Effect Of KDM6A On Expression Of MiR-204 And MiR-211 In Chondrogenic Differentiation Of Periodontal Ligament Stem Cells

Objective Cartilage tissue has limited ability of self-healing,and cannot be completely repaired after tissue injury.Traditional treatment methods cannot achieve satisfactory clinical results.In recent years,mesenchymal stem cells differentiation has provided an effective strategy for the repair and regeneration of cartilage.There is evidence that epigenetic regulation,including histone posttranslational modification and Micro RNA play an important role in the regulation of mesenchymal stem cell differentiation.However,there are few studies on the interaction between epigenetics and mi RNA to regulate the chondrogenic differentiation of mesenchymal stem cells.This study intends to investigate the effects of KDM6 A on the expression of Micro RNA204 and Micro RNA211 during the chondrogenic differentiation by changing the level of histone H3 lysine 27 trimethylation(H3K27me3)in periodontal ligament stem cells.The basic research evidence is provided to promote the clinical transformation of cartilage tissue regeneration.Method1.The premolar and the complete third molars were collected,and the periodontal ligament stem cells were isolated and cultured by enzyme digestion.2.Transfection of the lentivirus to establish a stable KDM6 A knockout cell line.3.PDLSCs treated with EZH2 inhibitors and PDLSCs knockdown of KDM6 A were cultured and chondrogenesis,respectively.Evaluated the Chondrogenic differentiation ability of them by Alcian blue staining and proteoglycan quantitative experiments.Immunofluorescence staining showed the expression of H3K27me3 in the periodontal ligament stem cells.4.The expression of transcription factor RUNX2 and mi R-204,mi R-211 in PDLSCs were detected by Real-Time PCR,which were treated with EZH2 inhibitors or knockdown of KDM6 A.The quantitative data were statistically analyzed by s Single factor analysis of variance.When the P value was less than 0.05,the representative was statistically significant.Result1.After two weeks of chondrogenic differentiation,The Alcian blue staining and proteoglycan quantitative analysis showed that the production of proteoglycan is low in PDLSCs knockdown of KDM6 A,the production of proteoglycan is nosignificant change in PDLSCs treated with EZH2 inhibitors compared with the control group.2.The expression of H3K27me3 in PDLSCs was increased after knockdown KDM6 A and the expression of H3K27me3 was reduced after EZH2 inhibitor treatment by immunofluorescence3.RT-PCR results showed that Runx2 expression increased in the third days of chondrogenic differentiation,decreased in 1,2 and 3 weeks of the normal PDLSCs.The expression of Runx2 was significantly reduced at 0,3,and 2 weeks compared with the control group after knockdown KDM6 A.The expression of Runx2 in PDLSCs increased at 3 days and 3 weeks,decreased at 2 weeks after treatment with EZH2 inhibitor.The difference was statistically significant.(P <0.05,n=3).During 3 weeks of chondrogenic differentiation,real-time PCR showed that the mi R-204 and mi R-211 in the normal PDLSC group decreased slightly at first weeks in the chondrogenic differentiation,and increased in 2 and 3weeks.After knockdown of KDM6 A,the levels of mi R-204 and mi R-211 were decreased compared with the control group(P< 0.05,n=3).The levels of mi R-204 and mi R-211 increased slightly after treatment with EZH2 inhibitors,and the difference was not statistically significant.Conclusions1.KDM6 A promotes periodontal ligament stem cells chondrogenic differentiation by reducing the level of H3K27 trimethylation.2.KDM6 A promotes the expression of mi R-204 and mi R-211 in the chondrogenic differentiation of periodontal ligament stem cells,which may be one of the mechanisms regulating the chondrogenic differentiation of periodontal ligament stem cells.