Patients and specimens
This study was approved by the Ethics Committee of the North Sichuan Medical College. All specimens were collected after obtaining informed consent from patients. Primary OSCC tissue samples and matching adjacent normal mucosa specimens were collected from 15 patients undergoing surgery at North Sichuan Medical College, China. All collected tissues were stored at − 80 °C until use. The pathological or normal state of the samples was confirmed by histopathological and clinical examination.
Tissue microarrays (TMAss)
The TMA technique was used to analyze SCRN1 expression in cancerous and adjacent tissues according to a previous method21. TMA sections were examined and scored by independent observers who were blinded to the clinical outcomes. Both cancerous and adjacent tissues were selected for comparative analysis. The intensity of the staining signal was measured and documented using Image‐Pro Plus 6.0 image analysis software (Media Cybernetics, Inc.). The mean densitometry of the digital image (× 500) was used to represent SCRN1 staining intensity. The signal densities in tissue areas from five randomly selected fields were determined blindly and subjected to statistical analysis.
Cell culture and cell transfection
Human oral squamous cell carcinoma cell lines, HSC3, HSC6(human oral squamous carcinoma cells), SCC15, SCC25 (tongue squamous carcinoma cells) were obtained from GuangZhou Jennio Biotech Co.,Ltd (GuangZhou, China), UM1, UM2 (oral carcinoma cells) were obtained from ATCC (Manassas, VA, USA).Six human oral squamous cell carcinoma cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM, HYCLONE, USA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin, and normal oral keratinocytes (NOKs) were grown in keratinocyte growth medium. All the cells were maintained at 37 °C in a 5% CO2 humidified incubator. Mycoplasma testing has been done for the cell lines used, the cell lines used have been authenticated.
The sh-SCRN1 plasmids and empty vector were designed and synthesized by GeneCopoeia (https://www.genecopoeia.com), and the viruses were generated using protocols from GeneCopoeia. HSC3 and SCC15 cells (2 × 105/well) were seeded in 6-well plates and grown for 24 h until they reached 60–70% confluency. The cells were then transfected with diluted virus. After 12 h, the medium was replaced with fresh complement medium, and the cells were harvested after 72 h of transfection.
For the TGF-β stimulation assays, the cells were harvested after 48 h of treatment with recombinant human transforming growth factor-beta 1 (10 ng/ml).
Western blot assay
The cells were lysed in RIPA lysis buffer (CWBIO, China) supplemented with protease inhibitors. Total proteins were fractionated using SDS-PAGE, transferred onto PVDF membranes (Millipore, USA), and examined with the corresponding primary antibodies using a chemiluminescence kit (Millipore, USA). The following primary antibodies were used in this study: anti-SCRN1 antibody (Abcam, ab105355, USA), anti-GAPDH antibody (Earthox, E021010, USA), anti-Smad3 antibody (HuaAn Biotechnology, ET1607, China), and anti-phospho-Smad3 antibody (S423/S425) (HuaAn Biotechnology, ET1609, China).
Quantitative real-time PCR (qPCR)
Total RNA from cells was extracted using an EZNA Total RNA Kit (Omega, USA), and total RNA was reverse transcribed to cDNA in strict accordance with the manufacturer’s instructions (PrimeScript™ RT Reagent Kit, TaKaRa, Japan); the cDNA was then amplified with SYBR (Roche) by qPCR. The sequences of the primers were as follows: SCRN1 (forward: 5′-GGATGGTCTGGTGGTATTTGG-3′ and reverse: 5′-CCTTGGAACTTGGTCGATTG-3′) and GAPDH (forward: 5′-GACTCATGACCACAGTCCATGC-3′ and reverse: 5′-AGAGGCAGGGATGATGTTCTG-3′). The relative mRNA levels of SCRN1 were normalized to GAPDH reference gene expression and calculated via the 2−∆∆CT method.
Cell counting kit-8 (CCK-8) assay
The cells were seeded in 96-well plates (1000 cells/well) for the cell proliferation assay (CCK-8 kit, Yeasen, China). After culturing for 24, 48, 72 and 96 h, 10 μl of CCK-8 solution was added to each well. After adding the CCK-8 reagent, the cells were cultured for an additional 1–4 h, and the OD values were measured at 450 nm.
Transwell migration and invasion assays
Cells (2 × 105) were seeded into the upper chambers of 24-well Transwell plates containing serum-free medium. Medium supplemented with 10% FBS was added to the lower chambers. Before the Transwell invasion assays were performed, a mixture of Matrigel (BD Biosciences, USA) and medium was spread on the upper surface of the chambers and incubated at 37 °C overnight. The cells were then allowed to incubate for 24 h at 37 °C with 5% CO2. The cells were fixed and stained with crystal violet (0.1%). The cells that migrated from the upper chamber to the lower chamber were counted, and the average number of cells in five random regions was considered the final result.
Enzyme-linked immunosorbent assay (ELISA)
The MMP-2/MMP-9 concentrations in the supernatants were determined by ELISA using a commercially available kit (Abcam, Cambridge, UK) in accordance with the manufacturer’s specifications. The absorbance was read by a microplate reader at 450 nm. The concentration of each sample was measured based on the regression equation established using serial dilutions of standards.
Determination of MMP2/MMP9 activity by gelatin zymography analysis
Equal amounts of total proteins were separated on 10% SDS–polyacrylamide gels containing 0.1% gelatin. The gels were washed two times with 2.5% Triton-X 100 for 20 min and incubated for 12 h at 37 °C in developing buffer consisting of 150 mM NaCl, 5 mM CaCl2 and 50 mM Tris pH 8.0. After staining with 0.5 mg/ml Coomassie Brilliant Blue R-250 in 10% acetic acid and 25% methanol for 2 h with gentle agitation, the gels were destained for an additional 2 h in 8% acetic acid and 4% methanol before being photographed.
For continuous variables, the mean and standard deviation were used to describe baseline characteristics. Baseline characteristics between two different groups were compared by t tests for continuous variables and by chi‐square tests for categorical variables. All statistical analyses were performed by using GraphPad Prism 6.0 and Stata/MP 14.0. All the data are presented as the mean ± standard deviation (SD) from three independent experiments. A two-sided P ≤ 0.05 indicated statistical significance.
Ethics approval and consent to participate
All procedures performed in the current study were approved by the Ethics Committee of North Sichuan Medical College. Written informed consent was obtained from all patients or their families.