Role Of CircNFKB1 In Cartilage Degeneration Of Osteoarthritis And Its Mechanism

BackgroundOsteoarthritis(OA)is a common disease of the musculoskeletal system in China and an important cause of disability in the elderly.OA is a total joint disease with pathological changes including articular cartilage degeneration,subchondral bone thickening,bone redundancy,synovial inflammation,ligament degeneration,and knee meniscus and joint capsule hypertrophy.Cartilage degeneration is the core pathological change of OA,and the molecular mechanism of action regulating cartilage degeneration is unclear.Circular RNA(circRNA)is a covalently closed single-stranded RNA molecule produced by reverse splicing of the precursor mRNA of eukaryotic gene exons.Some circRNAs exert potentially important functions at the molecular level through different modes of action in physiological and pathological conditions.Recent studies have revealed that a few circRNAs play important regulatory roles in the progression of OA.However,studies related to circRNA regulation of chondrocyte degeneration are currently inadequate and need to be further explored in depth.MethodsWe isolated and extracted cultured human primary chondrocytes from human articular cartilage tissue and identified human chondrocytes using toluidine blue staining and type Ⅱ collagen immunofluorescence staining.The differentially expressed circRNA was detected by RNA-seq technique after IL-1β stimulation of human primary chondrocytes.qRT-PCR,in situ hybridization and FISH experiments were used to detect the expression of circNFKB1 in human cartilage tissues and chondrocytes.The effect of circNFKB1 on cartilage extracellular matrix metabolism in in vitro cytological assays was investigated using qRT-PCR,Western Blot,and immunofluorescence assays.The molecular mechanism of action of circNFKB1 was explored by RNA pull down+MS,Western Blot and FISH experiments.Finally,the effect of circNFKB1 on OA in vivo was investigated by animal experiments with intracavitary injection of circNFKB1 overexpressing adenovirus into the knee joint of mice.ResultsWe successfully isolated and extracted cultured human primary chondrocytes,and identified and screened a differentially expressed circRNA,named circNFKB1,by RNA-seq technique.circNFKB1 expression was significantly increased in IL-1βstimulated human chondrocytes and human OA cartilage tissue.Knockdown of circNFKB1 inhibited chondrocyte degeneration;overexpression of circNFKB1 promoted chondrocyte degeneration.Molecular mechanism studies showed that circNFKB1 can bind to enolase-1(ENO1)and promote the transcription of NFKB1 by ENO1.increased transcription of NFKB1 is responsible for increased NFKB1 expression and thus activation of NF-κB pathway,which ultimately causes chondrocyte degeneration.Also,intracavitary injection of circNFKB1 overexpressing adenovirus into the knee joint of mice could promote the degeneration of articular cartilage and the progression of OA in mice.ConclusionsThis study identifies circNFKB1 as a possible new potential target for OA therapy and also provides new insights into the molecular regulation of NF-κB signaling pathway in OA.