CircSFMBT2-OA alleviates chondrocyte apoptosis and extracellular matrix degradation through repressing NF-κB/NLRP3 inflammasome activation

Abstract

Background: Intra-articular inflammation and cartilage degradation are the major pathological characteristics of osteoarthritis (OA). Mounting studies have revealed that circular RNAs (circRNAs) act as an important regulatory role in inflammatory diseases and are frequently dys-expressed in OA cartilage tissues.

Objective: Here, a dys-regulated cicrRNA (has_circ_0017636, termed circSFMBT2-OA) was identified, and its role in regulating lipopolysaccharide (LPS)-induced chondrocyte injury was next investigated.

Methods: CHON-001 chondrocytes were treated with LPS, and then the levels of circSFMBT2-OA, cartilage-related genes, and pro-inflammatory cytokines were measured using quantitative real-time PCR (qRT-PCR) and Western blot analysis. CHON-001 cell viability, proliferation, and apoptosis were assayed using Cell Counting Kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EDU), and terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay, respectively.

Results: CircSFMBT2-OA level was significantly down-regulated in OA cartilage tissues and LPS-treated CHON-001 cells. Functionally, circSFMBT2-OA overexpression accelerated cell proliferation, and suppressed cell apoptosis, pro-inflammatory cytokines production, matrix-degrading enzymes expression, and ECM degradation in CHON-001 cells. Inversely, circSFMBT2-OA depletion decreased cell viability and increased matrix-degrading enzymes expression and ECM degradation. Mechanistically, circSFMBT2-OA inhibited LPS-induced NF-κB/NOD-like receptor family pyrin domain containing protein 3 (NLRP3) inflammasome activation in CHON-001 cells. Consequently, NLRP3 activator reversed the effect of circSFMBT2-OA on repressing LPS-induced CHON-001 cell injury.

Conclusion: These data reveal a vital effect of a novel circSFMBT2-OA on repressing OA progression and provide a promising target to treat OA.

Keywords: Chondrocyte injury; Inflammation; NLRP3; Osteoarthritis; circSFMBT2-OA.

Fig. 1

CircSFMBT2-OA was down-regulated in OA cartilages. (A) Dys-regulated circRNAs in chondrocytes after IL-1β treatment were represented by volcano plots (prob ≥0.8, log2 |FC| ≥ 1). (B) Dys-regulated circRNAs between OA and normal cartilage tissues were represented by volcano plots (p < 0.05, |FC| ≥2). (C) Venn diagram analysis revealed 25 circRNAs appeared in GSE107009 and PRJAN516555 datasets. (D) CHON-001 cells were treated with LPS (5 μg/mL) or IL-1β (10 ng/mL) and relative circRNAs level was assessed after 24 h using qRT-PCR analysis. (E) qRT-PCR analysis of circSFMBT2-OA level in OA cartilage (n = 11) and normal cartilage (n = 9). *p < 0.05, **p < 0.01.

Fig. 2

Characterization of circSFMBT2-OA. (A) Schematic diagram illustrating the circularization of exon 3–7 of SFMBT2 to generate circSFMBT2-OA. Convergent primers (purple triangles) were applied to amplify linear sequence of circSFMBT2-OA, and Divergent primers (red triangles) were applied to amplify back-splicing site of circSFMBT2-OA. CircSFMBT2-OA was assessed using qRT-PCR, followed by Sanger sequencing. (B) cDNA was synthesized using Convergent primers and Divergent primers, respectively, and then CircSFMBT2-OA was assessed using qRT-PCR in gDNA and cDNA. (C) qRT-PCR analysis of circSFMBT2-OA and SFMBT2 levels after RNase R treatment. (D) cDNA was synthesized using Oligo dT primers and random primers, respectively, and then circSFMBT2-OA and SFMBT2 levels were assessed using qRT-PCR. (E) FISH were performed to measure the cellular localization of circSFMBT2-OA in CHON-001 cells. CircSFMBT2-OA-probes were dyed green and the nucleus was stained blue. **p < 0.01, ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

CircSFMBT2-OA decreased LPS-induced CHON-001 cell injury. (A) CHON-001 cells were transfected with pcDNA-circSFMBT2-OA for 48 h, and then circSFMBT2-OA and SFMBT2 levels were assessed using qRT-PCR. CHON-001 cells were treated with LPS (5 μg/mL) after circSFMBT2-OA overexpression, and then TUNEL assay (B) and quantitative analysis (C) was assessed. (D) CHON-001 cells were treated with LPS (5 μg/mL) after circSFMBT2-OA overexpression, and then cell viability was assessed using CCK-8. CHON-001 cells were treated with LPS (5 μg/mL) after circSFMBT2-OA overexpression, and then Edu assay (E) and quantitative analysis (F) was assessed. Western blot (G) and quantitative analysis (H) of MMP-13, ADAMTS-5, and Collagen II protein expression in CHON-001 cells after treated with indicated reagents. (I) qRT-PCR analysis of TNF-α, IL-1β, and IL-6 mRNA level in CHON-001 cells after treated with indicated reagents. **p < 0.01.

Fig. 4

CircSFMBT2-OA attenuated NF-κB/NLRP3 activation Western blot (A) and quantitative analysis (B) of p-p65 level in CHON-001 cells after treated with LPS (5 μg/mL) in the presence or absence of circSFMBT2-OA overexpression. Western blot (C) and quantitative analysis (D) of NLRP3 and cleaved caspase-1 protein expression in CHON-001 cells after treated with indicated reagents. (E) qRT-PCR analysis of IL-18 and IL-1β mRNA level in CHON-001 cells after treated with indicated reagents. **p < 0.01.

Fig. 5

CircSFMBT2-OA attenuated CHON-001 cell injury through repressing NLRP3 activation Western blot (A) and quantitative analysis (B) of MMP-13, ADAMTS-5, and Collagen II protein expression in circSFMBT2-OA-overexpressed CHON-001 cells in the presence or absence of Nigericin (10 μM). (C) qRT-PCR analysis of IL-18 and IL-1β mRNA level in CHON-001 cells after treatment with indicated reagents. (D) qRT-PCR analysis of circSFMBT2-OA level in CHON-001 cells after treated with si-circSFMBT2-OA. Western blot (E) and quantitative analysis (F) of NLRP3 and cleaved caspase-1 protein expression in circSFMBT2-OA-depleted CHON-001 cells treated with MCC950 (8 μM). (G) CHON-001 cells were treated with si-circSFMBT2-OA and MCC950 (8 μM), and then cell viability was assessed using CCK-8. (H) qRT-PCR analysis of MMP-13, ADAMTS-5, and Collagen II mRNA level in CHON-001 cells after treated with indicated reagents. *p < 0.05, **p < 0.01.