Patients and specimens
Sixteen patients with primary OS (age, 10–54 years; 10 males/6 females) who underwent complete resection between May 2017 and January 2020 were recruited at the Department of Orthopedics of the Second Hospital of Jilin University. The enrolled patients were not treated with preoperative chemotherapy or radiotherapy. OS tissues and matched adjacent tissues were collected simultaneously and immediately frozen in liquid nitrogen. The Enneking staging system was used to classify the OS tumors [18]. The study was approved by the ethics committee of the Second Hospital of Jilin University (2016.169). All enrolled patients provided a signed written informed consent.
CircRNA and mRNA sequencing
High-throughput sequencing of circRNA and mRNA in four pairs of OS tissues and adjacent tissues from patients with OS was performed at CloudSeq Biotech, Inc. (Shanghai, China). Briefly, total RNA was used to remove ribosomal RNAs using NEBNext® rRNA Depletion Kit (New England Biolabs, Ipswich, MA, USA). RNA libraries were constructed using ribosomal RNA-depleted RNAs with the TruSeq Stranded Total RNA Library Prep Kit (Illumina, San Diego, CA, USA). Libraries were controlled for quality and quantified using a BioAnalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). Library sequencing was performed on an Illumina Novaseq 6000 instrument with 150-base pair paired-end reads. The paired-end reads were quality controlled based on Q30. After 3′ adaptor-trimming, low-quality reads were removed using the cutadapt software (v1.9.3). High-quality trimmed reads were used to analyze the circRNAs and mRNAs by aligning the reads to the reference genome/transcriptome using the STAR software (v2.5.1b), and circRNAs were detected and identified using the DCC software (v0.4.4). edgeR software (v3.16.5) was used to normalize the data and analyze differentially expressed circRNAs. The hisat2 software (v2.0.4) was used to compare high-quality reads to the human reference genome (UCSC HG19). Using the GTF gene annotation file, HTSeq software (v0.9.1) was used to obtain the original count, and edgeR was used to standardize and calculate the fold-change and P-value between the two sample sets to screen for differentially expressed mRNAs.
Cell culture and RNA extraction
The human osteoblast cell line hFOB1.19 and OS cell lines MG63, HOS, and U2OS were acquired from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All cell lines were cultured in Dulbecco’s modified Eagle medium (Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Grand Island, NY, USA). Total RNA was isolated using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.
Quantitative real-time polymerase chain reaction
The RNA was reverse-transcribed into cDNA using an All-in-one First-Strand cDNA Synthesis Kit (GeneCopoeia, Rockville, MD, USA). Quantitative reverse-transcription polymerase chain reaction (RT-qPCR; ABI 7500, Applied Biosystems, Foster City, CA, USA) was performed using Power SYBR® Green PCR Master Mix (Applied Biosystems). GAPDH and U6 were used as internal references. RNA expression was quantified and fold-changes in expression were determined using the 2–△△Ct method. All experiments were performed in triplicate. The primer sequences used in this study are listed in Supplementary Table 1.
Cell transfection
The circ_0001174 small interfering RNA (si-circ_0001174), miR-186-5p mimic, miR-186-5p inhibitor, and negative control were synthesized by GenePharma (Shanghai, China). Lipofectamine 3000 (Thermo Fisher Scientific) was used for cell transfection according to the manufacturer's protocol. Cells were collected at 48 h after transfection for RT-qPCR analysis. Each experiment was performed in triplicate.
Cell counting kit-8 assay
MG63 cells were seeded at a density of 3000 cells/100 µL into each well of a 96-well plate, and their proliferation ability was detected using the Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s protocol. The optical density was measured at 450 nm.
Wound healing assay
Cells (1 × 106) were seeded into each well of a 6-well plate and cultured for 24 h to 100% confluence. The cells were scratched with a 200-μL sterile pipette tip, washed with serum-free medium, and cultured in serum-free medium for 24 h. Images were taken immediately after wounding and at 24 h post-stimulation. The migration rate was quantified using image analysis software (ImageJ, V1.48, NIH, Bethesda, MD, USA) and calculated based on the movement of cells from the position of their initial placement to the final distance, using the following equation: (initial distance – final distance)/initial distance × 100.
Cell migration and invasion assays
A transwell (8-mm-pore size, Millipore, Billerica, MA, USA) was used to evaluate cell migration and invasion ability. For cell migration assays, the cells (3 × 104 cells in 200 μL serum-free medium) were seeded into the upper chamber, and 400 μL complete medium was added to the lower chamber. After 24 h of culture, the cells were collected. For cell invasion assays, 100 μL Matrigel (BD Biosciences, Franklin Lakes, N, USA) was first added to the chamber. The cells (3 × 104 cells in 200 μL serum-free medium) were seeded into the upper chamber, and 400 μL complete medium was added to the lower chamber. After 48 h of culture, the cells were collected. The bottom of the upper membrane was fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The cells were quantified by counting five randomly selected fields under a microscope. The cells were counted using ImageJ software [19].
Cell colony formation assay
Cells (1 × 103) were seeded into each well of a 6-well plate and cultured for 10 days until colonies were clearly observed. After fixing the cells with 4% paraformaldehyde, the colonies were stained with 0.1% crystal violet solution and washed twice with phosphate-buffered saline. The colonies were counted using the ImageJ software.
Western blotting
Total proteins were extracted from cells and tissues using 100 μL lysis buffer. The protein concentrations in the total cellular lysates were quantified using a bicinchoninic acid protein assay kit (Beyotime, Shanghai, China). Equal amounts of protein were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes via electroblotting. After blocking with 5% skimmed milk, the membrane was incubated with the primary antibodies anti-MACC1 and anti-β-Actin at 4 °C overnight, followed by incubation with anti-rabbit horseradish peroxidase-conjugated secondary antibody (Boster, Wuhan, China). Signals were detected using an enhanced chemiluminescence detection system.
Bioinformatics analysis
Hierarchical clustering was performed using Euclidean distance and average linkage clustering based on the circRNA and mRNA expression profiles. Gene Ontology (GO, http://www.geneontology.org/) [20.21] and Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg/) [22] analyses were performed to identify the roles and related pathways of differentially expressed genes between OS and paracancerous tissues. circBase (http://www.circbase.org) [23] and the Database for Annotation, Visualization, and Integrated Discovery (https://david.ncifcrf.gov/) [24] were used to annotate the differentially expressed circRNAs and mRNAs. The circRNA-miRNA interactions were predicted using circinteractome (https://circinteractome.irp.nia.nih.gov/) [25]. miRNA interaction with mRNAs was predicted using Diana tools [26], miRDB [27], and TargetScan Human 8.0 [28].
Statistical analysis
All statistical analyses were performed using SPSS version 20.0 software (SPSS, Inc., Chicago, IL, USA). Differentially expressed circRNAs and mRNAs that significantly differed between the two groups were identified when the change in the threshold values was |fold-change| ≥ 2. Comparisons between groups were performed using unpaired Student’s t-test. Fisher’s exact test was used to evaluate the significance of GO terms. The association between miRNA and circRNA or mRNA expression was analyzed using Spearman’s correlation coefficient. The chi-square test was performed to determine the correlation between the expression of circ_0001174 and clinical characteristics of patients with OS. Statistical significance was set at P < 0.05.