Fig 1: circ0072568 serves as a sponge for miR-382-5p, and miR-382-5p directly targets TOP1. (A) Subcellular fractionation for circ0072568 in the cytoplasm and nucleus of chondrocytes. (B) Left panel, Venn diagram illustrating miRNAs targeted by circ0072568; right panel, prediction binding sites of circ0072568 and miR-382-5p. (C) Expression of miR-545-3p and miR-382-5p in the cartilage tissues of patients with OA and normal controls (n=10). (D) Expression of miR-545-3p and miR-382-5p in chondrocytes isolated from normal articular cartilage tissues with or without IL-1ß stimulation. (E) Luciferase activity in chondrocytes co-transfected with circ0072568-wt or -mut vectors and miR-382-5p or miR-NC. (F) Reverse transcription-quantitative PCR analysis of circ0072568 and miR-382-5p enrichment in RIP precipitates of anti-Ago2 in chondrocytes. (G) Expression of miR-382-5p in chondrocytes transfected with oe-circ0072568 or si-circ0072568. (H) Expression of miR-382-5p in IL-1ß-treated chondrocytes transfected with or without oe-circ0072568. (I) Analysis of the correlation between circ0072568 and miR-382-5p in cartilage tissues from patients with OA (n=10). *P<0.05, vs. respective control.
Fig 2: circ0072568 protects chondrocytes from IL-1ß-induced inflammation and extracellular matrix degradation via the miR-382-5p/TOP1 axis. The expression of TOP1 (A) mRNA and (B) protein in chondrocytes transfected with si-TOP1. The levels of (C) IL-6, (D) TNF-a and (E) MCP-1 in the supernatant of chondrocytes were measured using ELISA. (F-H) The expression of collagen II and MMP-13 was examined using western blot analysis; n=3. *P<0.05. OA, osteoarthritis; IL, interleukin; MMP-13, matrix metalloproteinase 13; MCP-1, monocyte chemoattractant protein-1.
Fig 3: miR-382-5p directly targets TOP1. (A) Left panel, Venn diagram illustrating mRNAs targeted by miR-382-5p; right panel, prediction binding sites of miR-382-5p and TOP1 mRNA. (B) Luciferase activity in chondrocytes co-transfected with TOP1-wt or -mut vectors and miR-382-5p or miR-NC. (C) Expression of miR-382-5p in chondrocytes co-transfected with miR-382-5p mimic and oe-circ0072568. Expression of TOP1 mRNA (D) and protein (E) in chondrocytes co-transfected with miR-382-5p mimic and oe-circ0072568. Expression of TOP1 (F) mRNA and (G) protein in chondrocytes co-transfected with miR-382-5p inhibitor and si-circ0072568. Expression of TOP1 (H) mRNA and (I) protein in IL-1ß-stimulated chondrocytes co-transfected with miR-382-5p mimic and oe-circ0072568; n=3. *P<0.05. OA, osteoarthritis; IL, interleukin; TOP1, topoisomerase 1.
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