 °C overnight. The immunocomplex was washed 5 times, and boiled in 2× SDS loading buffer for 10 min. The coprecipitation was performed on SDS-PAGE gel and blotted with the specific antibody.

For immunofluorescence staining, cells were cultured on coverslips washed 3 times with PBS, and fixed with 4% paraformaldehyde for 30 min. The glass coverslips permeabilized with 0.5% Triton X-100 at room temperature for 20 min, then blocked with 3% BSA in PBS for 1 h at room temperature and incubated with primary antibody at 4 °C overnight. The coverslips were washed 3 times with PBS and incubated with the corresponding fluorescent secondary antibody conjugated with fluorochrome for 1 h at 37 °C. The cell nuclei were stained with DAPI. Finally, cell images were observed and captured with fluorescence microscopy (Leica, Wetzlar, Germany).

Sirius red collagen detection

Collagen content in cell supernatant was detected using the Sirius Red Total Collagen Detection Kit (#9062, Chondrex, WA, USA) according to the manufacturer’s instructions. Briefly, the fibroblast culture medium was incubated with concentrating solution (#90626, Chondrex, WA, USA) at 4 °C overnight. After centrifugation at 10 000 r/min for 3 min, the precipitate was collected and dissolved in 0.05 mol/L acetic acid. Then the blank, standard and sample solutions were incubated with Sirius Red Solution. Finally, removed the supernatant and dissolved in the extraction buffer. Read the optical density values at 510–550 nm and the collagen concentration (μg/mL) in test samples was calculated using regression analysis.

Three-dimensional collagen gels

The treated fibroblast resuspended in 5× DMEM were mixed with 3 mg/mL rat tail type I collagen (Cellmatrix, Nitta Gelatin, Japan) at a ratio of 2:1. The mixture was seeded in 24-well plates at a cell density of 5 × 104per well. After collagen coagulation for 30 min at 37 °C, the edges of the gel separated from the wall of the well, and 0.5 mL culture medium was added. The gels were photographed by 48 h and the gel area was measured using ImageJ software (NIH, Bethesda, MD, USA).

Animal studies

Male BALB/c mice at 4 weeks of age were purchased from Hunan SJA Laboratory Animal Co. Ltd. (China) and housed in animal facilities at the Department of Laboratory Animals of Central South University. Animals were treated humanely and with regard for alleviation of suffering according to a protocol approved by the Institutional Animal Care and Use Committee of the School of Basic Medicine Science, Central South University (2022-KT66). To verify the expression of miR-17-5p in tissues, mice were exposed to PBS or Bleomycin 1 mg/mL (Yeasen, Shanghai, China) as described previously. Briefly, twelve mice were randomly divided into two groups. Every other day, we used a 29-gauge insulin syringe (Ultra-Fine, BD) to inject drug treatment into the bilateral buccal mucosa (30 μL) and back skin (50 μL) of the mouse for 6 weeks. For inhibition of miR-17-5p profibrotic effects, twenty-four mice were divided into four groups (PBS, Bleomycin + control, Bleomycin + miR-17-5p agomir, Bleomycin + miR-17-5p agomir + SB525334). After 6 weeks of Bleomycin treatment, miR-17-5p and TGF-β inhibitor SB525334 were administered concurrently for 2 weeks, respectively. Mice in the miR-17-5p agomir and control groups (5nmol, RiBoBio, China) were dosed with buccal mucosa injection 3 times a week for 2 weeks. SB525334 compound was dosed with intraperitoneal injection (10 mg/kg, SB525334, Selleck) daily for 2 weeks, After the mice were sacrificed, the buccal mucosa, back skin and blood sample obtained for the subsequent experiments.

Dual-luciferase reporter assay

TargetScan was used to predict the binding sites for miR-17-5p in Smad7. The 3’UTR sequence of Smad7, which was predicted to harbor the miR-17-5p seed region, or a mutant sequence, was inserted into the SacI and XbaI sites of the pmirGLO luciferase vector (Sangon Biotech, Shanghai, China). HEK293T cells were seeded in 24-well plates and co-transfected by DNA transfection reagent (Neofect biotech, Beijing, China) with pmirGLO vectors containing wild-type or mutant 3’-UTR of Smad7 and miR-17-5p mimics or miR-17-5p mimic-NC to examine the miRNA binding ability. About 48 h later, the cells were washed and lysed with the lysis buffer from the Dual-Luciferase Reporter Gene Assay Kit (Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions. Finally, the luciferase and renilla activity was detected by a chemiluminescence instrument (Promega, USA). Relative luciferase activity was normalized with renilla luciferase activity and then compared with those of the respective control.

Pathological tissue staining

The obtained buccal mucosa and skin tissue of the mouse was fixed (4% paraformaldehyde), dehydrated, and embedded in paraffin and sectioned for staining with HE, Masson’s trichrome (NJJCBIO, Nanjing, China) and Sirius red (Solarbio, Beijing, China) to assess the degree of fibrosis. The experimental procedures of tissue section staining were according to the reagent instructions. The collagen volume fraction area was read by ImageJ software.