ASXL2 Is Required For Osteogenic Differentiation Of Periodontal Ligament Stem Cells By Regulating H2AK119 Ubiquitination And H3K27 Trimethylation

Objective: Periodontitis is a common infectious disease in oral cavity initiated by plaque biofilm.Periodontal pathogen and their products directly or indirectly damage periodontal supporting tissue,and ultimately lead to tooth loss.Periodontal infection endangers the overall health and quality of life of patients.Therefore,controlling inflammation and repairing periodontal tissue defect play a pivotal role in periodontitis treatment.However,how to find an effective way to promote periodontal tissue regeneration is the key problem to be solved.In the previous study,we found that epigenetic modification can significantly affect the osteogenic differentiation of periodontal ligament stem cells(PDLSCs),which provides a possibility for the clinical application of PDLSCs in the treatment of periodontal diseases.The purpose of this study was to explore the regulation of ASXL2 on the osteogenic differentiation of PDLSCs and its epigenetic mechanism,and to provide new strategy for clinical translation of periodontal tissue regeneration.Methods:1.Design and synthesize sh RNA targeting ASXL2,construct lentiviral vector and package lentivirus then transfect PDLSCs.2.Identification of PDLSCs cell surface markers by flow cytometry.3.The proliferation activity and apoptosis of PDLSCs were detected by CCK8 and Annexin V method,respectively.4.Assessment of osteogenic differentiation ability by alkaline phosphatase staining and alizarin red staining.5.Expression of osteogenic genes were detected by RT-PCR.6.Western blot analysis was performed to assess changes of osteogenic protein and histone modificatons.7.RNA-seq analysis was performed to analyze the regulation mechanism of ASXL2 on the self-renewal and differentiation ability of human PDLSCs.Results:1.Alkaline phosphatase staining and Alkaline phosphatase activity was decreased in ASXL2 sh PDLSCs.The Alizarin red staining and the calcium ion concentration was significantly reduced in ASXL2 sh PDLSCs.2.Real-time RT-PCR showed that osteogenic related genes COL1A1,RUNX2,ALP,OCN and adipogenic related genes DGAT1 were significantly decreased in ASXL2 sh PDLSCs after osteogenic or adipogenic induction in vitro.3.Western blot results showed that the expression of OCN in ASXL2 sh PDLSCs decreased after osteogenic induction for 14 days.4.Western blot showed that the expression of H2AK119 ub and H3K27me3 increased and H3K4me3 decreased after osteogenic induction in ASXL2 sh PDLSCs.5.KEGG enrichment analysis showed that differentially expressed genes(DEGs)enriched in osteogenic related signaling pathways after ASXL2 knockdown.GO analysis showed that DEGs are involved in various molecular functions and cell components and biological processes.Conclusions:1.ASXL2 is required in osteogenic differentiation capability of proliferation of PDLSCs in vitro and inhibits the apoptosis of periodontal ligament stem cells.2.ASXL2 is required in the osteogenic differentiation of PDLSCs in vitro.3.ASXL2 may promote the transcription of osteogenic genes by down-regulating H2AK119 ub and H3K27me3 modification levels in the osteogenic gene promoter region and up-regulating H3K4me3 modification levels in the osteogenic gene promoter region.4.ASXL2 may regulate fate determination of PDLSCs by affecting multiple osteogenic signaling pathways,cellular components and molecular functions.