This study was approved by the Ethical Committee of the Affiliated Hospital of Stomatology, Nanjing Medical University (permission number PJ2016−034−001) and the Institutional Review Board of Nanjing Medical University (permission number 2014−132). Participants signed informed consent voluntarily and we conducted all procedures according to the Declaration of Helsinki.
We collected tissue samples from 15 healthy controls and 30 OLP patients (15 NEOLP patients and 15 EOLP patients) for culturing fibroblasts. All participants were recruited from the Department of Oral Medicine of the Affiliated Hospital of Stomatology at Nanjing Medical University from November 2019 to May 2021. The OLP diagnosis was established based on clinical and histopathological characteristics according to the criteria made by the WHO in 1978 and van der Meij et al. in 2003 . Normal tissue samples were collected from gingival tissues around the extracted teeth, and tissue samples of OLP groups were collected from buccal mucosa. The subjects had neither any systemic disorders nor any soft tissue lesions. Meanwhile, they had not received any treatment within 6 months. Smokers and severe alcoholics were excluded. The clinical features were displayed in Table 1. Fifteen tissue samples were collected from healthy controls, NEOLP group and EOLP group respectively for IHC following the above requirements. The clinical features were displayed in Table2.
Fresh specimens were washed three times in sterile phosphate-buffered saline (PBS) (Gibco) containing 5% penicillin-streptomycin-glutamine (100X) (Gibco) and minced into fragments. These fragments were cultured in DMEM medium (Invitrogen, USA) supplemented with 10% fetal bovine serum (Gibco) and 1% antibiotics, at 37 °C with 5% CO2. OLP AFs and normal mucosal fibroblasts (NFs) were used for experiments at 3-6 generations.
Cells were seeded on cover slips at approximately 70 % confluence overnight. For immunofluorescence processing, they were fixed and permeabilized, and then stained with primary antibody overnight at 4 °C after blocking with 3% BSA for 30 min at room temperature. Cells were then incubated with secondary antibodyies for 60 min in dark at room temperature. The nuclei were counterstained with DAPI (C1005, Beyotime, China) for 5 min and coverslips were imaged and mounted under a fluorescent microscope. Antibodies are listed in Table S2.
Total RNA of the NFs or OLP AFs was extracted using Trizol Reagent (Invitrogen, USA). Next, cDNA was obtained using a PrimeScript RT reagent kit (Takara, Japan). Real-time RT-PCR was performed using TB Green Premix Ex Taq II (Takara). U6 and GAPDH were used as internal controls for miR-155-5p and FAP-α relatively. The 2‑ΔΔCt method was used for relative quantification. Primers are listed in Table S1.
miRNA and siRNA (Small interfering RNAs) transfection
All the compounds were purchased from RiboBio (Guangzhou, China). Following the manufacturer's protocol of the riboFECT™ CP Transfection Kit (RiboBio), miR-155-5p mimic (50 nmol l−1), miR-155-5p inhibitor (125 nmol l−1) and negative control (NC) were transfected into control NFs and OLP AFs respectively. Si-SOCS1 (siRNA#1 and siRNA#2) (100 nmol l−1) and siRNA NC were transfected into NFs. Associated fibroblasts were used for the specified experiments at 48-72 h after transfection.
Western blotting assay
Protein samples were extracted with RIPA buffer (Beyotime, China) containing 10 mM protease inhibitor PMSF (Beyotime) on ice. Next, the protein was separated by 12.5% SDS-PAGE and transferred to a PVDF membrane (Millipore, USA). Afterwards, the membranes were incubated with 5% non-fat milk, followed corresponding primary antibody overnight at 4 °C and corresponding secondary antibodies for 2 h at 37°C. The blots were examined using an ECL system (NCM Biotech, China) and quantified by Image J (National Institutes of Health, USA). Antibodies are listed in Table S2.
The tissue samples were standardly fixed and embedded. The mucosal sections were repaired with sodium citrate, and then blocked with 1% BSA/PBS at 37˚C for 30 min. According to the manufacturer's protocols, add anti-SOCS1 primary antibody and incubate overnight at 4 °C. Follow with anti-goat-HRP and culture at 37℃ for 30 minutes. After that, add DAB (MXB Biotechnologies) chromogenic reagent, counterstain with hematoxylin, and dehydration. The strength of IHC staining was quantified by ImageJ. Antibodies are listed in Table S2.
Enzyme-linked immunosorbent assay (ELISA)
Fibroblasts from relevant groups were seeded and placed in serum-free Dulbecco's modified Eagle's medium without antibiotics after 24h. Those cells were transfected as described above for additional 48-72 h in culture medium. Then medium was then refreshed. The levels of IL-6 and IL-8 in supernatant were measured by using ELISA kits (Multi Sciences) after 24h.
miRNA target prediction
Through TargetScan (www.targetscan.org), a bioinformatics prediction program employed to predict miRNA targets, we identified the potential target of miR-155-5p.
Dual-Luciferase Reporter Assay
According to the manufacturer’s instructions, the pLUC firefly luciferase vectors contained wild type or mutant SOCS1 sequence were co-transfected into HEK 293T cells together with firefly luciferase plasmid pGL3 (RiboBio) and miR-155-5p transfection reagent. Dual-Luciferase Reporter Assay System (Vazyme, China) measured the luciferase activity after 48 h transfection.
Data are expressed as a mean ± standard deviation of three independent experiments. Statistical analysis was performed using the GraphPad Prism 8.0.2 (San Diego, CA, USA). The Student’s t test was used for statistical analysis with 2 groups. A one-way analysis of variance was performed to detect the data comparison of more than two groups. Pearson’s correlation coefficient was applied for detecting the correlations among miR-155-5p and FAP-α. p < 0.05 was considered significant.