2.1 Cells and reagents
Human immortalized nasopharyngeal epithelial cells (NP69) and NPC cell line 6-10B were maintained in our laboratory. RPMI 1640 culture medium was purchased from Wuhan Seth Biological Reagent Management Department; fetal bovine serum (FBS) was purchased from American Invitrogen Co., Ltd.; phosphate-buffered saline (PBS), dimethyl sulfoxide, and 0.25% trypsin were from Hangzhou Jinuo Biology Co., Ltd.; the CCK-8 kit for cell proliferation and toxicity detection was purchased from Tongren Institute of Chemistry, Japan; the apoptosis detection mitochondrial membrane potential detection (JC-1) kit was purchased from Shanghai Biyuntian Biotechnology Co., Ltd; and the objective protein antibodies were from Cell Signaling. The equipment used included a carbon dioxide incubator (Thermo Scientific, USA), inverted microscope (Leica Instrument Co., Ltd., Germany), automatic microscope (Olympus, Japan), automatic enzyme labeling instrument (Bio-Red, USA), and Odyssey two-color infrared laser imaging system (LI-COR, USA).
2.2 Cell culture
NP69 and 6-10B cells were cultured in RPMI 1640 medium containing 10% FBS (with 1% penicillin and streptomycin) at 37℃ in a 5% CO2 incubator.
2.3 Construction of S100A14 low-expression and overexpression NPC cell lines
A GV492/GV493 lentivirus packaging system was used to package an S100A14 virus and gcGFP control virus, which were used to transfect 6-10B cells in six-well plates. After 48 h, 1 µg/ml puromycin was added until all the parent cells had died. The knockdown and overexpression efficiency of S100A14 was detected by western blotting.
2.4 Cell viability analysis
S100A14-knockdown 6-10B cells and control 6-10B cells were digested and inoculated in 96-well plates at 2000 cells per well, with five parallel wells in each group. After the cells were attached, a working solution of Cell Counting Kit-8 (CCK-8) reagent and medium was prepared at a ratio of 1:9. The original medium in each well was replaced with 100 µl of this solution, and the cells were incubated at 37 ℃ for 1, 2, or 3 h. The absorbance (optical density; OD) value was measured at a wavelength of 450 nm with an enzyme-labeling instrument. Taking the OD value on the 0th day as the origin, the OD value was measured three times every 24 h, taking the average of four measurements per time, and a cell growth curve was constructed.
2.5 Early apoptosis detection and mitochondrial membrane potential detection
Two groups of shS100A14 cells and negative control cells were inoculated in six-well plates with three holes in each group. Experimental treatment was carried out when the cells were in the logarithmic growth phase. The CCCP (10 mM) in the kit was added to the cell culture medium at a ratio of 1:1000; this culture medium was then added to the first well of each of the three groups for 20 minutes as a positive control. A JC-1 dye working solution and JC-1 dye buffer were prepared, and the latter was placed in an ice bath. Then, the culture medium was removed, cells were washed once with PBS, and 1 ml culture medium and 1 ml working solution were added and mixed well. This preparation was placed in a cell incubator for 20 min at 37 ℃. The supernatant was removed, cells were washed twice with buffer solution, and 2 ml cell culture medium was added before observation under a fluorescence microscope.
2.6 Cell cycle experiments
Three groups of cells were seeded in six-well plates (control group, shS100A14-1 group, and shS100A14-3 group) and cultured until the cells were in the logarithmic growth phase. Cells were rinsed with precooled PBS and fixed with fixing buffer at 4 ℃ for 25 min. Then, they were slowly dripped into 5 ml 75% cold ethanol, maintained at − 20 ℃ for at least 2 h, and washed once with PBS or staining buffer. Finally, the cells were resuscitated with 500 µl PI/RNase staining buffer, incubated at room temperature for 15 min, and subjected to flow analysis within 1 h.
2.7 In vitro invasion and migration assays
For the scratch wound-healing motility assay, cells were grown to confluence in six-well culture plates and ‘scratch’ wounds were created using 1 ml pipette tips. The cells were then incubated for 24 h and washed twice with PBS. The ratio of the size of the gap after 24 h to the size of the gap at the start of the experiment was taken as the relative migration. Data representative of three independent experiments are presented.
For the transwell invasion assay, cells were seeded onto the upper chambers of transwell inserts precoated with Matrigel (2.5 mg/mL; BD Biosciences, San Jose, USA 354230) in RPMI-1640 containing 2% FBS (1.0 × 105 cells/well), and 600 µl of RPMI-1640 supplemented with 10% FBS was added to the lower chambers. After 48 h, the membranes were swabbed clean. Invading cells were stained with crystal violet, and the numbers of cells in three or four fields on each filter were counted under a light microscope. Data representative of three independent experiments are presented.
2.8 Flow cytometry apoptosis analysis
A 100 µl cell suspension containing 1 × 105 cells was prepared. Then, 5 µl annexin V-FITC and 10 µl propidium iodide (20 µg/ml) were added, and cells were incubated for 15 min in the dark at room temperature. The percentage of apoptotic cells was analyzed using a FACS Caliber flow cytometer (BD Biosciences, USA). Each experiment was repeated three times.
2.9 Western blotting experiments
Western blotting was used to quantitatively evaluate protein expression. Total protein was extracted with Radio Immunoprecipitation Assay (RIPA) lysis buffer. Nuclear and cytoplasmic proteins were extracted using the corresponding extraction reagents according to the manufacturer’s instructions. The amounts of protein measured by the enzyme-labeling instrument were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. After sealing, the imprinted proteins were incubated with the designated primary antibody (in which S100A14 had been incubated at 4 ℃ for 36 h). After washing and incubation with secondary antibodies, the Odyssey system was used to visualize the proteins. Primary antibodies against S100A14, GAPDH, β-actin,, B cell lymphoma (Bcl)2, Bcl2-associated X protein (Bax), B-cell lymphoma-extra large (Bcl-xl), Bcl2-associated agonist of cell death (Bad), poly(ADP-ribose) polymerase (PARP), caspase-3 and caspase-9 were from American Cell Signaling.
2.10 Database analysis
The Cancer Genome Atlas (TCGA) data for head and neck squamous cell carcinoma (HNSC) were collected using the GEPIA and UALCAN tools to analyze the relationship between S100A14 expression and tumor grade. The relationship between S100A14 expression and metastasis-free survival of patients with HNSC was analyzed using the PROGgeneV2 online database.
2.11 Statistical methods
GraphPad Prism 5 (La Jolla, CA, USA) and SPSS 20.0 were used for statistical analyses. The results are presented as the mean ± SD (standard deviation) of three independent experiments. The independent samples t-test was used to compare control and S100A14-knockdown or S100A14-overexpressing cells. Analysis of variance (ANOVA) was used in the CCK-8 experiment, and single-factor ANOVA was used to compare protein expression in multiple groups. Other experiments used the t-test method. P < 0.05 was defined as significant.