Patients and specimens
The clinical specimens were collected from glioma patients at the First Affiliated Hospital of China Medical University. Normal brain tissues were obtained from patients suffering from cerebral injury who were underwent internal decompression. Thyroid tissue from patients with thyroiditis was used as a positive control group because the protein expression of S100A12 had been well reported in thyroiditis. The patients were written consent and with the approval of the ethics committee of the China Medical University. Glioma samples were immediately fresh frozen in liquid nitrogen and then stored at -80℃ until further analysis.
A172, U373, U118, U251 and U87 were obtained from Chinese Academy of Sciences (Shanghai, China). The cells were cultured with Modifed Eagle’s Medium (DMEM), supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin, 100 mg/ml streptomycin). The cell lines were incubated in 37℃, 5% CO2 saturation.
The glioma specimens and normal brain tissues were embedded in paraffin and then sliced to 4μm sections. Rabbit polyclonal anti-S100A12 (1:200; Abcam, Cambridge, UK) and biotinylated goat anti-rabbit immunoglobulin G were used as primary and secondary antibodies. Then counterstaining with diaminobenzidine, sections were inspected under microscope.
The shRNA vectors were purchased from GeneChem Company (Shanghai,China).The S100A12#1 sequence was 5'-CGACTTTCAAGAATTCATA-3',the S100A12#2 sequence was 5'- GGATGCTAATCAAGATGAA -3' and the shRNA control (shNC) sequence was 5'- TTCTCCGAACGTGTCACGT -3'.
Cell proliferation analyses
The cell viability was determined by MTT assay. Cells were seeded on 96-well plates at 1 d, 2 d and 3d after transfection, then incubated in the medium containing 100 mg/0.1 ml of MTT (Sigma Aldrich) for 4 h and incubated at 37℃. After removing the medium, the blue crystal layer attached to the surface of the material was dissolved using dimethyl sulfoxide (DMSO). The OD value was measured at 490 nm by enzyme immunoassay instrument.
Clony formation assay
Cells were seeded into 6 well plates at a density of 500 cells/well and cultured at 37℃,5% CO2 for 14 days. Cells were then fixed in 4% paraformaldehyde and stained with crystal violet solution. Colonies from 3 independent groups were counted and the data were presented as mean ± standard deviation (SD).
Cell invasion and migration assays
Transwell assay was used to evaluate the cell migration and invasion of glioma cell lines. For migration assay, 5×104 cells were seeded in the upper chamber with serum-free culture medium (200μl), and the lower chamber was filled with 10% FBS medium. After culturing for 24 h, the cells were fixed with 4% paraformaldehyde and stained with gemsa for 15min. The images were acquired under microscope and migrated cells were counted in 3 random fields. The method of invasion assay was similar to the migration analysis, while the upper chamber was coated with matrigel (BD Biosciences, San Jose, CA).
Flow cytometry assay
After 72 hours of transfection, cells were collected and subjected to ﬂow cytometry. Cell apoptosis was quantifed using Annexin V-ﬂuorescein isothiocyanate apoptosis detection kit I (BD Biosciences, San Jose, CA, USA). Cell apoptosis analysis was performed using a Flow Cytometry System (BD Bioscience, Bedford, MA, USA).
Western blot analysis
Proteins from glioma cells were homogenized in cold PBS containing 0.05% Triton X-100 and protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Protein samples were electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gels (Sigma), transferred onto PVDF membranes according to standard protocols, and then blocked with 5% dried skimmed milk in TBST for at least 1 h. The membranes were incubated overnight at 4°C with the following antibodies: anti-S100A12, anti-E-cadherin, anti-N-cadherin (1:1000, Abcam, Cambridge, MA, USA), anti-cleaved caspase 3, anti-Bcl2, anti-Bax (1:1000, Cell Signaling Technologies), and anti-GAPDH (1:1000, Abcam), and then incubated with horseradish peroxidase-conjugated secondary antibodies (1:1000, Abcam) at room temperature for 1 h after washing 3 times using TBST. Blots were washed 3 times again and developed using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech). Immunoblot band quantification was calculated by means of a Bio-Rad calibrated densitometer (GS-800) using the vendor’s software (Bio-Rad Laboratories); GAPDH was used as an internal reference for analyses.
Statistical analyses were performed using the SPSS version 17.0 and GraphPad Prism version 5.0. The comparisons among multiple groups were conducted by the one-way analysis of variance (ANOVA). Pearson correlation analysis was applied for correlation analysis. Kaplan Meier analysis was used to construct the survival curves of the high expression group and the low expression group, and log rank test was used to compare the survival differences between the groups. A value of P <0.05 indicated that the difference was statistically significant, and a value of P < 0.01 showed that the statistics were of highly significant difference.