Clinical specimens
A total of 24 patients (11 male and 13 female; 14-51 years old) histologically diagnosed as OS (14 distal femur and 10 proximal tibia) were screened from our hospital between January 2016 and January 2018. Paraffin-embedded OS tissues, and adjacent normal tissues were collected form these patients prior to administering any treatment. This study was approved by the local Institutional Review Board, and informed consents were obtained from all subjects.
Immunohistochemistry (IHC)
Paraffin-embedded tissues were sliced at 5 μm, dewaxed in xylene, dehydrated with graded ethanol, incubated in 0.3% H2O2 for 15 min, and incubated in 10 mM ethylene diamine tetraacetic acid (EDTA) for 15 min under microwave irradiation. The sections were blocked with 10% bovine serum albumi (BSA) for 30 min, and incubated with primary antibody (anti-ERβ, 1:100, Cell Signaling, USA) for 3 h at 37°C. Then the sections were washed with PBS for 5 times, and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1: 1000, Cell Signaling) for 1h at 37°C. Followed by staining with diaminobenzidine (DAB), dehydration with graded ethanol and vitrification with dimethylbenzene, positive stained cells (yellow-brown or brown) were observed under microscope (Olympus, Japan), and counted in five randomly selected fields.
Cell culture and treatments
Human OS cell line U2-OS and human osteoblast cell line hFOB1.19 (preserved in our laboratory) were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cells were maintained in a humidified incubator containing at 37°C with 5% CO2.
U2-OS cells in logarithmic growth phase were randomly divided into four groups: si-ERβ, U2-OS cells transfected with si-ERβ (Santa Cruz Biotechnology, USA) for 48 h; NC-ERβ, U2-OS cells transfected with siRNA-negative control-ERβ for 48 h; si-ERβ + FH535, U2-OS cells transfected with siRNA-ERβ and treated with 20 μmol/L FH535 (an inhibitor of Wnt signaling) (Sigma, USA) for 48 h; Blank, U2-OS cells without transfection and treatment. Cell transfection was performed by using lipfectamine 2000 (Thermo Fisher Scientific, USA) according to the manufacturer's instruction.
Quantitative real-time PCR (qRT-PCR)
Total RNAs were extracted from specific tissues, and cells by using RNApreppure tissue kit (TIANGEN, China), and RNApreppure cell kit (TIANGEN), respectively. cDNA was synthesized by using PrimeScript RT reagent kit (Takara, China). The special primers were used as followed: ERβ-F, 5′-GCCGCCCCATGTGCTGAT-3′; ERβ-R, 5′-GGACCCCGTGATGGAGGACTT-3′; β-catenin-F, 5′-TGAGGACAAGCCACAAGATTAC-3′; β-catenin-R, 5′- TCCACCAGAGTGAAAAGAACG-3′. GAPDH was used as an internal control (GAPDH-F, 5'-GAGTCAACGGATTTGGTCGT-3'; GAPDH-R: 5'- TTGATTTTGGAGGGATCTC-3'). The PCR program included 95°C for 10 min, 40 cycles of 95°C for 10 s, 60°C for 20s, and 72°C for 34s. The relative expression levels of target genes were calculated using the 2-ΔΔCt method 17.
Western blot
Cells of different groups were lysed in RIPA Lysis buffer (Thermo Fisher Scientific). Total proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidenefluoride (PVDF) membrane. The membrane was blocked with 5% skim milk in Tris Buffered Saline Tween (TBST) for 1 h, and incubated with primary antibody (anti-ERβ, -MMP-7, -MMP-9, and -β-catenin; 1: 100, Cell Signaling) overnight at 4°C. After washed with TBST for three times, the membrane was incubated with HRP-conjugated secondary antibody (1:1000, Abcam, England) for 2 h at 25°C. The protein bands were visualized and quantified using a Gel Imaging System (Thermo Fisher Scientific).
Immunofluorescence
Cells of different groups were fixed in 4% paraformaldehyde for 20 min at 4°C, and permeated in 0.1% Triton X-100 (MP Biomedicals, USA) for 5 min. Then cells were blocked with 5% BSA for 30 min, and incubated with primary antibody (anti-β-catenin, 1:100, Abcam) overnight at 4°C. After washed with PBS for 5 times, cells were incubated with Alexa Fluor 488-conjugated secondary antibody (1:500, Abcam) for 1h at 37°C. Followed by staining with DAPI (4,6-diamino-2-phenylindole), positive stained cells (green fluorescence) were observed under fluorescence microscope (Olympus).
Scratch assay
Cells of different groups were seeded at a density of 0.5×106/well in 6-well plates, and cultured overnight (more than 90% confluence). A wound track at approximately 5 mm was scored in each dish with a pipette head, and the debris was removed by 3 times of washing with phosphate buffer saline (PBS). After 48h of culturing, the scratch healing state was observed under microscope (Olympus).
Transwell assay
Transwell assay was performed by using transwell chambers (Corning, USA). Cells of different groups were seeded at a density of 0.1×105/µL in the upper chamber (pre-coated with Matrigel). A total of 600 µL DMEM containing 100 ng/mL stromal cell-derived factor-1 (SDF-1) were placed in the lower chamber. After incubated at 37°C for 24 h, cells on the upper chamber were removed with cotton swabs. Cells on the lower chamber were fixed in formaldehyde for 30min and stained with 0.1% crystal violet for 20min. Positive stained cells were observed under microscope (Olympus).
Establishment of subcutaneous tumor-bearing model and orthotopic transplantation model in mice
Four-week-old specific pathogen free (SPF) mice (male, 20-25g) were purchased from the Medical College of Shanghai Jiaotong University (Shanghai, China). Mice were feeding at 25°C and 50% humidity with free access to water and food. A total of 100μl U2-OS cells in different groups (si-ERβ, NC-ERβ, si-ERβ + FH535, and Blank) were subcutaneously injected into the posterior limb of each mouse at a density of 0.1×108 cells/ml (subcutaneous tumor-bearing model). Mice were killed by cervical dislocation, and the tumor volumes were measured by vernier caliper every 5 days. After the injection for 20 days, small pieces of tumor tissues were transplanted into the liver of healthy mice (orthotopic transplantation model). Five weeks later, the metastatic tumors in liver tissues were counted, and observed by Hematoxylin-Eosin (HE) staining. All animal experiments were approved by the local Institutional Review Board.
HE staining
The liver tissues of mice were fixed in 10% formaldehyde, and embedded in paraffin. The tissue sections at 5μm were dewaxed in xylene, rehydrated in graded ethanol, and stained with Hematoxylin for 5 min and Eosin (Beyotime) for 2 min. After dehydrated with graded ethanol and vitrificated with dimethylbenzene, the tissues were observed under microscope (Olympus).
Statistical analyses
All experiments were performed in triplicate, and all data were expressed as mean ± Standard Deviation (SD). Statistical analysis was performed by SPSS version 17.0 (SPSS Inc., Chicago, IL). Comparison between different groups was determined by Student’s t test (two groups) and one-way ANOVA (more than two groups). A p-value less than 0.05 was considered to be significantly different.