The Effect Of KDM6A On Chondrogenic Differentiation Of Human Periodontal Ligament Stem Cells

Objectives:Cartilage defect is frequently caused by inflammation,trauma,tumor,etc.And,the damaged cartilage cannot fully regenerate due to the intrinsic healing capacity to reconstitute its integrated matrix and regenerate native surface.Recently,stem cell induced tissue regeneration has emerged as a promising strategy for cartilage regeneration or repair.Recently,a new population of MSCs has been isolated from dental and craniofacial tissues,and numerous studies demonstrate that dental stem cells showed superior proliferation capacity and multipotent differentiation potential compared with bone marrow mesenchymal stem cell.The mechanism of MSC differentiation is complicated,and covalent histone modifications play an important role.Previous study showed that the level of H3K27me3 on SOX9 promoter was significantly increased.KDM6 A and KDM6 B are the main histone lysine demethylases,and KDM6 A has been demonstrated critical in the regulation of cell fates.However,whether KDM6 A is involved in cartilage formation remains unclear.Content:In this study,PDLSCs were employed to investigate the function of KDM6 A and the change of miRNAs in this process.Our results showed that depletion of KDM6 A repressed the chondrogenic differentiation potentials in PDLSCs by increasing the histone H3K27me3 level and decreasing the H3K4me3 level.Methods:Healthy periodontal ligament(PDL)was obtained from patients(age 16-20 years)who had no history of periodontal disease and exhibited a relatively healthy periodontium.KDM6 A shRNA was transfected into PDLSCs by lentivirus,and knockdown was confirmed via real-time RT-PCR.The chondrogenic differentiation potential of PDLSCs was assessed by Alcian blue staining and quantify synthesis.SOX9,Col2a1,ACAN and miRNAs(miR-29 a,miR-204,miR-211)were detected by real time RT-PCR.Immunofluorescence and western blot were performed to demonstrate H3K27me3 and H3K4me3 levels during chondrogenesis.The inhibitor of H3K27 methyltransferases(EZH2)was used to examine the influence on chondrogenesis of PDLSCs after knockdown of KDM6 A.Results:The results showed that depletion of KDM6 A had no effect on cell viability and apoptosis of PDLSCs.The Alcian blue and Sirius red staining results showed that production of proteoglycans and collagen in PDLSCs was decreased after knockdown of KDM6 A.Western blot showed that depletion of KDM6 A resulted in decreased expression of SOX9.And real-time PCR showed that depletion of KDM6 A inhibited the expression of SOX9,Col2a1,ACAN,and immunofluorescence showed that knockdown of KDM6 A resulted in increased H3K27me3 and decreased H3K4me3 levels.EZH2 inhibitor rescued the chondrogenic potential of PDLSCs after knockdown of KDM6 A by regulating H3K27me3.Conclusions:KDM6A is required in chondrogenic differentiation of PDLSCs,and plays a positive role.The mechanism may be that KDM6 A affects the level of histone methylation in genes involved in chondrogenesis,such as SOX9.Application of EZH2 inhibitor(EPZ-6438)may reverse or partially reverse the level of histone methylation caused by KDM6 A knockdown.It could be inferred that upregulation of KDM6 A or application of EZH2 inhibitor be of far-reaching significance for the future therapies in cartilage tissue destruction such as osteoarthritis.