Screening And Analysis Of Epigenetic Enzymes Related To Periodontitis

Periodontitis is a common multiple infectious disease which is caused by bacteria and it often encroaches the gingiva, parodontium and alveolar bone. If not treated in time, it will lead to the exfoliation of teeth. Now it has become the main reason for tooth exfoliation in adults in our country and its diagnosis and treatment have become a topic with widespread concern. Currently, the diagnosis and assessment of Periodontitis are usually based on the clinical symptoms, oral history and anamnesis of patients. However, some researches have shown that using the same treatments for patients with the same clinical manifestation, the prognoses are not consistent. In addition to genetic predisposition, the factors related to diet, smoking, environment, and bacteria also can affect oral health by influencing genes that involved in the inflammatory response. The above factors are all correlated with epigenetics, which reminds us that epigenetics may be involved in the occurrence and development occurrence of Periodontitis.Epigenetics is proposed relatively to genetics. There are no changes in DNA sequences, but the gene expressions change heritably. It’s kind of changes in the heritable substances other than the genetic information and can be inherited stably in the development and proliferation of cells. Epigenetics includes DNA methylation, histone modifications and chromatin remodeling and other aspects, while DNA methylation and histone modifications are two main ways of Epigenetics. Now some studies suggest that there is a correlation between Epigenetics and many diseases such as cancer, cardiovascular disease, neurodegenerative diseases and rheumatoid arthritis. Recently, more and more studies indicate that Epigenetics is correlated with periodontal disease, and influence the occurrence and development of Periodontitis by regulating cytokines. However, the relation between Epigenetics and Periodontitis is still unclear and its mechanism in the pathogenesis of Periodontitis needs further study. In our reaearch, we aim to screen out the main epigenetic enzymes that related to periodontitis and study its functions. This will be helpful to interpret the development of periodontitis and provide theory for the diagnosis and molecular targeted therapiesObjectiveThis study aims at screening out the epigenetic related enzymes that change obviously in the process of periodontitis from i-PDLSCs and PDLSCs of the same patients and studying the effect of these enzymes on the proliferation of PDLSCs, providing evidence to interpret the relations between epigenetics and periodontitis.Methods1. The isolation, culture and identification of i-PDLSCs and PDLSCsThe i-PDLSCs and PDLSCs were isolated respectively from extracted teeth of periodontitis and the healthy third molar teeth of the same patients through collagenase treatment. Then the cells were purified by limiting dilution method. Stem cell-related antigens were analyzed by flow cytometry, proliferation were evaluated by MTT and Colony-Forming Units(CFU) rates and multi-differentiation potentials were assessed by osteogenic and adipogenic induction.2. The screen of epigenetic enzymes related to periodontitisThe total number of epigenetic enzymes that had been reported was 72(chart 1). The total RNA obtained from i-PDLSCs and PDLSCs were analyzed by RT-PCR and the enzymes that changed obviously were screened out. Then the PDLSCs were treated with inflammatory factor TNF-α,and the RNA of control groups and experimental groups were collected to give RT-PCR analysis to find out the epigenetic enzymes that changed patently. Finally, the target epigenetic enzymes that associated with periodontitis would be choose according to screening results, previous reports and related research in our group.3. The effect of KAT2 A on the proliferation of PDLSCsIn order to study the effect of epigenetic enzymes on PDLSCs, the expression of KAT2 A in i-PDLSCs and PDLSCs were assessed by WB. Then we silenced the expression of KAT2 A and checked the results. Once the silence worked, we compared the proliferation of cells in groups before and after silence by the analysis of MTT, cell proliferation cycle and expression of CCD1, CCE1 and E2F1 that related to reproduction.Results1. The teeth from 12 patients were collected for cell culture and the cells of 8for i-PDLSCs, 10 for h PDLSCs and 7 for both were cultured successfully. Adherent cells appeared at 3-7 days, shaped like spindle and formed colonies. Then the cells were purified by limiting dilution method cultured for enlargement. The flow cytometry analysis showed that stem cell-related antigens CD105,CD146 were positive and CD31,CD31 were negative. Osteogenic and adipogenic inductions approved the multi-lineage potentials of PDLSCs. These all showed that the cells we isolated and cultured were highly purified functional PDLSCs.2. The total RNA of i-PDLSCs and PDLSCs were analyzed by RT-PCR to assess the expression of epigenetic enzymes and the results showed different expression of enzymes in groups of i-PDLSCs and PDLSCs. According to the results of RT-PCR and related study of our research group, we confirmed that the family of histone acetyltransferase was the important target and its expression was higher in PDLSCs than that in i-PDLSCs.The analysis of data from all the samples suggested that the expression of KAT2A、ELP3、GTF3C4、HAT1、NCOA3、KAT6A, KAT6 B had the same regular patterns in all the samples, the expression of KAT2A、ELP3、HAT1、KAT6A and KAT6 B were higher in groups of PDLSCs and lower in groups of i-PDLSCs. At the same time, NCOA3 and GTF3C4 showed a low level in groups of PDLSCs and a high level in groups of i-PDLSCs. The screening results were in line with above ones when PDLSCs were stimulated with TNF-α. That was to say, the expression of histone acetyltransferase decreased in inflammatory environment. To sum up, the expression of KAT2 A, KAT6 A and KAT6 B had similar trend in all the tests : they showed high-expression in groups of PDLSCs and control groups without TNF-α stimulation, while they showed low-expression in groups of i-PDLSCs and experimental groups with TNF-α stimulation.3. KAT2 A was chosen as the epigenetic enzymes that related to periodontitis according to the screening results and related reports. After silencing the expression of KAT2 A bysi-RNA, the data from WB and RT-PCR showed the silence efficiency was up to 50%. Then we assessed the proliferation of these cells by MTT, cell proliferation cycle analysis and expression of proliferation related genes and all the results indicated decrease in proliferative ability.Conclusion1. PDLSCs and i-PDLSCs were isolated and cultured successfully. The flow cytometry analysis and results of osteogenic and adipogenic induction both indicated their mesenchymal stem cells’ characterization. 2. Inflammatory conditions could affect the expression of epigenetic enzymes. Most of the histone acetyltransferase showed decreased expression in inflammatory environments and the expression of KAT2A、KAT6A and KAT6 B had the same trend in all the tests. 3. Histone acetyltransferase KAT2 A had relations with the proliferative capacity of PDLSCs and silence of KAT2 A could decrease proliferation.