Ethics statement
The current study has got the approval of the ethics committee of Guizhou Medical University (No. 2001221).
Clinical tissue samples
Fifty-eight OSCC tissues and the adjacent noncancerous tissues were obtained from patients with primary OSCC undergoing surgical resection. No patients received chemotherapy or radiotherapy prior to surgery. All specimens were confirmed by two pathologists. The written informed consents were signed by every patient and the current study has got the approval of the ethics committee of Guizhou Medical University. All tissue samples were rapidly placed at −80°C for further study. The OSCC patients were divided into PDIA6 high expression and low expression groups based on the expression medium of PDIA6 at mRNA level.
Cell lines and culture method
Two human OSCC cell lines, SCC9 and Cal27 were all purchased from ATCC (Manassas, VA, USA). SCC9 cells were seeded in Eagle's medium and Ham's F12 medium, and Cal27 cells were placed in DMEM, with 10% fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin. All cells were kept in a humidified atmosphere with 5% CO2 with a stationary temperature of 37°C. Cell culture medium and FBS were purchased from Invitrogen (Carlsbad, CA, USA).
Lentivirus and small interfering RNAs (siRNAs)
The lentivirus vectors applied to overexpress PDIA6 (called OE-PDIA6), and the siRNA used to downregulate PDIA6 (called as si-PDIA6) and their negative controls (OE-NC, si-NC) were all purchased from Shanghai GenePharma Co., LTD (Shanghai, China). The lentivirus vectors were introduced into cells via cell infection by using polybrene, and si-PDIA6 and si-NC were introduced into cells via cell transfection using Lipofectamine 2000 reagent (Invitrogen). The infected cells were incubated in G418 (100 μg/ml) for 14 days to establish the stable cell lines which were used in the in vivo assay.
Real-time quantitative polymerase chain reaction (qPCR)
Total RNA was extracted from tissues and cells by using TRIzol reagent (Life Technologies, Waltham, MA, USA) referring to the manufacturer’s manual. Then, the cDNA was produced using a PrimeScriptTM 1st Strand cDNA Synthesis Kit (Takara, Dalian, China). After that, the cDNA was served as substrate for qPCR with SYBR Premix Ex Taq (Takara) on an ABI 7900 system (Applied Biosystems, Foster City, CA, USA). The 2−ΔΔCt method was applied to analyze the relative levels of mRNAs after being normalized to that of the expression level of β-ACTIN. The sequences used in this experiment were listed in Table 1.
Western blotting assay
Total protein was obtained from cells using the RIPA lysis buffer (Beyotime, Jiangsu, China), supplemented with 1% (v/v) protease inhibitors (Beyotime). Then, 30 μg protein sample obtained from every group was separated by 10% sodium dodecyl ulfate-polyacrylamide gel electrophoresis, followed by transformation into the polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were then successively incubated with 5% fat-free milk for 1 hour at room temperature and proved with the first antibodies, including PDIA6 (1:2,000 dilution; No. ab11432, Abcam, Cambridge, MA, USA), and β-actin (No. ab8226, Abcam, 1:5,000 dilution) overnight at 4°C. After that, the membranes were probed with secondary antibodies (Abcam) for 1 hour. Protein signals were detected using iBright CL750 (Thermo Fisher Scientific, MA, USA) after incubation with ECL (Thermo Fisher Scientific).
CCK-8 assay
The proliferation assay was performed with the Cell Counting Kit-8 (CCK-8) assay. First, the OSCC cells (2500 cells for each well) were seeded in 96-well plates, allowed to adhere, transfected and then cultured at 37 °C for 0, 24, 48 and 72 hours. Then, 10 μL CCK-8 solution (Abcam) was added to each well and allowed to incubate for further 4 hours at 37°C. The OD values at 450 nm were determined using a spectrophotometer (BioTek Instruments, Winooski, VT, USA).
Transwell chamber assay
Cell metastasis and invasiveness were assessed with Transwell chambers (8 μm, Corning, NY, USA) in 24 well plates using uncoated or Matrigel-(BD Biosciences, San Jose, USA) coated membranes. In briefly, OSCC cells in culture medium with 1% FBS were seeded in the upper well with 1×105 cells for each well, and 600 μL cell culture medium with 10% FBS was added in the lower chambers. After 24 hour or 48 hours of incubation, the cells did not penetrate the membrane on the upper chamber were removed with a cotton swab. Cells in the lower membrane were fixed with methanol and stained with 0.1% crystal violet solution. After washing with PBS for 4-5 times, the numbers of stained cells in 6 random fields were counted under an inverted microscope.
Detection of lactate production, glucose consumptionand ATP levels
OSCC cells were inoculated in 6-well plates at a density of 2×105 cells and allowed to attach. Following 24 hours of incubation, the medium was collected to measure lactate production and glucose consumption with commercial kits from Nanjing Jiancheng Biotech (Jiangsu, China; A019) and Applygen Co., LTD (Beijing, China; No. E1010) in the light of the manufacturer’s descriptions, respectively. ATP levels were tested using an ATP Colorimetric/Fluorometric Assay Kit (Sigma-Aldrich, MO, USA) in the light of manufacturer’s instructions.
Tumor xenograft
Male NOD/SCID mice aged 5–6 weeks obtained from Wuhan Huaguenke Biotechnology Co. LTD (Hubei, China) were randomly divided into OE-NC and OE-PDIA6 groups (n=5 for each group). This animal assay has got the approval of the Experiment Ethics Committee of Guizhou Medical University. Approximately 5×106 SCC9 cells with OE-NC or OE-PDIA6 stable transfection were suspended in 200 μl of PBS and then subcutaneously injected into the armpit of mice. The tumors were measured to assess tumor formation every week. Tumor volume = length × width2/2. The mice were euthanized 4 weeks after injection, and the tumor xenografts were harvested and weighed.
Statistical analysis
Data are expressed as mean ± standard deviation (SD) from 3 independent experiments. SPSS software (version 23.0, SPSS Inc., Chicago, IL, USA) was applied for statistical analyses. Correlations were determined by Pearson correlation analysis. Two-side t tests and one-way ANOVA with Bonferroni post-hoc tests were used to compare differences between two and ≥ 3 groups. The value of p < 0.05 was considered as statistically significant.