| Background and objectives:With the accomplishment of Human Genome Project, study of gene functions has become the focus of life sciences. Epigenetics,investigating heritable gene function changes that don't lead to changes in the sequence of nuclear DNA, will become one of the crucial fields, providing when, where and how to use genetic information to determine the activation or silence of genes. Its mechanisms include DNA methylation and histone modifications (such as methylation, acetylation, phosphorylation, ubiquitination and glycosylation), which determine regional chromatin structure; DNA methylation is the final exhibition of every epigenetic modification.In the mammal, DNA methylation only occurs on cytosine residues found within cytosine and guanine (CpG) dinucleotide and involves methylation in the fifth carbon of DNA pyrimidine ring, resulting in the formation of 5-methylcytosine. Methylation of CpG islands, located in the promoter and sometimes extending to the first extron of genes, is closely correlated with silence of genes.Immune system is regarded as a proper model for studying the epigenetic regulation mechanism. Immune cell differentiation and function are associated with epigenetic modification. Related cytosine genes can be methylated to stimulate the differentiation of CD4+T cell subsets. Many autoimmune diseases are involved in the activation of autoreactive lymphocytes and polarization of CD4+T cell subsets, thus turning on the epigenetic regulation to the gene expression.Rheumatoid Arthritis (RA) is a chronic, inflammatory and autoimmune disease, with its pathogenesis remaining unclear. Studies suggested that CD4+T cell subsets played an important role in the occurrence and development of RA. Many studies found imbalance of T-helper 1/T-helper 2 (Th1/Th2) in RA, and abnormal increase of Th1 cells and cytokines. Some research, with flow cytometry assay, detected that regulated T (Treg) cells in active RA were lower than those of healthy individuals, and T-helper 17 (Th17)-secreted IL-17 increased in both blood and synovial fluid of RA patients. In GPI-induced arthritis, GPI-specific CD4+T cells were found to be differentiated to Th1 and Th17 cells, but not Th2 cells. These findings gave clues that the imbalance of cytokines of Th1, Th2, Treg and Th17 cells might be the reason of progressive inflammation in RA. Recently hypomethylation of T cell genome and hypermethylation of death receptor 3 gene (DR3) were detected in RA patients. However, there was no report about the DNA methylation status of cytokines secreted by Th1, Th2, Treg and Th17 in RA patients. The DNA methylation profiling, as a remarkable character in mammal, were filtered to diagnose RA at its earliest stage.Epigenetic changes, without changing DNA sequence, bring hope to clinical therapy of RA. The inhibitor for DNA methyltransferase, 5-azacytidine (5-azaC), can demethylate the methylated DNA and restore transcription of genes. High dose homocystine (Hcy) can mediat DNA methylation and suppress gene expression. Therefore changing the methylation status by 5-azaC and Hcy can promote or suppress gene activity, which is helpful to alleviate RA symptom and find new target for the therapy.The study based on the imbalance of cytokines secreted by Th1, Th2, Treg and Th17 cells, analyzed the relationship between DNA methylation status and mRNA expression of IL-1β, TNF-α, IFN-γ, IL-4, IL-10 and IL-17 in PBMC of RA, and made the methylation profiling of inflammation-related genes in RA to identify the biomarkers that could be used to diagnose RA at its earliest stage. Moreover, peripheral blood mononuclear cell (PBMCs) of RA were treated with 5-azaC or Hcy in vitro, in order to explore roles of methylation in the occurrence and development of RA, and to get more information about RA's early diagnosis, development, prognosis estimation and new therapeutic target. Methods:1 Observing the imbalance of cytokines secreted by Th1, Th2, Treg and Th17 cells in peripheral blood of RA patients.Peripheral blood was obtained from 64 patients with active RA and 30 healthy individuals, and RA judgment was consistent with the revised criteria of the American College of Rheumatology (ACR) in 1987. The patients were divided into two groups according to the EULAR criterion: early RA (persistence≤2 years, n=34) and chronic RA ( persistence > 2 years,n=30); the ratio of male to female was 1: 3 and related clinical data were noted. PBMCs were isolated by Ficoll density gradient centrifugation, and mRNA and protein expression of IL-1β,TNF-α,IFN-γ,IL-4,IL-10 and IL-17 were evaluated with RT-PCR and ELISA respectively. Then relationship between the expression and disease active index (erythrocyte sedimentation rate, C reactive protein, rheumatic factor, time of morning stiff, number of arthritic joints) was analyzed.2 Methylation of inflammation related genes in RAExtracting the genome DNA from PBMCs of RA patients and healthy individuals, and modifying the DNA by sodium bisulfite. The cytosine bases of unmethylated DNA, were converted to uracils after sodium bisulfite, while methylated cytosines were not affected. Therefore, the treatment resulted in a sequence difference between methylated and unmethylated DNA. Subsequent PCR amplification, using primers specific for methylated or unmethylated DNA designed by MethPrimer software, could determine the methylation status of a given DNA sequence. The methylation status of IL-1β, TNF-α, IFN-γ, IL-4, IL-10 and IL-17 genes were detected by Methylation-specific PCR (MSP), and then the methylated and unmethylated PCR products were sequenced. Moreover, we analyzed the relationship between DNA methylation status and mRNA expression, the relationship between DNA methylation status and disease active index (erythrocyte sedimentation rate, C reactive protein, rheumatic factor, time of morning stiff, Number of arthritic joints). The methylation profiling of inflammation related genes in RA was made to identify the biomarkers that could shed some light on the diagnosis of RA at its earliest stage. Finally the roles of DNA methylation in RA development were investigated.3 Exploring the reversal role of 5-azaC in DNA methylation and transcriptional regulation of IL-4 and IL-10 genes.PBMCs of RA were cultured in vitro and treated with 1μmol/L, 5μmol/L and 10μmol/L 5-azaC for 3 days or 5μmol/L 5-azaC for 1day, 2 days, 3 days and 4 days, respectively. The methylation status of IL-4 and IL-10 genes, whose promoters were hypermethylated in RA, were tested by MSP before and after 5-azaC treatment, and their mRNA and protein expressions were evaluated with RT-PCR and ELISA, respectively. Then the relationship between DNA methylation and gene transcription was discussed. The binding activity of IL-10 promoter and p-CREB before 5μmol/L 5-azaC treatment and 2 days after the treatment was examined by chromatin immunoprecipitation (CHIP). The methylation percentage of 5 CpG sites in IL-10 promoter was calculated by Pyrosequencing method. Meanwhile, MeCP2,DNMT1 and mbd2 mRNA changes were detected by RT-PCR. 4 Examining the influence of Hcy on DNA methylation and mRNA expression of IL-1β, TNFαand IL-17 genes.PBMCs of RA were cultured in vitro and treated with both 50μmol/L, 100μmol/L, 200μmol/L Hcy for 3 days and 200μmol/L Hcy for 1day, 2 days and 3 days. The methylation status of IL-1β, TNFαand IL-17 genes, whose promoters were hypomethylated in RA, were detected by MSP before and after Hcy treatment, and the mRNA and protein changes of those genes were evaluated with RT-PCR and ELISA respectively. MeCP2,DNMT1 and mbd2 mRNA changes were tested by RT-PCR as well. Then the relationship between DNA methylation and gene transcription was studied.5 Genetics investigation.Peripheral bloods were got from 7 standard paternity families (biological father, mother and child), from which genome DNA was extracted. The DNA was modified by sodium bisulfite. MSP was used to examine the methylation status of IL-1β, TNF-α, IFN-γ, IL-4, IL-10 and IL-17 genes. We explored the inheritable way from parents to children and discuss the possible generation mechanism of DNA methylation.Results:1 The mRNA and protein levels of inflammatory cytokines such as IL-1β, TNF-α, IFN-γand IL-17 were significantly higher in the RA patients than the healthy control (P <0.05), in early RA than chronic RA (P <0.05). In contrast, anti-inflammatory cytokines such as IL-4,IL-10 proved the opposite. There were statistical correlations between mRNA level of IL-1 and the number of arthritic joints (r=0.581, P <0.05), between mRNA and protein levels of IFN-γand ESR (r=0.466, P <0.05) (r=0.471, P <0.05), between the mRNA level of IL-4 and persistence (r=0.468, P <0.05).2 IL-1β, IL-10, TNF-αand IL-17 promoter methylation and their mRNA expression showed a strong negative correlation (r=-0.401 , P <0.05) (r=-0.579,P <0.01) (r=-0.452,P <0.05) (r=-0.414,P <0.05). However, there were no statistical correlations between IFN-γ, IL-4 promoter methylation and their mRNA expression (r=-0.017, P >0.05) (r=-0.154, P >0.05).3 Compared with the healthy control, IL-10 promoter was hypermethylated (6.67% versus 50%, P <0.05), but IL-1β, TNF-αand IL-17 promoters were hypomethylated in RA (82.61% versus 33.33%, 50%versus 6.67%, 85.42% versus 4 6.67%; P <0.05). They casted some light on the diagnosis of RA at its earliest stage.4 There were no significant difference between IL-4 and IL-10 methylation status before and after 1μmol/L 5-azaC treatment, but changes became distinct when PBMCs are treated with 5μmol/L and 10μmol/L 5-azaC. The mRNA and protein levels of IL-4 and IL-10 increased when PBMCs were treated with 1μmol/L and 5μmol/L 5-azaC, but droped with 10μmol/L 5-azaC treatment. The binding activity between IL-10 promoter and p-CREB increased 2 days after 5μmol/L 5-azaC treatment, and -62, -41, -39, -9 CpG sites in IL-10 promoter were reversed, but +222 CpG site did not decline significantly.5 200μmol/L Homocysteine (Hcy) could mediate aberrant DNA methylation of IL-1β, TNF-αand IL-17 genes and repressed their mRNA expression. Although 50μmol/L, 100μmol/L Hcy didn't influence DNA methylation status, they could promote their mRNA expression.6 The mRNA levels of mbd2 increased when treated with 5-azaC, but the mRNA levels of MeCP2, DNMT1 decreased; Hcy treatment produced the opposite result. The methylation status and mRNA levels of IL-4, IL-10, IL-1β, TNF-αand IL-17 weren't correlated with the mRNA levels of mbd2, MeCP2 and DNMT1 (P>0.05).7 The inheriting way of methylation status of IL-1β, TNF-α, IFN-γ, IL-4, IL-10 and IL-17 was different from Mendel law and genetic imprinting genes which are parentally dependent and inherited from parents to children.Conclusions:1 Among early RA patients, Th1 and Th17 type cytokines prevailed; so did Th2 and Treg type cytokines in chronic RA patients. With the progress of RA, IL-1β, TNF-α, IFN-γ, IL-17 protein levels declined while IL-4, IL-10 protein levels increased little by little. These indicated there were imbalances of Th1/Th2, Th17/Treg type cytokines in RA peripheral blood at the early and developing stage.2 DNA methylation took part in the imbalances of Th1/Th2, Th17/Treg type cytokines in RA process. Hypermethylation of IL-10 gene and hypomethylation of IL-1β, TNF-αand IL-17 genes suggested a possibility of RA.3 The upstream region such as -62, -41, -39, -9 CpG positions in IL-10 promoter might be crucial regulation sequences.4 5-azaC or Hcy could reverse DNA methylation to restore or inhibit their expression. The method to interfere with lymphocyte inflammation related cytokines genes methylation in vitro was explored to provide experimental data for seeking new therapeutic target and establishing method of selfcell recycle into body.
|
PDF Full Text Request