Clinical osteosarcoma samples
Clinical osteosarcoma samples were acquired from patients who received partial or radical tumorectomy in the Department of orthopedics, Putuo people`s Hospital, Tongji University, ShangHai, China.50 osteosarcoma samples were collected, and they all were paired with adjacent normal tissues. Fresh osteosarcoma tissue samples (n = 50) were obtained from 28 patients with primary osteosarcoma without metastases and 22 patients with metastases. Of 28 patients with primary osteosarcoma, 19 were recurrence-free and 9 were recurrent patients.Part of the matched tissues were frozen in liquid nitrogen or -80℃ freezer for RNA extraction or Western Blot.The remaining tissues were fixed in 10% formalin at room temperature for 24 hours,and then embedded in paraffin for immunohistochemistry.These patients had not received preoperative assisted anti-cancer treatment. This study and experimental procedures were approved by the Human Research Ethics Committee of Tongji University (ShangHai,China), and written informed consent was obtained from all the patients involved.
Cell lines and Cell culture
Human osteosarcoma cell lines (U2OS) and the normal osteoblastic cell line, hFOB, were purchased from the Shanghai Institute for Biological Sciences, Chinese Academy of Sciences.All cells were cultured with Eagle's Minimum Essential Medium (cat.no. 30-2003; ATCC) containing 10% heat-inactivated fetal bovine serum in an incubator (37˚C, 5% CO2).
Immunohistochemistry (IHC) and evaluation of the results
The collected OS tissues and paired adjacent normal tissues were fixed in formalin and then embedded in paraffin.For IHC, paraffin-embedded sections were dewaxed in xylene and rehydrated in ethanol. Subsequently, after deparaffinization,rehydration,blocking with hydrogen peroxide and antigen retrieval, these sections were incubated with primary rabbit anti-BTAF1 polyclonal antibodies (A12251,ABclonal; Wuhan, China) overnight at 4℃. After washing three times with PBS, they were incubated with HRP-conjugated anti-rabbit secondary antibodies for 2 hours at room temperature. DAB was used for c5oloration, and dark brown was regarded as positive. Appropriate positive and negative controls were included in each run, and the absence of a primary antibody was used as a negative control. Two pathologists, blinded to clinical information, independently assessed BTAF1 staining under an Olympus CX31 microscope (Olympus, Center Valley, PA). The positive rate of tumor cells ranged from 0 to 100%. Staining intensity was defined as: negative, 0; weak, 1; mild, 2; and intense, 3. Finally, each case was given a weighted score (% × staining intensity) on a scale of 0 (0 × 0) to 3 (100% × 3).
Transient transfection assays
The BTAF1-short hairpin RNA (shRNA1, shRNA2) plasmids was constructed and purchased in Shandong Vigene Biosciences Company, China. Cell lines U2OS were seeded in 6-well plates at 50–70% confluence when being transfected.3µg BTAF1-short hairpin RNA (shRNA) plasmids per well was added to the cells using Lipofectamine™ 2000 reagents (Thermo Fisher Scientific; Waltham,USA).24–48 hours after transfection, the cells were collected for efficiency determination and subsequent assays. All steps are carried out in accordance with the manufacturer's instructions.
Cell proliferation assays
the BTAF1-short hairpin RNA (shRNA) plasmids were purchased from GECEM biotechnology, Shanghai, China. 1 × 105 U2OS cells were inoculated in six-well plates and transfected with 5 ng BTAF1-shRNA1, BTAF1-shRNA2 and vector plasmid per well using Lipofectamine™ 2000 reagent (Invitrogen, Karlsruhe,Germany) according to the manufacturer’s instructions.48 h after transfection, U2OS cells were added to 5 96-well plates at a density of 2000 cells per well, and cell viability was measured at 0, 24, 48, 72 and 96 h, respectively. Cell proliferation rate was measured by using cell count Kit − 8 (CCK-8) according to the manufacturer's protocol. Specifically, 10 µ L CCK-8 solution and 100 µ L of the above cell culture medium were added to each well, and the optical density of each well was measured at 450 nm after 4 h.Each experiment was repeated three times independently.
Quantitative real-time-polymerase chain reaction (qRT-PCR)
According to the manufacturer’s agreement, use TRizol reagent (Invitrogen, Carlsbad, CA) to extract total RNA from OS tissues and cell lines. 1µg enriched tissue or cell RNA was used to synthesize cDNA through reverse transcription,which was accomplished by TaqMan® Reverse Transcription Reagents(Applied Biosystems Inc.; Thermo Fisher Scientifc, Inc.). The procedure was based on the protocol provided by Invitrogen. The Hieff™ qPCR SYBR Green Master Mix (Thermo; Massachusetts, USA) was used for qPCR analysis by ABI ViiA7 qPCR System (Applied Biosystems; Foster, CA).specific primers were purchased from RiboBio (Guangzhou, China), they were listed as follows: Forward primer 5`-CGCTCAGCTCTCTGGAAACT-3`,and reverse primer 5`-AAGGCGATCTAGCCTGGAGA-3`.
Western blotting
Whole-cell lysates (P0013B, Beyotime, Shanghai, China) were collected and prepared from BTAF1-shRNA or empty vector transfected cells All the proteins of the processed tissues or cells were lysed on ice in RIPA protein lysis buffer with 1 mM PMSF (phenylmethylsulfonyl fluoride). then the impurities were removed after centrifugation at 15,000 rpm at 4°C. Subsequently, the protein concentration of the solution was measured with PierceTM BCA protein assay kit (Thermo; Massachusetts, USA). The calculated 30µg protein per well was separated in a 10% SDS-PAGE gel and transferred to a polyvinylidene fluoride (PVDF) membrane at 100 V for 1–2 hours. The PVDF membranes were washed thrice for 5 min each, and then blocked in PBST for 2 hours at room temperature. The blocked membranes were washed,and cut into different strips according to the molecular weight of target proteins, then incubated with primary antibodies against target proteins overnight at 4℃. The next day, the membranes were incubated with secondary antibodies (1:1000,Invitrogen) for 1 hour at room temperature.Blots were developed using the enhanced chemiluminescence method (Pierce™ ECL Western Blotting Substrate;Thermo Fisher Scientifc, Inc.).The primary antibodies used included anti-BTAF1,anti-E-cadherin, anti-Vimentin, anti-α-actin,anti-N-cadherin and anti-Snail1.
Tumor xenografts in nude mice
BALB/C-nu/nu nude mice were purchased from the National Resource Center for Rodent Laboratory Animal of China.Each group included 5 nude mice, each of which was inoculated subcutaneously with 5.0x106 cells(BTAF1 knockdown and control tumor cells).Mice were kept in sterile animal facilities and their tumor growth was monitored daily.The tumor volume was measured weekly with vernier calipers and calculated according to the formula, V = length*width2*0.25.2 months later the mice were sacrificed, tumors were dissected for histological examination. All data are expressed as mean ± SE. The animal experiments were approved by the Institutional Animal Care and Use Committee of Tongji University and met all regulatory guidelines.
Statistical analysis
Significant differences between the experimental groups and controls were assessed using Student's t test. Survival information was assessed by Kaplan-Meier analysis, and differences were assessed using log-rank tests. χ2 tests were used to assess the significant correlation between BTAF1 expression and clinicopathological parameters of OS patients.Survival curve analysis was used to evaluate the diagnostic value of BTAF1.A Cox proportional hazard regression model was used for univariate and multivariate analysis. All values are reported as means ± the standard deviation (SD), P < 0.05 was statistically significant. The asterisks indicate that the data are significantly different from the controls, P < 0.05, *; p < 0.01, **; p < 0.001, ***; p < 0.0001, ****.GraphPad Prism 5.0 and SPSS 18.0 software were used for all statistical analysis.