Human tissue samples
Sixty-five cases of PTC tissue confirmed by pathology and 30 cases of normal thyroid tissue adjacent to PTC tissue were collected from patients undergoing surgical treatment Shanghai General Hospital and Shanghai Seventh People’s Hospital, between February 2003 and February 2006. All participants did not receive any preoperative treatment. All the tissues were dissected, then immediately frozen in liquid nitrogen, and stored at − 80 degree °C for future treatment. The histological sections of samples were reviewed by two pathologiststogether to verify the diagnosis. The patients were divided to two group according to the EZH2 expression, high group means the EZH2 expression is higher than the median, and low group means the EZH2 expression is equal to or lower than median. This study was approved by the Medical Ethics Committee of Shanghai General Hospital and all the research works were carried out in accordance with the Helsinki declaration. All participants signed written informed consent before participating in this study.
Cells, cell culture
Human PTC cell line K1 was purchased from the American Type Culture Collection(ATCC Catalogue No.92030501), and PTC cell line W3 was a kind gift from Dr. Robert Gagel (MD Anderson Cancer Center, University of Texas). Human thyroid cell line (human thyroid follicular epithelial) Nthy-ori 3–1 was purchased from the European Collection of Animal Cell Cultures (ECACC Catalogue No. 90011609). No further authentications were performed by the authors, except for the exclusion of mycoplasma infection. K1 cells, W3 cells and Nthyori 3–1 cells were cultured in DMEM-Ham’s F12-MCDB 105 (2:1:1) (Invitrogen), DMEM, and RPMI-1640 (Invitrogen) medium respectively, all supplemented with 10% fetal bovine serum (FBS) (Gibco), 100 μg/mL streptomycin, and 100 U/mL penicillin.
RNA extraction and quantitative real time PCR (qPCR)
Total RNA was isolated from tissue samples and cells using the TRIzol (Invitrogen), and reverse transcribed. Followed by qPCR with Power SYBR Green PCR Master Mix (Eppendorf), each gene relative expression levels were calculated and normalized to β-actin as an endogenous control using the 2-△△CT method. All reactions were performed in triplicates.
Immunohistochemistry
Tissue specimens were fixed in 10% neutralized formalin and embedded in paraffin blocks. Sections were subjected to routine deparaffinization and rehydration. Antigen retrieval was performed by microwavingin 0.01 mol/L citrate buffer for 10 min. After inhibition of endogenous peroxidase activity for 20 min with methanol containing 3% hydrogen peroxide, sections were blocked with 2% BSA in PBS. After three PBS washes, the specimens were reacted overnight at 4 °C with EZH2 and ERα antibody (Abcam). The sections were then counterstained with hematoxylin and mounted. IHC staining was independently examined by two clinical pathologists who were unaware of the patient outcome. Interpretation and evaluation of IHC results was as described previously [16].
Western blotting
The total proteins were separated by standard SDS-PAGE. Equal amounts of protein were transferred to a polyvinylidene difluoride membrane (Millipore), immunoblotted with first antibodies against ERα (Abcam) or ERβ (Abcam), and visualized with horseradish peroxidase–conjugated secondary antibodies. The GAPDH antibody was purchased from Sigma-Aldrich, and antibodies against p38 PK kinase, phospho (p)-p38 MAPK, ERK1/2 andphospho-ERK1/2were acquired from Cell Signaling technology.
Cell proliferation assays
Cell proliferation was measured with Cell Counting Kit-8 (CCK-8) (Dojindo Laboratories). Cells were seeded in triplicate in 96-well plates at a density of 5000 cells/well. Each condition was repeated three times. All the cells were harvested at the designated times after treatment.
Cell migration assay
Cells were suspended in serum-free medium at 5 × 105 cells/mL and seeded into the upper chambers of Transwell chamber (Millipore). RPMI-1640 medium containing 10% FBS was added to the lower chamber. Cells were allowed to migrate for 12 h at 37 °C. The nonmigrating cells were gently removed from the upper surface of the membrane. The fixed and stained migrated cells, adherent to the lower surface of the membrane were photographed using an inverted light microscope and counted manually using 5 randomly selected areas. Each experiment was repeated three times.
Animals and tumor model
Female nude mice (6–8 weeks) were purchased from Shanghai Laboratory Animal Center at the Chinese Academy of Sciences. Mice were housed in a specific pathogen-free facility at the Shanghai Jiao Tong University School of Medicine. All animal procedures were approved by the Animal Welfare & Ethics Committee of Shanghai Jiao Tong University School of Medicine. Five animals were used in each group. Mice were injected subcutaneously into the left flanks with 6 × 106 K1 or W3 control or EZH2 knockdown cells suspended in PBS. Tumor volumes were estimated using the formula (length × width2)/2. Tumor were measured every 4 days.
Statistical analysis
SPSS 19.0 software was used for statistical analysis. Relationship between staining intensity and Clinicopathology was assessed using χ2-test and two-sided Fisher’ exact test. All the data are expressed as means s.e.m. for at least three separate experiments, using an independent t test to perform comparisons of two independent groups.
