Study population and clinical examinations
Thirty-five patients with NIP and 30 patients with SNSCC (sinonasal squamous cell carcinoma) who were admitted to the Second Affiliated Hospital of Dalian Medical University from June 2016 to March 2017 and 30 control patients (based on normal mucosa at the back of the inferior turbinate) were selected. The 20 cases of NIP, 20 cases of SNSCC, and 20 control cases were used to detect the O-GlcNAc protein expression with immunohistochemistry and western blotting. The 10 cases of NIP, 15 cases of SNSCC, and the 10 control cases were used to detect the ogt gene expressions with RT-qPCR.
Inclusion criteria were (1) NIP confirmed by pathology before or after surgery, excluding specimens containing a large amount of necrotic tissue or with a large number of infiltrated inflammatory cells, (2) patients in the SNSCC group needing to be diagnosed as NIP before malignant transformation, (3) patients generally in good condition undergoing routine examination and comprehensive evaluation before surgery and having no surgical contraindications, and (4) patients confirmed to have squamous cell carcinoma and not having undergone any physical, chemical, or immunological antitumor treatment before surgery. All patients were approved by the Ethics Committee of Dalian Medical University (2009 No. 006), giving informed consent.
SCC6 and CNE-E1 cells (American Type Culture Collection) were grown in Dulbecco's Modified Eagle Medium (Invitrogen) supplemented by 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin. The cells were maintained at 37°C under 5% CO2 in humidified air. The cells (106) were seeded onto 6-well plates. When the cells reached 80% confluence, they were washed three times with phosphate-buffered saline (PBS) and subsequently starved of serum for 3 h before progestogen treatment.
The tissue was treated by conventional methods and embedded in paraffin, serially sectioned, sliced to a thickness of 3 µm, and the attached sections and reagents required for the experiment were placed on a fully automated immunohistochemical staining instrument. O-GlcNAc antibody dilution was 1:100 (Abeam). Immunohistochemistry images were taken using a Nikon microscope and a CCD image sensor. All sections were photographed in the same light-intensity room using the same aperture, the same light source, and the same magnification (400×). Each slice was randomly photographed with five fields of view. The image was analyzed by the US Image-Pro Plus 6.0 image analysis system. The immunohistochemical results of each group were analyzed. After standardization, the area-weighted average optical density (IOD/area) was used as the analysis standard.
RNA Isolation and RT-qPCR
The tissues were combined, and total RNA was isolated using Trizol (Takara Biotechnology). The cDNA was formulated from 100 ng of total RNA using the PrimeScript RT reagent Kit (Takara Biotechnology) and subjected to quantitative PCR (qPCR) using SYBR Green in an RT-qPCR thermal cycler. Relative quantification was used to analyze the RT-qPCR data. The threshold cycle threshold (CT) was used to record gene expression. The PCR conditions were as follows: DNA denaturation at 95°C, followed by 35 cycles at 94°C for 30 s, 56°C for 30 s, and 72°C for 30 s. The CT was determined by monitoring the incorporation of SYBR Green I (Takara Biotechnology) into the amplified product with fluorescence detection. The expression of CK8 was normalized according to that of gapdh. The following specific primers were designed: gapdh (XM_001256799.2) forward, 5′-GTGAAGGTCGGAGTCAACG-3′, reverse, 5′-TGAGGTCAATGAAGGGGTC-3′, ogt (XM_017029908.1) forward, 5′-CGGGAATCACCCTACTTCACACC-3′, reverse, 5′-CCGCCATCACCTTCACTCGAAA-3′, oga (XM_017015586.1) forward, 5′-TCCCCAGAGATGTCCATGCAAG-3′, reverse, 5′-TCCTTTGGGTCCATGCTCGTA-3′.
According to the instructions in the protein extraction kit, the entire process was carried out on ice. The protein concentration in the sample was detected using the QuantiPro BCA assay kit instructions (KeyGen Biotech). The protein was subjected to 10% polyacrylamide gel electrophoresis. After electrophoresis, the protein was transferred to a polyvinylidene fluoride membrane, and a 1:1,000 dilution of rabbit antihuman O-GlcNAc antibody was used. The primary antibody was left at room temperature for 3 h, and then TBST was added. All antibodies were diluted with TBST and washed three times with TBST for 10 min. The membrane was incubated with 1:2,500 horseradish peroxidase-conjugated antibody for 1 h at 4°C. The membrane was then placed in an imaging system (Bio-Rad) and ECL luminescent solution was added.
Short, Interfering RNA (siRNA) Knockdown Experiments
The SCC6 cells and CNE-E1 cells were seeded in 6-well plates. For the knockdown experiments, siRNA targeted the ogt and oga gene (ogt-siRNA and oga-siRNA; 200 nmol/well) and a negative control siRNA were purchased from GenePharma. The SCC6 cells and CNE-E1 were transfected with the si-OGT and si-OGA Xfect RNA Transfection Reagent (TaKaRa). The transfection efficiency was determined with RT-qPCR.
The cells were seeded in 96-well plates at 100 µL per well (n = 103 cells) and cultured for 24 h before 10 µL of a Cell-Counting Kit-8 (CCK-8) solution was added to each well. The plates were incubated for 4 h. Absorbance (OD) at 450 nm was measured.
Transwell Invasion Assay
A permeable filter of a Transwell system (Corning Inc) was used to study the invasion capacity of cells. The inside compartment of the Transwell inserts was coated with Matrigel (BD Biosciences) at 37°C for 3 h and then blocked with 1% PBS solution for 30 min at room temperature. The SCC6 and CNE-E1 cells (105/well) were loaded in the upper chamber in a culture medium for 24 h. Cell migration to the other side of the membrane was induced by 20% FBS-containing medium in the lower chamber. The cells were fixed in methanol for 30 min and stained with 0.5% Crystal Violet for 20 min. After the cells on the upper side of the top chamber were gently removed, the migrated cells were photographed and counted using ImageJ software (National Institutes of Health).
Wound Healing Assay
SCC6 or CNE-E1 cells were seeded in 6-well plates for 24 h after pretreatment (knockdown or overexpression of O-GlcNAc). The cells were then wounded by removing a 700 µm-wide strip with a standard 200 µL pipette tip. Wound healing was quantified by measuring the migratory distance of cells.
The results were graphically depicted as the mean ± standard deviation. One-way ANOVA was performed (SPSS 20.0 for Windows) to detect statistically significant differences. A P-value < 0.05 was considered statistically significant.