The Role Of Inflammation-related Noncoding RNA In Osteoarthritic Cartilage Degradation

BackgroundOsteoarthritis(OA)is the most common joint disabling disease in China,and knee OA is more common.Its pathological features include articular cartilage degradation,synovitis,osteophyte formation,subchondral bone remodeling and other changes in joint structure.The pathogenesis of knee OA is complex and there are many subtypes of diseases,among which high inflammatory response type of knee OA is a common clinical subtype.The activation of inflammatory signaling pathway mediated by multiple pathogenic factors plays an important role in cartilage degradation.However,the specific molecular mechanism of its occurrence and development remains unclear.Noncoding RNAs(ncRNAs)are a class of RNA transcripts that have no protein-coding capacities,including long noncoding RNA(lncRNA)and microRNA(miRNA).Recent studies have confirmed that ncRNAs are widely involved in the pathogenesis of a variety of bone and joint diseases and have important biological functions-However,are there lncRNA and miRNA specifically targeting inflammatory signaling pathway in the progression of knee OA?There is still a lack of further research on this issue.MethodsWe isolated and purified human and mouse primary chondrocytes from articular cartilage tissue of human and suckling mice,and identified the chondrocytes by collagen Ⅱ immunofluorescence staining and toluidine blue staining.High throughput transcriptome sequencing was used to detect the expression profile of lncRNA in human primary chondrocytes with inflammatory cytokines(IL-1β and TNF-α)stimulation.The expression levels of lncPILA and miR-214-3p in chondrocytes and cartilage tissues were detected by qRT-PCR and in situ hybridization.Western blotting,immunofluorescence,qRT-PCR and other techniques were used to study the effects of lncPILA and miR-214-3p on cartilage extracellular matrix metabolism and apoptosis in vitro.The specific mechanisms of lncPILA and miR-214-3p were explored by RNA pulldown,immunoprecipitation,luciferase and other methods.Finally,a mouse OA model was established to explore the effects of lncPILA and miR-214-3p on OA in vivo.ResultsWe successfully isolated and cultured human and mouse primary chondrocytes.A novel lncRNA,named lncPILA,was identified by high-throughput sequencing.The expression of lncPILA was significantly up-regulated in human chondrocytes with inflammatory cytokines stimulation and OA cartilage tissues,while the expression of miR-214-3p was significantly down-regulated.Knockdown of lncPILA can inhibit the activation of NF-κB pathway and chondrocyte degradation.Overexpression of lncPILA significantly activated the NF-κB pathway and promoted chondrocyte degradation.Moreover,knockdown of miR-214-3p can promote chondrocyte degradation,while overexpression of miR-214-3p can inhibit chondrocyte degradation.Mechanistically,lncPILA interacted with the protein arginine methyltransferase PRMT1,promoting PRMT1-mediated arginine methylation of DExH-box helicase 9(DHX9).Increased arginine methylation of DHX9 enhanced the transcription of transforming growth factor β-activated kinase 1(TAK1),which eventually promoted NF-κB pathway activation.The protective effect of miR-214-3p downregulated the nuclear factor kappa B kinase β(IKKβ)expression and led to the dysfunction of NF-κB signaling pathway.Furthermore,intra-articular injection of adenovirus-lncPILA or miR-214-3p antagomir in mouse joints triggered spontaneous cartilage loss and OA development while miRNA-214-3p agomir alleviated OA in the experimental mouse models.ConclusionsThis study provides new insights into the complex regulation of NF κB signaling pathway and a potentially new therapeutic target of lncPILA and miR-214-3p in OA.