The Mechanism Research Of Long Non-coding RNA DANCR Enhances Chondrogenic Differentiation And Proliferation Of Human Synovium-derived Mesenchymal Stem Cell

Background:Cartilage damage has a very limited capacity for repair and cause osteoarthritis,as the articular cartilage lacks nerve distribution and blood vessel supply of its own characteristics.Although a variety of cartilage injury treatment has been applied to clinical,but still lack of an effective treatment.SMSCs has become the most ideal cartilage repair seed cell because of some advantages.Previous studies have demonstrated that lncRNAs play important roles in biological processes,including cell differentiation,proliferation and migration.However,there are few reports on the study of lncRNA in promoting the proliferation and differentiation of SMSCs.Objective:To explore the mechanism research of long non-coding RNA DANCR enhances chondrogenic differentiation and proliferation of human synovial-derived mesenchymal stem cells.Method:Knee joint synovial tissues of healthy New Zealand White Rabbits were obtained under sterile condition,synovial-derivedmesenchymal stem cells was isolated in vitro;after purified by limited dilution method,the synovial-derivedmesenchymal stem cells were cultured and proliferated in vitro,and then multi-directional osteogenic/adipogenic/chondrogenic differentiation abilities appraisal were conducted under certain conditions.Full-length DANCR cDNA was inserted into pcDNA3.1 mammalian expression vector(pcDNA3.1-DANCR).Cell viability was estimated at 1,2,3,4,5,6 and 7 days after DANCR transfection using the CCK-8 assay.The passage 3 cells were cultured in the chondriogenic medium for 14 days.And the cells were examined by toluidine blue staining.The chondrogenic-specific marker genes Aggrecan,Type II collagen(Col2)and Sox9 were determined by Real-time PCR.The corresponding target gene was constructed by the construction of plasmid,lentivirus transfection,siRNA transfection.Using BLAST,we find that DANCR associated with myc,STAT3 and Smad3 mRNA and regulated their stability.RNA immunoprecipitation and Real-time PCR to confirm the correlation between DANCR and myc,STAT3 and Smad3,and to explore the mechanism of myc,STAT3 and Smad3 in chondrogenic differentiation and proliferation of human synovium-derived mesenchymal stem cellResults:The obtained cultured synovial-derivedmesenchymal stem cells were in uniform morphology,and distributed in flat or swirl shape,besides,under certain induction conditions,it can be differentiated to osteogenesis,adipogenesis and chondrogenesis different directions.CCK-8 assay and toluidine blue staining shows overexpressing DANCR significantly promoted proliferation in SMSCs.The aggregates from DANCR-SMSCs had a greater amount of toluidine blue staining than control group.In addition,we detected the expression of chondrogenic specific marker genes,such as Col2,Sox9 and Aggrecan.our data suggested that DANCR directly interacted with myc,Smad3 and STAT3 mRNA and regulated their stability.Finally,we found that the promotion of SMSCs proliferation induced by DANCR was depended on myc,and DANCR activated chondrogenesis of SMSCs via Smad3 and STAT3.Conclusion:The results showed that In vitro culture of SMSCs cells were homogeneous,spindle-shaped distribution,and has the ability to multi-directional differentiation.LncRNA DANCR could promote proliferation and chondrogenesis of SMSCs.our data suggested that DANCR directly interacted with myc,Smad3 and STAT3 mRNA and regulated their stability.The results showed that the promotion of SMSCs proliferation induced by DANCR was depended on myc,and DANCR activated chondrogenesis of SMSCs via Smad3 and STAT3.