Gene expression profile
The GOLM1 expression data in HNSCC used in this study were derived from the gene expression interactive analysis website GEPIA (http://gepia.cancer-pku.cn/), while the RNA sequencing data obtained by GEPIA for analysis were from the TCGA and GTEx databases.
Immunohistochemical staining of LSCC tissues
We collected 60 samples of LSCC tissues surgically removed from LSCC patients who were hospitalized in the First Affiliated Hospital of China Medical University, and collected 30 samples of normal tissues adjacent to the cancerous tissue as controls. All specimens were obtained after the provision of informed consent from the patients before resection, and this study was reviewed by the Ethics Committee of China Medical University.
All specimens were fixed in formalin, embedded in paraffin, serially sectioned at a thickness of 4 μm, and subjected to SP immunohistochemical staining. Strictly following the instructions of the SP kit (ZSGB Bio, Beijing, China), the sections were deparaffinized and hydrated, exposed to 3% H2O2 to eliminate endogenous enzyme activity, and subjected to a high-temperature and high-pressure method for antigen retrieval. They were exposed to the primary anti-GOLM1 monoclonal antibody (BOSTER,Wuhan, China; working concentration 1:200) overnight at 4°C, incubated with secondary antibody, and exposed to DAB(3,3’-Diaminobenzidine, DAB)(BOSTER) for color development, followed by dehydration, transparency, and mounting.
GOLM1 was found to be expressed centrally around the nucleus. Five high-powered fields of cancer nests (malignant tumors) or cell aggregation areas (non-malignant tumors) were randomly selected under a microscope (Nikon DS-F12). The average value of the proportion of GOLM1-positive cells in each section was used as the positive cell index (labeling index, LI). The results are expressed as mean±standard deviation, with LI ≥0.25 being considered positive.
Cell line culture
The human laryngeal squamous cell line TU686 (Fenghui Bio, Changsha, China) and human oral normal squamous epithelial cell line NOK (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were used in this study. The cells were seeded in RPMI-1640 medium (Solarbio, China) containing 10% FBS and cultured in an incubator at 37°C and 5% CO2.
siRNA and overexpression vector construction
siRNA targeting GOLM1, negative control siRNA, GOLM1 overexpression vectors, and their negative controls were designed and synthesized by Wanleibio (Shenyang, China). TU868 cells in logarithmic growth phase were transfected with LipofectamineTM 3000 (Invitrogen, USA) when the cell density reached 60%–70%. After transfection for 48 h, qRT-PCR and Western blotting were used to determine the transfection efficiency. GOLM1-overexpressing cells were treated with Wnt/β-catenin inhibitor (20 μM ICG001) halfway through the 48 h of transfection, and again 24 h after that treatment.
Total cell RNA or nuclear RNA was extracted using the UV spectrophotometer NANO 2000 (Thermo Scientific, USA) to determine the concentration of RNA in each sample. Each RNA sample was reverse-transcribed with Super M-MLV reverse transcriptase (BioTeke, Beijing, China) to the corresponding cDNA under reaction conditions of 37℃ for 15 min and then 85℃ for 5 s, 4℃ +∞. Then, cDNA samples were subjected to qRT-PCR using the SYBR qPCR Master Mix system (BioTeke) in an Exicycler 96 fluorescence quantitative PCR instrument (Bioneer, South Korea). The reaction conditions were 95℃ for 30 s, followed by 40 cycles of 95℃ for 5 s and 60℃ for 34 s, and then melting curve analysis at 65–95℃. The results were calculated by the 2ΔΔCt method and expressed as relative levels using GAPDH as an internal reference gene. See Table 1 for the real-time PCR primer sequences.
Total protein or nuclear protein was extracted using the Nuclear and Cytoplasmic Protein Extraction Kit (Wanleibio). Then, the protein concentration in each cell lysate sample was determined by the BCA (Bicinchoninic acid, BCA) method. Proteins were heated at 100℃ for 10 min to denature them and then electrophoresed on SDS-PAGE gels. Proteins were subsequently transferred to a membrane at 4℃ with 200 mA constant current for 1 h, followed by blocking with 5% skimmed milk at room temperature for 1 h. After the primary antibody had been incubated overnight at 4°C and the secondary antibody had been incubated at 37°C for 45 min, an ultrasensitive ECL chemiluminescence kit (Wanleibio) was used for luminescence development, the film was scanned, and a gel image processing system (Gel-Pro- Analyzer software) was used for analysis with the optical density value of the target band. The antibodies are shown in Table 2.
CCK-8 detection of cell proliferation
The cells were digested with trypsin and resuspended as a cell suspension at a concentration of 5×104/mL. This suspension was seeded in a 96-well plate at 100 μL per well, with five replicate wells being set in each group. After regular culture for a certain period of time (24, 48, or 72 h), 10 μL of CCK-8 (Wanleibio) solution was added to each well, followed by incubation for 2 h, after which the optical density (OD) value at 450 nm was measured with a microplate reader (Bioteke).
A Transwell chamber (Corning, USA) was placed in a 24-well plate, precoated with Matrigel (Corning) to prepare a single cell suspension (1×105/ml), 800 μl of culture medium containing 10% FBS was added to the lower chamber, and 200 μl of cell suspension was added to the upper chamber, giving cell density of 3×104/well. After culturing in groups for 24 h the cells were fixed with 4% paraformaldehyde (Aladdin, China) for 20 min, stained with 0.5% crystal violet (Amresco, USA) for 5 min, rinsed with distilled water, and the cells in the lower layer of the microporous membrane were counted under a microscope (OLYMPUS, Japan; 200×).
Wound healing assay
The cells in each group were scratched with a 200 μl pipette tip and photographed under a microscope (Olympus; 100×) after the transfected cells had reached confluence . After adding serum-free and antibiotic-free medium and continuing the culture at 37℃ for 24 h, another image of each wound was taken and the cell migration distance was calculated.
The cells were centrifuged at 300g for 5 min and washed with phosphate buffered saline(PBS). Next, the cells were incubated with ice-cold 75% alcohol at 20℃ overnight for fixation. Then, the cells were centrifuged and incubated with RNase A at 37℃ for 30 min, followed by staining with propidium iodide at 4℃ for 30 min. After this staining, the cells were analyzed using a NovoCyte flow cytometer (ACEA Biosciences Inc., USA).
The statistical software programs GraphPad Prism 7.0 and SPSS 22.0 were used for statistical analysis, and the data are expressed as mean ± SD. Chi-squared test or Fisher’s exact probability method was used for counted data. Kaplan-Meier survival curve and Cox regression analysis were used for survival analysis, while the log-rank test was used to determine significance. Spearman’s rank method was used for correlation analysis. The measured data were compared between two groups by t-test, while comparisons between multiple groups were performed by one-way analysis of variance. All differences were considered statistically significant at P<0.05, and the experiments was replicated more than three times.