A total of 36 primary osteosarcoma tissue samples and adjacent non-cancerous bone tissue samples were obtained from the Department of Orthopedics, Affiliated Hospital of Weifang Medical University (Weifang, China) between January 2009 and February 2013. The patients included 23 males and 13 females, with a median age of 35.8 years and an age range of 10 to 56 years old. The histological grade was assessed according to the tumor-node-metastasis (TNM) system (10). None of the patients received any treatment prior to surgery. The removed fresh tissues were immediately placed in liquid nitrogen and then stored at −80°C for RNA analysis. The clinicopathological data are summarized in Table I. Written informed consent was obtained from all patients and the study was approved by the Ethics Committee of Affiliated Hospital of Weifang Medical University.

Two human osteosarcoma cell lines (MG-63 and U2OS) and a normal human osteoblastic cell line hFOB were obtained from Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The hFOB cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The human OS cells were cultured in RPMI-1640 medium supplemented with 10% FBS (both Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 mg/ml streptomycin (Thermo Fisher Scientific, Inc.). Cells were incubated at 37°C with 5% CO₂ in a humidified chamber.

siRNA targeting has-circ_001569 (5′-GCATCGTGCAGGACTGGAA-3′) and si-negative control (NC; 5′-UUCUCCGAACGUGUCACGUTT-3′) was constructed and purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). Cells were transiently transfected with 100 nmol/l oligoribonucleotides using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Human hsa_circ_001569 cDNA was synthesized and cloned into pLVX-IRESneo by GeneCopoeia, Inc. (Rockville, MD, USA). Cells were transfected with the oligonucleotides at room temperature at a final concentration of 20 nmol/l using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Cells were harvested following cell transfection at 48 h.

Total RNA was extracted from tissues and cells using TRIzol reagent (Thermo Fisher Scientific, Inc.). Reverse Transcription kit (Guangzhou RiboBio Co., Ltd.) was used to reverse RNA to cDNA, according to the manufacturer's protocols. RT-qPCR was performed using a SYBR Premix Ex TaqTM II kit (Takara Biotechnology Co., Ltd., Dalian, China) on an ABI7500 system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 10 sec, 57°C for 20 sec and 72°C for 15 sec. The mRNA fold-change of circ_001569 was calculated by the 2^−ΔΔCq method (11) and was normalized to GAPDH mRNA expression. The primer sequences were as follows: circ_001569 forward, 5′-TCCCCTGAACATTCTCCCCAT-3′ and reverse, 5′-GAAAGCACTTGGTGAAGTCGG-3′; and GAPDH forward, 5′-CACCGTAGCCTTCCGAGTA-3′ and reverse, 5′-GCCCTTGATGAGCTGTTGA-3′.

Cell proliferation capacity was evaluated using the Cell Counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) according to the manufacturer's protocol. Transfected cells were seeded onto 96-well plates (2,000 cells/well) and were incubated overnight at 37°C with 5% CO₂ in a humidified chamber. The proliferation rate of osteosarcoma cells was detected every 24 h until 72 h. Each well was supplemented with 10 µl CCK-8 regent and cells were incubated for 2 h. Next, cell proliferation was measured at 450 nm using a Multiskan MK3 spectrophotometer.

The transfected osteosarcoma cells were plated at 500 cells/well onto 6-well plates. After cells were cultured at 37°C with 5% CO₂ in a humidified chamber for two weeks, the cell colonies were fixed with 4% paraformaldehyde at room temperature for 20 min and stained with 0.1% crystal violet at room temperature for 20 min, and the images of cell colonies were captured and cell colonies were calculated.

Total protein was extracted using lysis buffer (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). A BCA kit (Tiangen Biotech Co., Ltd., Beijing, China) was used to detect the concentration of the proteins. Equal protein (30 µg) was separated on 8–12% SDS-PAGE gel and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Following blocking with 5% skimmed milk at room temperature for 1 h, the membrane was incubated with antibodies including p-GSK-3β (cat. no. 9336; 1:1,000), GSK-3β (cat. no. 9315; 1:2,000), β-catenin (cat. no. 9562; 1:1,000) and GAPDH (cat. no. 5174; 1:1,000; all Cell Signaling Technology, Inc., Danvers, MA, USA) overnight at 4°C and then incubated with horseradish peroxidase-conjugated goat anti-rabbit antibody (cat. no. 10285-1-AP; 1:1,000; ProteinTech Groups, Inc., Chicago, IL, USA) at room temperature for 1 h. The membrane was detected with an enhanced chemiluminescence detection system (Nanjing KeyGen Biotech Co., Ltd.). GAPDH was used as an internal control.

The cells were transfected with si-NC and si-circ_001569 or pLVX-IRESneo-vector (vector) and pLVX-IRESneo-circ_001569 (circ_001569). Cell viability was detected using CCK-8, according to the manufacturer's protocol. The IC50 values were defined as the chemotherapeutic agent concentration producing 50% inhibition of cell proliferation. Absorbance was detected at 450 nm.

All data in the study are presented as the mean ± standard deviation and were analyzed using SPSS 19.0 software package (IBM Corp., Armonk, NY, USA). Student's t-test was applied to compare the differences between two groups. For comparisons among 3 or more groups, one-way analysis of variance, followed by the Student-Newman-Keuls post hoc test, was used. P<0.05 was considered to indicate a statistically significant difference.

To investigate the clinical significance of circ_001569 expression in osteosarcoma, the present study examined the expression of circ_001569 by RT-qPCR in 36 primary osteosarcoma tissues and adjacent non-cancerous bone tissues. As demonstrated in Fig. 1A, circ_001569 expression was significantly elevated in osteosarcoma tissues compared with adjacent non-cancerous bone tissues (P<0.05). Next, the association between circ_001569 expression and clinicopathological factors was analyzed. The results of the present study demonstrated that circ_001569 expression was associated with distant metastasis (Fig. 1B; Table I, P<0.05) and TNM stage (Fig. 1C; Table I, P<0.05), but was not associated with any others factors in osteosarcoma patients (Table I, P>0.05). In addition, the expression of circ_001569 was detected in two human osteosarcoma cell lines (U2OS and MG-63) and an osteoblast cell line (hFOB) by RT-qPCR analyses. The results revealed that expression of circ_001569 was higher in osteosarcoma cell lines than that in hFOB cell lines (Fig. 1D). These results indicated that circ_001569 expression was upregulated in osteosarcoma.

To evaluate the effects of circ_001569 on cell proliferation, a loss-function assay was performed in U2OS cells and a gain-function assay was performed in MG-63 cells according to their expression of circ_001569 (Fig. 2A and B). CCK-8 assay results revealed that the cell proliferation capacity was significantly inhibited following circ_001569 knockdown compared with the negative control group in U2OS cells, but was notably enhanced following circ_001569 overexpression in MG-63 cells, compared with the control group (Fig. 2C and D). Cell colony formation assay revealed that the number of colonies formed in the circ_001569 knockdown group was smaller than that in the negative control group in U2OS cells, but was more when circ_001569 was overexpressed in MG-63 cells (Fig. 2E and F). Therefore, these results indicated that upregulation of circ_001569 could enhance cell proliferation in vitro.

Combination of cisplatin with doxorubicin and methotrexate is frequently used for osteosarcoma treatment (12,13). Furthermore, the present study evaluated whether circ_001569 expression affected conventional chemotherapy of osteosarcoma. The results indicated that inhibition of circ_001569 activity in U2OS cells resulted in a significant decrease in the calculated IC50 value for cisplatin, doxorubicin or methotrexate, compared with the control group (Fig. 3A). However, overexpression of circ_001569 activity in MG-63 cells enhanced the IC 50 value for cisplatin, doxorubicin or methotrexate (Fig. 3B). These results indicated that upregulation of circ_001569 promoted osteosarcoma cell resistance towards chemotherapy.

Activation of Wnt/β-catenin signaling is associated with chemotherapy resistance in osteosarcoma (14). Furthermore, western blot analysis revealed that circ_001569 knockdown in U2OS cells decreased the expression levels of p-GSK-3β and β-catenin, but increased GSK-3β expression levels (Fig. 3C). However, circ_001569 overexpression increased the expression levels of p-GSK-3β and β-catenin in MG-63 cells, but decreased GSK-3β expression levels (Fig. 3D). These results indicated that circ_001569 overexpression activated the Wnt/β-catenin signaling pathway.

Next, the present study demonstrated whether circ_001569 overexpression enhanced the resistance of osteosarcoma to cisplatin, doxorubicin or methotrexate by activating the Wnt/β-catenin signaling pathway. As demonstrated in Fig. 4A and B, the Wnt/β-catenin inhibitor, XAV939, or Wnt/β-catenin agonist, LiCl, could reduce or increase the IC50 value for cisplatin, doxorubicin or methotrexate. It was further demonstrated that circ_001569 knockdown resulted in a decreased IC50 value for cisplatin, doxorubicin or methotrexate, compared with the negative control. However, this chemotherapy sensitivity was only rescued by treatment with the Wnt/β-catenin agonist, LiCl in the cisplatin-treated group, compared with the control group (Fig. 4C). Additionally, it was demonstrated that circ_001569 overexpression exhibited an increased IC50 value for cisplatin, doxorubicin or methotrexate, compared with the negative control. However, this chemotherapy resistance was also rescued by treatment with the Wnt/β-catenin agonist, LiCl, in the cisplatin-treated group, compared with the control group (Fig. 4D). Therefore, our aforementioned results indicated that circ_001569 could enhance cisplatin resistance of OS cells via activating the Wnt/β-catenin pathway.