Clinical samples
Forty OS tissues and adjacent normal tissues were obtained from OS patients who received surgery at Weifang People’s Hospital. This research was ratified by the Ethics Committee of Weifang People’s Hospital. All participants signed written informed consent. All tissues were immediately frozen in liquid nitrogen and then stored at -80°C.
Cell culture
Two OS cell lines (U2OS and HOS) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). DOX-resistant cell lines (U2OS/DOX and HOS/DOX) were produced by U2OS and HOS cells exposed to gradient doses of doxorubicin (DOX) (Solarbio, Beijing, China). All cells were incubated in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco).
Cell transfection
Small interfering RNA (siRNA) against circ_0002060 (si-circ_0002060#1, si-circ_0002060#2 and si-circ_0002060#3), the siRNA control (si-NC), miR-198 mimics (miR-198), the mimics control (miR-NC), circ_0002060 overexpression vector (circ_0002060), the empty vector (pCD-ciR), miR-198 inhibitor (anti-miR-198), the inhibitor control (anti-miR-NC), ABCB1 overexpression vector (ABCB1) and the empty vector (pcDNA) were commercially obtained from GenePharma (Shanghai, China). The vectors and oligonucleotides were transfected into U2OS/DOX and HOS/DOX cells using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA).
Quantitative real-time polymerase chain reaction (qRT-PCR)
After extracting RNA with Trizol (Invitrogen), RNA was reversely transcribed using HiScript II One Step RT-PCR Kit (Vazyme, Nanjing, China) or miRNA 1st Strand cDNA Synthesis Kit (Vazyme). Then, qRT-PCR was carried out using AceQ qPCR SYBR Green Master Mix (Vazyme). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and U6 were taken as internal controls. Primers were shown below: miR-198-F: 5’-GGTCCAGAGGGGAGAT-3’, miR-198-R: 5’-GAATACCTCGGACCCTGC-3’; ABCB1-F: 5’-CCCATCATTGCAATAGCAGG-3’, ABCB1-R: 5’-GTTCAAACTTCTGCTCCTGA-3’; GAPDH-F: 5’-TGCACCACCAACTGCTTAGC-3’, GAPDH-R: 5’-GGCATGGACTGTGGTCATGAG-3’; U6-F: 5’-CTCGCTTCGGCAGCACA-3’, U6-R: 5’-AACGCTTCACGAATTTGCGT-3’; 18sRNA-F: 5’-AAACGGCTACCACATCCA-3’, 18sRNA-R: 5’-CACCAGACTTGCCCCTCCA-3’. The primers of circ_0002060 were purchased from GenePharma.
Cell Counting Kit-8 (CCK-8) assay
Cells (3×103) were plated into 96-well plates and incubated with escalating doses of DOX for indicated time. Subsequently, cells were reacted with 10 μL CCK-8 solution (Solarbio) for 2 h. Next, a Multi-Mode Reader (BioTek, Burlington, VT, USA) was used to measure the optical density at 450 nm to assess cell viability. The half-maximal inhibitory concentration (IC50) of DOX is the concentration at which cell viability is reduced to 50%.
Colony formation assay
The treated cells were seeded into six-well plates and cultured in DMEM medium containing 10% FBS at 37°C for 14 days. After staining with 0.5% crystal violet, colonies were imaged and counted at least three times.
Flow cytometry
The treated cells were inoculated into six-well plates and washed with cold PBS. Then, AnnexinV-fluorescein isothiocyanate (AnnexinV-FITC)/Propidium Iodide (PI) Apoptosis Detection kit (Invitrogen) was used to detect cell apoptosis. Final, Attune NxT Flow Cytometer (Thermo Fisher Scientific, Waltham, MA, USA) was utilized to monitor the apoptosis rate.
Western blot assay
After lysing cells with RIPA buffer (Solarbio), the extracted proteins were separated by polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Then, the membranes were probed with primary antibodies against caspase 3 (ab13847, Abcam, Cambridge, UK), B-cell lymphoma 2 (Bcl-2) (ab196495, Abcam), Bcl-2 associated X protein (Bax) (ab53154, Abcam), ABCB1 (ab129450, Abcam) and GAPDH (ab9485, Abcam). Next, the membranes interacted with secondary antibody (ab7090, Abcam). The signal intensity was measured using the enhanced chemiluminescence system (Millipore).
Xenograft tumor experiment
U2OS/DOX cells were transfected with the lentivirus carrying circ_0002060 short hairpin RNA (sh-circ_0002060) or the negative control (sh-NC). Subsequently, stably transfected cells (5×106) were subcutaneously injected into the right-back of BALB/c nude mice (n=6 per group). Then, DOX (15 mg/kg) was intravenously administered to nude mice twice a week. Tumor volume was monitored every week. After five weeks, the mice were sacrificed, and xenografts were weighted. The xenograft assay was ratified by the Animal Research Committee of Weifang People’s Hospital.
Dual-luciferase reporter assay
The sequences of circ_0002060 or ABCB1 3’UTR containing miR-198 binding sites or mutant were amplified and then inserted into pmirGLO vector (Promega, Madison, WI, USA), named circ_0002060-wt, circ_0002060-mut, ABCB1 3’UTR-wt or ABCB1 3’UTR-mut reporter. Then, the corresponding luciferase reporter and miR-198 or miR-NC were co-transfected into U2OS/DOX and HOS/DOX. Finally, the luciferase intensity was monitored using a Dual-Luciferase Reporter Assay Kit (Promega).
RNA immunoprecipitation (RIP) assay
RIP analysis was carried out using EZ-Magna RIP kit (Millipore). Briefly, U2OS/DOX and HOS/DOX cells were lysed by RIP lysis buffer. Then, cell lysates were incubated with magnetic beads conjugated with Ago2 antibody or IgG antibody. The enrichment of circ_0002060, miR-198 or ABCB1 was measured by qRT-PCR.
RNA pull-down assay
Biotin-labeled circ_0002060 probe (Bio-circ_0002060) and the control probe (Bio-NC) were purchased from GenePharma. Briefly, biotinylated probes were reacted with M-280 Streptavidin Dynabeads (Invitrogen) at 37°C for 2 h to construct probe-coated beads. Next, the cells were lysed and incubated with probe-coated beads at 4°C for 3 h. Finally, the expression of miR-198 was measured by qRT-PCR.
Statistical analysis
All data were exhibited as mean ± standard deviation using Graphpad Prism 7.0 software (GraphPad, San Diego, CA, USA). Differences were tested by Student’s t-test or one-way analysis of variance. When P <0.05, the difference was considered statistically significant.