Study On The Relationship Between DNA Methylation And DNA Demethylase In Tuberculosis Patients

Pulmonary tuberculosis (PTB) is one of the chronic inflammatory diseases caused by Mycobacterium tuberculosis (M.tb). It is closely related with the body’s cells immune response. During the development of PTB, the cell mediated response plays an important role. In the process of mycobacterium tuberculosis infection, antigen presenting cells (APC) present the antigen of mycobacterium tuberculosis by double ways of MHC molecules complex and CD28-CD80 to T cells to activate them. And at the same time, the APCs secret IL-12、IL-1 and so on in order to further stimulate immune cells to release the efficient molecular TNF-α which could kill the mycobacterium tuberculosis.DNA methylation is a kind of important form of epigenetic modifications in many organisms, either in animals or in plants. It has important biological influence on the development and the occurrence of diseases. DNA methylation realizes its modification function mainly through raising and lowering its methylation level and subsequently affects the gene expression. DNA methylation is mainly through DNA methyltransferases (mainly including DNMT1, DNMT3A and DNMT3B) to raise its DNA methylation level, and it is mainly through DNA demethylases (mainly includingTET1、TET2、TET3、 TDG) to lower its methylation level. The two aspects of regulation togetherly maintain the level of DNA methylation.In our early work, we preliminarily illuminate that DNA methylation level is downregulated in the tuberculosis (TB) infection using the method of genome DNA methylation chip. In order to illuminate the molecular mechanisms of DNA methylation adjustment and find out the influence factors of downregulated DNA methylation trend, on the basis of previous research findings, our research firstly collect healthy controls (IGRA negtive) and active tuberculosis patients’ whole blood RNA to quantitatively detect four genes of DNA demethylases which could forwardly downregulate the DNA methylation level (mainly including TET1, TET2、TET3 and TDG) by means of Real-time PCR method. In this section, we purposely want to illustrate the relationship between the DNA methylation and the DNA demethylases.The results show that all the four genes (TET1、TET2, TET3, TDG, DNMT1) that downregulate DNA methylation level appear upexpressed in the active tuberculosis patients (p<0.05). In order to comprehensively discuss the influence effectors of low DNA methylation trend, we additionally detect the three members of DNA methyltransferases which could upregulate DNA methylation level. The result shows,in the TB group, only DNA methyltransferases 1 (DNMT1) is differentially upexpressed(p<0.05). In order to further validate our findings, subsequently we use H37Rv cracking antigen and positive stimulus (5azac) which could make the DNA methylation level drop to stimulate A549, Beas-2b two cell lines to conduct the validation experiments in vitro. And then we collect the stimulated cell DNA, RNA of different timepoints. Use the DNA samples to process methylation sensitive restriction analysis (MSRA) test in order to confirm the DNA methylation level after stimulation. The result of MSRA shows the H37Rv antigen and 5-azac positive stimulus can cause DNA methylation level’s drop as the genome DNA methylation chip shows, and it drops more as the time goes. In order to detect the expression of DNA demethylases and DNA methyltransferases, we make use of the RNA samples to test the genes by the method of Real-time PCR as mentioned before. The Real-time PCR detections reflect that the DNA demethylases are increased as the time grows in the H37Rv-sti groups of both A549 and Beas-2B. And on the fourth day, TET2 and TET3 are differently up-regulation expressed in the H37Rv-sti group of A549 cellline. And TDG is differently increased in the H37Rv-sti group of Beas-2B cellline. And the expression of DNMT3A、DNMT3B is differently increased in the H37Rv-sti group of A549 cell line. But DNMT3A is differently down expressioned in the H37Rv-sti group of Beas-2B cell line.In conclusion, DNA methylation is on the decline as the DNA demethylases are increasingly expressed in both active tuberculosis patients and H37Rv-sti cells. And as the time goes, Concerning the DNA methyltransferases, the results show the expression of DNA methylationtransferases is not specific. In a word, according to the above results we finally answered the following scientific questions. The occurrence and development of active tuberculosis disease are closely associated with the host low DNA methylation trend. The difference change of DNA methylation level in tuberculosis patients is closely related with DNA demethylases. And it is the chief enzymes to drop the of DNA methylation level.