Introduction

Osteosarcoma (OS), the most common primary malignant tumor of bone with peak incidence in children and adolescents, is highly invasive and tends to metastasize to the lung [1, 2]. Chemotherapy combined with complete surgical resection has been the standard treatment for most OS patients with a significantly improved 5-year disease-free survival rate to 70–80% [3]. But the survival rate of patients who suffer from chemoresistance or lung metastasis has declined to less than 20% [4, 5]. Therefore, it is essential to expand the molecular understanding of tumorigenesis and progression of OS to provide novel biomarkers or treatment targets for this refractory disease [6, 7].

With the development of deep-sequencing technology, many novel noncoding RNAs, including circular RNAs (circRNAs), have been identified and found to act a pivotal part in diverse cellular physiological and pathological processes [8, 9]. Unlike its homologous linear transcript, the characteristic of circRNA is that it has a closed-loop structure without a 3′ polyadenylate tail or 5′ cap, making it highly tolerant to RNase R [10]. In addition, many reports have demonstrated that circRNAs can regulate the expression of numerous genes at the pre-, transcriptional, and post-transcriptional levels through interacting with DNA, RNA or protein [8, 11]. Lately, some circRNAs have been discovered to take part in the progression of OS, such as circMYO10 [RNase R treatment and Actinomycin D

Total RNA of circ_0004674 and ADAM22 (10 μg) was incubated with RNase R (R0301, Geneseed, Guangzhou, China) at 37 °C for 60 min to analyze the RNase R resistance. Actinomycin D (2 mg/mL, 129935, Millipore, USA) was added to the cell culture medium, and the half-lives of circ_0004674 and ADAM22 were evaluated and analyzed. After treatment with actinomycin D or RNase R, the expression levels of circ_0004674 and ADAM22 were detected by qRT-PCR.

Plasmid construction and cell transfection

MG63/DXR and KH-OS/DXR cells were transiently transfected with siRNAs (or miR-142-5p mimicss, miR-142-5p inhibitor, miR-NC) using Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions after being seeded into 6-well plates overnight. Forty-eight hours after transfection, the cells were harvested to detect the knockout efficiency via qRT-PCR detection of the expression of circ_0004674 and ADAM22. Two different siRNAs against the junction site of circ_0004674 were designed and synthesized by GenePharma (Shanghai, China). The sequence of si-circ_0004674-1 was 5′-GTCGTGCTAAATCAATTCAGA-3′, that of si-circ_0004674-2 was 5′-GTGCTAAA TCAATTCAGATGT-3′ (si-circ_0004674-1 has the highest inhibition efficiency, and si-circ_0004674 mentioned in the article refers to si-circ_0004674-1) and the relative si-NC sequence was 5′-AAUUCUCCGAACGUGUCACGU-3′.

CCK-8 assay

Stably transfected MG63/DXR (or KH-OS/DXR) cells (5 × 103 cell/well) were seeded in 96-well plates, freshly prepared medium containing several final concentrations of doxorubicin (0, 2, 4, 8, 16, 32, and 64 μg/ml) was added to the wells with three replicate wells for each concentration. After incubation for another 48 h, cell viability was measured using Cell Counting Kit-8 (CCK-8, Do**do, Japan) according to the manufacturer’s instructions. The absorbance of each well was measured with a microplate reader set at 450 nm.

Flow cytometry

For the cell cycle assay, after 48 h of transfection, the cells were digested with trypsin, and the supernatant was discarded. Subsequently, the cells were washed with PBS, centrifuged, and fixed in 75% ethanol at 4 °C overnight. After washing twice with PBS, the cells were stained with propidium iodide (PI) at room temperature in the dark for 15 min before analysis.

For the cell apoptosis assay, after 48 h of transfection, doxorubicin (8 μg/ml) was added to the wells. After incubation for another 12 h, the Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, San Diego, USA) was used to stain cells according to the manufacturer’s protocol. The cell cycle and apoptosis were then analyzed by FACS scan flow cytometry using FlowJo software (BD Biosciences).

Transwell assay

For the invasion assays, a 24-well Transwell chamber with the upper chamber coated with Matrigel (354230, BD, USA) was used. A total of 5.0 × 104 stably transfected MG63/DXR (or KH-OS/DXR) cells in 200 µL of serum-free DMEM were seeded in the upper chamber, and 600 µl of medium containing 10% FBS was placed in the lower chamber. After incubation for 24 h, cells on the upper membrane surface were wiped off using a cotton swab, and the invading cells that had traversed the membrane were stained with crystal violet and counted.

Wound healing assay

A total of stably transfected MG63/DXR (or KH-OS/DXR) cells (5 × 105 cell/well) were seeded in 6-well plates. Wounds were then created on a monolayer of cells using a sterile pipette tip. Cells were further cultured with a medium containing 1% FBS for 48 h. The healing wounds were photographed twice at 0 h and 48 h after scratching, and then, the cell migration rate was calculated.

Xenograft tumor assay

Female nude (BALB/c) mice (4 weeks old) were purchased. Mice were divided into two groups according to the completely randomized method (N = 6/group). All procedures for the mouse experiments were approved by the Animal Experimental Ethics Committee of Shanghai Tenth People’s Hospital. MG63/DXR cells stably expressing sh-circ_0004674 or sh-NC were propagated, and 5 × 106 (100 µl) cells were inoculated into the medullary cavity of the right proximal tibia of mice. Tumor growth was examined at the indicated time points, and tumor volumes were measured. After 7 weeks, the mice were killed, and tumors were removed and weighed. The activity of Ki-67 and caspase3 in the tumors was measured by immunochemistry (IHC).

Bioinformatics prediction

The sequence of circ_0004674 was obtained from Circbase (http://www.circbase. org/cgi-bin/simplesearch.cgi), and visual graphics of circ_0004674 were obtained from CircPrimer, a software which is for annotating circRNAs and determining the specificity of circRNA primers. CircInteractome (https://circinteractome.nia.nih.gov/) was used to analyze the target miRNA of circ_0004674. All the downregulated miRNAs in OS previously reported were collected from the database of dbDEMC (database of Differentially Expressed MiRNAs in human Cancers, https://www.picb. ac.cn/dbDEMC/index.html) and miRCancer (microRNA Cancer Association Database, http://mircancer.ecu.edu/).

RNA immunoprecipitation (RIP)

RIP experiments were performed in MG63/DXR cells using an RIP Kit (Millipore, Billerica, MA) following its manufacturer’s protocol. Cells were lysed and incubated with RIP buffer containing A/G magnetic beads conjugated with Ago2 antibody (Abcam) and normal anti-IgG (Millipore) as a negative control. Finally, immunoprecipitated RNA was isolated and purified for qRT-PCR to analyze the expression levels.

Biotin-coupled probe pull-down assay

Biotin-coupled probe pull-down assays were performed to determine the interaction between circ_0004674 and miR-142-5p. Briefly, the miR-142-5p-WT or miR-142-5p- Mut probe was synthesized and biotinylated by GenePharma (Shanghai, China). RNA pull-down assays were carried out using the Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific, Waltham, MA) following its manufacturer’s protocols. Finally, the RNA binding protein complexes were washed and eluted for qPCR analysis of circ_0004674.

Dual-luciferase reporter assay

MG63/DXR cells were seeded at 5 × 104 cells/well in 24-well plates and allowed to settle overnight. The next day, cells were co-transfected with pmirGLO-circ_0004674 (or MCL1)-WT or-MUT reporter plasmids and miR-142-5p mimics. Twenty-four hours after transfection, 20 µl of the protein supernatant was added to 50 µl of the firefly luciferase substrate and mixed thoroughly. Then, the relative luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) and normalized against Renilla luciferase activity.

RNA fluorescence in situ hybridization (FISH)

Cy3-labeled circ_0004674, Cy5-labeled miR-142-5p, and DAPI-labeled U6 probes were obtained from GenePharma (Shanghai, China). RNA FISH was performed using a fluorescent in situ hybridization kit according to the manufacturer’s protocol (Thermo Fisher).

Western blot

For western blotting, the total protein was extracted using RIPA lysis buffer from cells (P0013, Beyotime, CA). Then, the protein loading buffer was added to the protein and mixed, and the mixture was boiled for 10 min. Next, a 30-μg protein sample was added to each well, electrophoresed, and subsequently transferred to a NC membrane (Immobilon-P Transfer Membrane, EMD Millipore Corporation, MA). The membrane was blocked with 5% skim milk for 2 h and then incubated with the primary antibody overnight at 4 °C, followed by incubation with the corresponding secondary antibody for 2 h at room temperature. The protein bands were visualized using an Odyssey scanner (LI-COR Biosciences, Lincoln, NE, USA). ImageJ software was used for semiquantitative analysis. The primary antibodies were MCL1 and β-actin.

Statistical analysis

All statistical analyses were performed using SPSS 22.0 software (IBM) and GraphPad Prism 7.0 (GraphPad Software Inc, San Diego, CA, USA) software. All the experiments in the current study were independently performed at least three times and data are presented as the means ± SEM. Student’s t test or one-way ANOVA was used to evaluate the differences between groups for continuous data. Chi-square test was used to compare the distribution of stage between high- and low-expression circ_0004674 level groups for categorical data. Overall survival was calculated by Kaplan–Meier survival analysis and compared using the log-rank test. The correlation of circ_0004674 and MCL1 mRNA expression level in OS tissues was determined by Spearman’s correlation. Then, p values of <0.05 were considered statistically significant.