The human study was approved by the Institutional Ethical Board of the First Affiliated Hospital of Zhengzhou University, Zhengzhou Central Affiliated Hospital to Zhengzhou University, the Affiliated Cancer Hospital of Zhengzhou University. All patients with OS prior to enrollment into the study signed written informed consents on enrolment. All of recruited patients were diagnosed between 2008-2013 were totally enrolled. Surgical removed samples included adjacent benign tissue and primary tumor were fixed in 10% formalin then embedded with paraffin for immunohistochemistry analyses.
Construction of tissue microarray (TMA)
We combined all OS samples from the above mentioned three hospital to create a relative large-scale cohort with more OS samples[16, 17]. The osteosarcoma TMA used in present study was constructed with 120 osteosarcoma specimens and 65 normal specimens.
Cell lines and cell culture
MG63, U2OS, and 143B were purchased from the American Type Culture Collection (Rockville, USA). Normal osteoblast cells HOBC and HFOB was purchased from Shanghai Institute for Biological Science (Shanghai, China). All cell lines were maintained at 37℃ in a humidified incubator (Thermo Fisher Scientific, USA) containing 5% CO2.
Immunohistochemical (IHC) and in situ hybridization (ISH)
IHC was performed as previous description. ISH was performed through digoxygenin (DIG)-labeled circ-ANKS1B ISH probes as described previously. circ-ANKS1B expression in OS tumor tissues and adjacent non-tumor tissues was detected by fluorescence. The intensities and proportion of circ-ANKS1B dyeing was calculated as following method: 1-3 scores were the low expression, while 3-5 scores were represented high expression.
Western blotting was performed as previous description . And the primary antibodies were as following: Ki-67 (Proteintech, 27309-1-AP), GAPDH (Proteintech, 10494-1-AP).
Quantitative real- time PCR
Total RNA was obtained via the method of Trizol (Life Technologies, USA) extraction. And the target cDNA was synthesized through the PrimeScript RT reagent Kit (Promega, USA). Quantitative real-time PCR reactions were performed using SYBR Green kit (ABI, USA). The relative gene expression was normalized to control using 2-ΔΔCt method. The primers used in this study were listed in Supplemental Table 1.
MG63 or U2OS cells (1 × 106 cells/well) were seeded into a 6-well plate. 24 h later, cells were transiently transfected with the miR-149 mimics or negative control (NC), shRNA targeting circ-ANKS1B or negative control through lipofectamine 3000 (Invitrogen, USA). The RNAi sequences used in this study were listed in Supplemental Table 2.
CCK-8, Cell invasion and Colony formation assay
CCK-8, cell migration and invasion assays were performed according to previously described method.
Dual luciferase reporter gene assay
PmirGLO, pmirGLO-circ-ANKS1B or pmirGLO-circ-ANKS1B-mut vectors, pmirGLO-Ki-67 or pmirGLO-Ki-67-mut vectors were cotransfected with miR-149 by Lipofectamine™ 2000 Transfection Reagent (Invitrogen); 24 hours later, the luciferase intensity was measured and normalized to the renilla luciferase intensity using the Dual-Luciferase® Reporter Assay System (Promega).
Male nude mice were ready for subcutaneously injection. U2OS cells stably transfected with sh-circ-ANKS1B or ctrl (5 × 106 cells) were inoculated subcutaneously into the flanks of the nude mice. Tumor size and volumes were measured every week. 4 weeks later, all the mice were sacrificed and the tumor weights were measured. All animal experiments were approved by the Animal Care Committee of the First Affiliated Hospital of Zhengzhou University. All experimental procedures involving animals were strictly followed in accordance with the Guide for the Care and Use of Laboratory Animals as described previously.
All of the statistical analyses were performed using GraphPad Prism software. Two-tailed Student’s t-tests were used to compare mean values of two groups. Two-way ANOVA was conducted to compare mean values of more than two groups. Pearson χ2 tests were adopted to analyze the association of circ-ANKS1B expression with clinicopathological variables. For the survival analysis, Kaplan-Meier plots and log-rank tests were used. Two-tailed Fisher’s exact test was performed as appropriate. p value < 0.05 was considered as statistically significant difference.