Mechanism Of IncRNA DANCR Regulating Apoptosis Of Chondrocytes In Osteoarthritis

Objective To identify the changed expression of lncRNA DANCR in OA chondrocytes and confirm the potential roles of DANCR in the progress of osteoarthritis.Methods OA cartilage tissues(experimental)were collected from patients who under total knee replacement surgery.Normal cartilage tissues(control)were obtained from patients with femoral neck fracture.The expression of DANCR in cartilage tissues was detected using quantitative real-time PCR.After cell transfection,the effects of DANCR inhibition on the apoptosis of OA chondrocytes were detected using the Annexin V-FITC/PI apoptosis kit and flow cytometry assay.Novel target of DANCR was then identified through gene array and bioinformatics analysis(satrBase v2.0).Then,the target was confirmed by luciferase reporter assay ? RNA immunoprecipitation assay and quantitative real-time PCR.Novel target of miRNA was identified through the online software(TargetScan)and confirmed by luciferase reporter assay ?RNA immunoprecipitation assay and quantitative real-time PCR.The effects of miRNA inhibitors were detected using the quantitative real-time PCR and Western blot assay.Results(1)The expression of DANCR was significantly increased in the OA group compared with the normal group by qRT-PCR assay.(2)Flow cystometric assay was used to determine the difference in cell apoptosis and the results showed that down-regulation of DANCR induced cell apoptosis in chondrocytes.(3)The online software(starBase v2.0)showed that miR-216a-5p contained putative binding sites with DANCR.The results of dual-luciferase reporter assay showed that miR-216a-5p mimics significantly decreased the luciferase activity of DANCR-WT reporter vector.In addition,the results of anti-Ago2 RIP assay showed that DANCR and miR-216a-5p were preferentially enriched in the Ago2 pellet.Furthermore,DANCR inhibition increased the expression of miR-216a-5p in chondrocytes.(4)We found JAK2 was a potential downstream target of miR-216a-5p through online bioinformatics software(TargetScan).Luciferase reporter assay showed that miR-216a-5p mimics significantly decreased the luciferase activity of JAK2-WT reporter vector.Furthermore,qRTPCR and Western Blot assay revealed that miR-216a-5p mimics led to signifycantly decrease of JAK2 and STAT3.(5)Function assays demonstrated that DANCR inhibition markedly decreased the mRNA and protein expression of JAK2 in OA chondrocytes,while miR-216a-5p inhibitors could abolish the inhibitory effects.Conclusion The expression of DANCR was increased in OA cartilage tissues.DANCR reduced cell apoptosis in OA chondrocytes through regulating miR-216a-5p/ JAK2/STAT3 signaling pathway.