Patients
A total of 59 formalin-fixed paraffin-embedded parotid carcinoma tissues from 2010 to 2015 were obtained from Department of Otorhinolaryngology of our institute. Besides, 15 cases of pleomorphic adenoma were also collected as control. Written patient consent was obtained and the study was approved by the Ethics Committee of our institute. All the samples had been rechecked for the confirmed pathological diagnosis by pathologists.
Immunohistochemistry
The paraffin-embedded SACC tissues were cut into 4mm-thick sections, dewaxed in xylene, rehydrated and graded in ethanol solutions. Antigen was retrieved by heating the tissue sections at 100ºC for 30 min in EDTA (1 mmol/L, pH = 9.0) solution, and the sections were later immersed in a 0.3% hydrogen peroxide solution for 30 min to block endogenous peroxidase activity. After rinsing with phosphate buffered saline (PBS) for 5 min, the tissue section was blocked with 3% BSA at room temperature for 30 min, and then incubated with rabbit anti-human NSD2 antibody (1: 200 dilution) at 4 ºC overnight. Then, the tissue section was incubated with HRP-labeled goat anti mouse secondary antibody, followed by incubation with diaminobenzene as the chromogen, and hematoxylin as the nuclear counterstain. After that, the sections were dehydrated, cleared and mounted and further photographed under a microscope.
Two pathologists with no knowledge of the patients’ information examined the stained sections independently. At high magnification, 10 representative views are selected. Then strained tumor cells were counted. Extremely strong positive, strong positive, positive and negative were considered when the number of strained tumor cells were no less than 75%, no less than 50%, no less than 10% and less than 10%, respectively. Results from the two pathologists were averaged and used for statistical analysis.
Cell culture
Human salivary adenoid cystic carcinoma cell lines SACC-2 were obtained from Chinese Academy of Sciences, Shanghai Institutes for Biological Sciences. The cells were cultured in DMEM supplemented with 10% FBS and incubated at standard culture conditions (5% CO2, 37ºC).
siRNA transfection in SACC-2 cells
For transfection, SACC-2 were seeded at each 1.5×105 cells/well in a 6-well dish and were transfected with 100 ng/L siRNA against NSD2 using Lipofectamine RNAi-MAX (Life Technologies, Carlsbad, USA) in accordance with the manufacturer′s instructions. NSD2 siRNA sequences were designed after selecting appropriate DNA targets as follows: NSD2: 5′-CATACATGAATGGGAAGCCTCTCTT-3′; NSD2 siRNA-2: 5′-CGACATAGGGAAGAGTACTCCTCAA-3′.
Western blot analysis
Proteins were prepared as described previously. Briefly, 1×107 cells were homogenized briefly in 200µl of lysis buffer containing (in mM) 20 Tris-HCl (pH = 7.4), 150 NaCl, 2.5 EDTA, 50 NaF, 0.1 Na4P2O7, 1 Na3VO4, 1 PMSF, 1 DTT, 0.02% (v/v) protease cocktail (Sigma-Aldrich, Missouri, USA), 1% (v/v) Triton X-100 and 10% (v/v) glycerol. The homogenates were centrifuged twice at 20,000 g at 4°C for 15 min, and the supernatants were saved as total proteins. Protein concentrations were determined by the BCA method. Equal amounts of proteins were separated by SDS-PAGE and transferred to a PVDF membrane (Bio-Rad, California, USA). Western blot analysis was performed under standard conditions with specific anti-NSD2 (Ab75359, Abcam, USA) antibody and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Ab8245, Abcam, USA). The immunoreaction was visualized using an enhanced chemiluminescent detection kit (Amersham, London, UK), exposed to X-ray film, and quantified by densitometry with a video documentation system (Gel Doc 2000, Bio-Rad, California, USA).
Cell viability assay
The cell viability was monitored using the Cell Counting Kit-8 (CCK8) (Dojindo Molecular Technologies, Kumamoto, Japan) according to the manufacturer’s protocol. All of the experiments were repeated at least three times.
Colony formation assay
The cells were counted and seeded into six-well plates at adensity of 500 cells per well and cultured for 14 d until the colonies were visible. The cells were washed with phosphate-buffered saline (PBS) and fixed with paraformaldehyde for 15 min. The colonies were stained with Giemsa's solution for 15 min and washed wittap water. After air-drying, the colonies with more than 50 cells were counted.
Apoptosis assay
Cells were centrifuged at 800 g at room temperature for 10 min and then colleted and incubated in 24-well plate, with 1.0×105 cells in each well. Then NSD2 siRNA was transfected into cells and after 72h cells were collected. Cell apoptosis was detected according to the manufacturer’s protocol of Annexin V/PI Detection Kit (Life Technologies, Carlsbad, USA) using flow cytometer (Thermo Fisher Scientific, Shanghai, China). All of the experiments were repeated at least three times.
Wound-healing assay
Cells were digested and seeded in 6-well culture plates with FBS deleted medium to inhibit cell proliferation. After being cultured to 80% confluence, the cells were scratched with a 200 ml pipette tip, washed with PBS, and replaced with FBS-free medium. Wounds were photographed under a microscope after scratching at 0 and 24 h, respectively.
Transwell for cell invasion assay
The filters of Transwell chambers were coated with 30 mL of basement Matrigel at a dilution of 1:10 by serum-free medium. Cells were counted (25000) and resuspended in 200 mL serum-free medium, and then seeded in the upper chamber. The lower chamber received 600 mL of 10% FBS medium as a chemoattractant. After 24 h of incubation, cells above the filter were removed. The invaded cells at the bottom of filter were fixed in 4% paraformaldehyde and stained with 1% crystal violet. The cells were photographed and counted under an Olympus fluorescence microscope.
Statistical analysis
All the statistical evaluations were analyzed by SPSS 13.0 software (SPSS Standard version 13.0, SPSS Inc.). All data were expressed as mean ± standard error. Data were analyzed for statistical significance by using one-way ANOVA. P < 0.05 was considered statistically significant.