Circular RNA circ_0008365 regulates SOX9 by targeting miR-338-3p to inhibit IL-1β-induced chondrocyte apoptosis and extracellular matrix degradation

Abstract

Background: Osteoarthritis (OA) is a chronic disease that involves chondrocyte injury and dysfunction. CircRNAs participate in OA progression, but the roles of circRNAs in the occurrence of OA are unclear. In this study, we explore the role of circ_0008365 in OA.

Methods: CHON-001 cells were treated with interleukin-1β (IL-1β) to construct an in vitro OA cell model. The levels of circ_0008365, SRY-related high mobility group-box gene9 (SOX9) mRNA, and microRNA-338-3p (miR-338-3p) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) assay. Western blot (WB) assay was used to measure protein levels. Cell Counting Kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EDU) assay, and flow cytometry analysis were used to detect cell viability, proliferation, and apoptosis, respectively. Dual-luciferase reporter assay, RNA pull-down assay, and RNA immunoprecipitation (RIP) assays were used to confirm the interaction between miR-338-3p with circ_0008365 or SOX9.

Results: Circ_0008365 expression was reduced in OA tissues and IL-1β-induced CHON-001 cells. Functionally, circ_0008365 inhibited viability, proliferation, and ECM degradation and promoted apoptosis of IL-1β-induced CHON-001 cells. Mechanistically, circ_0008365 acted as a sponge of miR-338-3p to regulate SOX9 expression, thus exerting its functions in IL-1β-induced CHON-001 cells. Moreover, exosomal circ_0008365 had great value in diagnosing OA.

Conclusion: Circ_0008365 alleviates IL-1β-induced CHON-001 cell damage through the miR-338-3p/SOX9 axis, which suggested that circ_0008365 might be a new therapeutic target for OA.

Keywords: Osteoarthritis; SOX9; circ_0008365; miR-338-3p.

Fig. 1

Circ_0008365 expression was decreased in OA cartilage tissues and IL-1β-induced CHON-001 cells. A Relative expression of circ_0008365 was detected in OA cartilage tissues (n = 25) and normal tissues (n = 20); B Relative expression of circ_0008365 was detected in IL-1β-induced CHON-001 cells; C The stability of circ_0008365 and SERPINE2 was analyzed by qRT-PCR in CHON-001 cells treated with or without RNase R; D The location of circ_0008365 was determined by cytoplasmic and nuclear RNA separation assay. **P < 0.01, ***P < 0.001

Fig. 2

Circ_0008365 overexpression inhibited IL-1β-induced CHON-001 cell damage. A The expression of circ_0008365 was detected by qRT-PCR in CHON-001 cells transfected with pcDNA or circ_0008356; B–F CHON-001 cells were divided into 4 groups, including con, IL-1β, IL-1β + pcDNA, and IL-1β + circ_0008356 groups; (B) CCK-8 assay was used to detect cell viability; C EDU assay was used to measure cell proliferation; D Flow cytometry analysis was performed to evaluate the apoptosis of cells; E, F Western blot assay was employed to detect the protein levels of proliferation-related PCNA, apoptosis-related pro-caspase-3 and cleaved caspase-3 and ECM-related protein (MMP13, ADAMTS5, COL2A1, and Aggrecan). **P < 0.01, ***P < 0.001

Fig. 3

MiR-338-3p was a target of circ_0008365. A The binding sequence between circ_0008365 and miR-338-3p was predicted by starbase online database; B The expression level of miR-338-3p was detected by qRT-PCR in CHON-001 cells transfected with miR-NC or miR-338-3p; C, D The luciferase activity in 293 T and CHON-001 cells co-transfected with miR-NC/miR-338-3p and WT-circ_0008365/MUT-circ_0008365 was detected by dual-luciferase reporter assay; E The enrichment of circ_0008365 in CHON-001 cells incubated with Bio-NC or Bio-miR-338-3p was detected by RNA pull-down assay; F The enrichments of circ_0008365 and miR-338-3p were measured by RIP assay; G The miR-338-3p expression in OA cartilage tissues (n = 25) and normal tissues (n = 20) was measured by qRT-PCR; H Pearson correlation analysis was carried out to reveal the relationship between miR-338-3p and circ_0008365 expression in OA tissues; I The expression of circ_0008365 in CHON-001 cells treated with IL-1β, IL-1β + pcDNA or IL-1β + circ_0008365 and control cells was detected by qRT-PCR assay. ***P < 0.001

Fig. 4

Circ_0008365 alleviated IL-1β-induced CHON-001 cell damage by binding to miR-338-3p. A–E IL-1β-induced CHON-001 cells were transfected with pcDNA, circ_0008365, circ_0008365 + miR-NC or circ_0008365 + miR-338-3p; A The viability of cells was examined by CCK-8; B EDU assay was conducted to detect cell proliferation; C The apoptosis of cells was measured by Flow cytometry analysis; D, E Western blot analysis was used to determine protein levels. **P < 0.01, ***P < 0.001

Fig. 5

SOX9 was a direct target of miR-338-3p. A Starbase online database was used to predict the binding sites between miR-338-3p and SOX9; B, C Relative luciferase activities were detected in 293 T and CHON-001 cells co-transfected with miR-NC or miR-338-3p and SOX9-3′UTR-WT or SOX9-3′UTR-MUT; D Western blot assays were used to examine the protein level of SOX9 in CHON-001 cells after miR-338-3p overexpression; E The knockdown efficiency of miR-338-3p inhibitor was detected by qRT-PCR; F The protein level of SOX9 was detected in CHON-001 cells after miR-338-3p knockdown; G The relative level of SOX9 was detected in OA cartilage tissues (n = 25) and normal tissues (n = 20) by qRT-PCR; H, I The correlation between SOX9 and miR-338-3p or circ_0008365 expression level in OA tissues was analyzed by Pearson correlation analysis; (J) The protein level of SOX9 was assessed in IL-1β-induced CHON-001 cells; (K) The protein level of SOX9 was measured by western blot in IL-1β-treated CHON-001 cells transfected with pcDNA, circ_0008365, circ_0008365 + miR-NC or circ_0008365 + miR-338-3p. **P < 0.01, ***P < 0.001

Fig. 6

Knockdown of miR-338-3p inhibited IL-1β-induced CHON-001 cell damage by regulating SOX9 expression. A The transfection efficiency of SOX9 overexpression vector was assessed by western blot analysis; B–F IL-1β-treated CHON-001 cells were transfected with anti-NC, anti-miR-338-3p, anti-miR-338-3p + si-NC or anti-miR-338-3p + si-SOX9; CCK-8 assay (B), EDU assay (C), and flow cytometry analysis assay (D) were used to examine cell viability, proliferation, and apoptosis of cells; E, F The protein levels of proliferation-related PCNA, apoptosis-related pro-caspase-3 and cleaved caspase-3 and ECM-related protein (MMP13, ADAMTS5, COL2A1, and Aggrecan) were detected by western blot analysis. **P < 0.01, ***P < 0.001

Fig. 7

Circ_0008365 was secreted by exosomes in the serum of OA patients. A Exosomes (indicated by red arrows) derived from serum of OA patients and normal people were detected by electron microscope; B The protein levels of CD63 and TSG101 were measured by western blot assay; C The relative expression of circ_0008365 was measured by qRT-PCR in exosome derived from serum of OA patients (n = 25) and healthy controls (n = 20); D The receiver operating characteristic (ROC) curve analysis for exosomal circ_0008365 in diagnosing OA. ***P < 0.001