MiR-17?92 Cluster Mediated HDAC9 Effects On The Osteogenetic Capability Of The Inflammatory PDLSCs

?Background? Periodontal disease is one of the commonest oral diseases which can lead to gingival bleeding, alveolar resorption, tooth loss and even periodontal festering at the advanced stage. But traditional treatments cannot cure alveolar absorption radically. At present, the emerging stem cell therapy brings new hope for the reconstruction of alveolar bone. When inflammation occurs, the genome sequences in cells have no change, but their expression is very different from that of normal cells. This phenomenon may results from the changes of epigenetic modifications. In present study, we focus on that how to facilate the osteogenic differentiation of periodontal ligament stem cells for treating alveolar resorption caused by periodontal diseases from the perspective of epigenetic inheritance. Epigenetic modification is the covalent modification of chromosome, which don't change the sequences of base pairs, but can lead to the stable changes of genetic phenotype[1].Histone acetylation belongs to epigenetic modification. Histone deacetylases(HDACs) can remove the acetylated group binding on histone lysine site, which hindersthe binding of transcription factors and DNA promoter. Consequently, the transcription process is repressed. Micro RNA(mi RNA) is endogenous non-coding small RNAs with the length of 19 to 25 nt, and it can complementatly pair with its targeted m RNA, and inhibit the translation of m RNA or degrade targeted m RNA directly, resulting in the negative regulation on the targeted gene expression. It was reported that HDAC inhibitor activated an apoptotic mechanism mediated by mi R-15 and let-7 [3]. This result indicates that HDACs may have effects on the mi RNA to regulate the cellular function. Liu Yali et al. have proved that TNF-?, mi R-17 and its target genes Smurf1 in the periodontal ligament stem cells formed a feedback regulation in the inflammatory microenvironment, which contributed to the osteogenic differentiation of the inflammatory periodontal ligament stem cells[4]. In the present study, we evaluated the influence of HDACs on the osteogenic differentiation of inflammatory periodontal ligament stem cells, moreover, we explored whether mi R-17-92 cluster could mediated the influence of HDAC on the osteogenic differentiation of inflammatory periodontal ligament stem cells.?Objective? Study that HDAC9 effects on the osteogenic differention of i-PDLSCs, and explore if the effects are mediated by mi R-17~92 cluster.?Methods? a) Detecting the differences of HDACs expression in the inflammatory periodontal ligament stem cells and the healthy periodontal ligament cells by q RT-PCR and Western Blotting. b) After transfection of HDAC9 si RNA into inflammatory periodontal ligament stem cells, evaluating the osteogenic capability of cells by q RT-PCR, Western Blotting and alizarin red staining methods. c) After transfection of mi R-20 a mimics, using q RT-PCR, Western Blotting methods and alizarin red staining method to identify the osteogenic capability of cells. a) After transfection of HDAC9 si RNA, using Taqman PCR method to detect the change of the expression of pri-mi R-17-92 family, and q RT-PCR method to test the expression of the mature pri-mi R-17-92 family members, and WesternBlotting method to observe the acetylation level of H3K9 and H4K16.?Results? a) The results have showed that the expression of HDAC9 in inflammatory periodontal ligament stem cells is higher than that in healthy periodontal ligament stem cells, and there was no significant difference in other HDACs. b) After silencing the expression of HDAC9, the results of q RT-PCR, Western Blotting and alizarin red staining tests have proven that the osteogenic capability of inflammatory periodontal ligament stem cells partially recovered. c) After transfection of mi R-20 a mimics, the osteogenic capability of inflammatory periodontal ligament stem cells increased. d) After silencing the expression of HDAC9, the expression of the pri-mi R-17-92 in inflammatory periodontal ligament stem cells was upregulated, and the expressions of the mature members including mi R-17, mi R-19, mi R-20 a, mi R-92 a were also upregulated. Whereas the mi R-18 expression showed no significant difference. The acetylation level of the H4K16 and H3K9 in inflammatory periodontal ligament stem cells improved.?Conclusion ? The expression of HDAC9 in inflammatory periodontal ligament stem cells was upregulated compared to healthy periodontal ligament stem cells, which may inhibit the expression of mi R-17-92 cluster, leading to the inhibition of the osteogenic differentiation.