Subjects information
KBD patients were diagnosed in accordance with the diagnostic criteria for KBD (WS/T 207-2010). Articular cartilages were obtained from KBD patients (n = 5, two females and three males, age range 55-70 years) undergoing joint replacement surgery. The normal article cartilages were obtained from control donors (n =5, two females and three males, age range 50-66 years) suffering from traffic or other accidents. Subjects with other bone and cartilage diseases, such as rheumatoid arthritis or osteoarthritis, were excluded by clinical and imaging studies. All cartilage specimens were taken from the same anatomical area. This study was approved by the Human Ethics Committee of Xi’an Jiaotong University, and performed according to the principles of the Declaration of Helsinki as revised in 1983. All subjects signed informed consent.
Chondrocyte collection and culture
Cartilages obtained from a KBD patients and normal controls were digested by trypsin and collagenase and then chondrocytes were isolated and collected. The main procedures are as shown below. After stripping fascia and perichondrium, the separated cartilage tissue was cut into 0.3-0.5 mm3 tissue blocks and washed with PBS. Then, the tissue blocks were digested by 0.25% of trypsin for 30 minutes and type Ⅱ collagenase at 37℃ for 6-8 hours. Chondrocytes were obtained by centrifugation and cultured at 37C in 5% CO2 in DMEM/F-12 medium containing 10% fetal bovine serum, 100 ml /mL penicillin and 100 mg/ml streptomycin. The chondrocytes were passaged when the fusion reached 80%, later chondrocytes were harvested using for experiments.
Total RNA extraction
Total RNA was extracted from cultured chondrocytes by using the Trizol reagent (Invitrogen Corporation, Gaithersburg, MD) and purified with mirVana miRNA isolation kit (Ambion, Austin, TX, USA) according to manufacturers’ protocol. The purity and concentration of RNA were determined by OD260/280 readings using a spectrophotometer (Nano Drop ND-1000). RNA integrity was determined by 1% formaldehyde denaturing gel electrophoresis. Only RNA extracts with RNA integrity number values > 6 underwent further analysis.
circRNA microarray analysis
The extracted RNAs were digested, dephosphorylated, denatured, expanded, and labeled with Cy3-dCTP following the instructions given by the product data sheet. Purified RNAs were hybridized to a microarray containing 170,340 human circRNA probes (CapitalBio Technology Human CircRNA Array v2; CapitalBio Corporation, Beijing, China). Finally, the array scan results of circRNAs were analyzed using Gene Spring software V13.0. (Agilent Technologies, Santa Clara, CA).
Microarray data acquisition
Raw data was extracted from the scanned images of microarrays by Genepix Pro 6.0 software (Axon; Molecular Devices, LLC, Sunnyvale, CA). Next, the original data was normalized and processed using the R software package. Finally, low-intensity substances were excluded. The circRNA array data was analyzed after data summarization, normalization and quality control by using the GeneSpring software V13.0 (Agilent). The data was log2 transformed and median centered by genes using the adjust data function of CLUSTER 3.0 software then further were analyzed hierarchical clustering between groups with average linkage. Finally, tree visualization was performed using Java Treeview (Stanford University School of Medicine, Stanford, CA, USA).
Bioinformatics analysis
To select the differentially expressed genes, we used threshold values of ≥ 2 and ≤ -2 fold change and a T-test P value = 0.05. Molecular function (MF), cellular component (CC) and biological pathway (BP) of differently expressed circRNAs targeting genes were analyzed using gene ontology (GO). Kyoto encyclopedia of genes and genomes (KEGG), reactome, panther, and biocyc packages were used to explore pathways related to circRNAs target genes.
Construction of the circRNA-miRNA Network
Based on the miRanda3.3 software, we explored the functional annotation of identified circRNAs and predicted the interaction of circRNA-miRNA. CircRNA-miRNA pairs were selected and the circRNA-miRNA interaction network was constructed by using the open source bioinformatics software Cytoscape.