Lead exposure in pheochromocytoma cells induces persistent changes in amyloid precursor protein gene methylation patterns

It has been suggested that lead (Pb) exposure in early life may increase amyloid precursor protein (APP) expression and promote the pathogenesis of Alzheimer's disease in old age. The current study examined whether the DNA methylation patterns of APP gene in rat pheochromocytoma (PC12) cells changed after Pb acetate exposure. Undifferentiated PC12 cells were exposed to three doses of Pb acetate (50, 250, and 500 nM) and one control for 2 days or 1 week. The methylation patterns of APP promoter and global DNA methylation were analyzed. The DNA methyltransferase 1 (DNMT1) expression and the level of amyloid β peptide (Aβ) were also investigated. The results showed that the exposure of the three concentrations of Pb acetate could make the APP promoter hypomethylated. The global DNA methylation level and the expression of DNMT1 were changed in the 500 nM group after 2 days exposure and in the 250 and 500 nM group after 7 days exposure. Thus, Pb may exert neurotoxic effects through mechanisms that alter the global and promoter methylation patterns of APP gene. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.     

DNMT1通过下调SOX1表达参与宫颈癌生长和转移的机制研究

目的 探讨DNA甲基转移酶1（DNMT1）通过调节性别决定区Y-框1（SOX1）表达对宫颈癌（CC）生长和转 移的影响。方法 采用亚硫酸氢盐定量PCR测定CC组织和细胞及CC癌旁组织和正常宫颈细胞中SOX1甲基化水 平,Western blot检测DNMT1、SOX1蛋白表达。将HeLa229和SW756细胞分为DNMT1-NC组、DNMT1-siRNA组和空 白组,采用亚硫酸氢盐定量 PCR 测定 SOX1 甲基化比例,Western blot 检测 DNMT1、SOX1、c-Myc、Cyclin D1 及 β- catenin蛋白表达,细胞克隆和裸鼠成瘤实验测定细胞增殖,Transwell实验测定细胞迁移和侵袭。结果 CC组织较癌 旁组织DNMT1表达水平及SOX1甲基化比例增高,SOX1蛋白表达降低（P＜0.05）;宫颈癌HeLa229、ME-180、SiHa、 SW756细胞较宫颈上皮Ect1/E6E7细胞DNMT1表达水平及SOX1甲基化比例增高,SOX1蛋白表达降低（P＜0.05）。 与空白组、DNMT1-NC组比较,DNMT1-siRNA组HeLa229和SW756细胞体外克隆形成数、迁移和侵袭数量减少,裸 鼠内瘤体质量减轻,DNMT1、c-Myc、Cyclin D1、核/质β-catenin蛋白表达及SOX1甲基化比例降低,SOX1蛋白表达增 加（P＜0.05）。结论 DNMT1在CC组织和细胞中高表达,可能通过维持SOX1启动区高甲基化并抑制其蛋白表达, 促进CC生长和转移。     

Mithramycin A inhibits DNA methyltransferase and metastasis potential of lung cancer cells

Abnormal CpG island hypermethylation of multiple tumor-suppressor genes (TSGs) can lead to the initiation and progression of human cancer. The cytosine of the CpG island on the promoter region is methylated by 5'-cytosine-methyltransferases (DNMTs). Pharmacologic inhibitors of CpG island methylation provide a rational approach to reactivate the TSGs in tumor cells and to restore the critical cellular pathways in cancer cells. Mithramycin A (MMA) is known to be a GC- and CG-rich DNA-binding agent. We sought to determine whether MMA could inhibit CpG island methylation and DNMT expression in lung cancer cells. We found that MMA reduced the CpG island methylation of antimetastasis TSGs, including SLIT2 and TIMP-3 genes, and was associated with the prevention of metastasis. When highly metastatic CL1-5 lung cancer cells were treated with low doses (10 nmol/l) of MMA for 14 days, they reexpressed mRNA levels for these genes. MMA also inhibited the invasion phenotypes of CL1-5 cells as indicated by its inhibition of cancer cell migration using wound-healing and transwell assays. Molecular docking of MMA onto the DNMT1 catalytic domain revealed that MMA might interact with the catalytic pocket of DNMT1. Western blots showed that DNMT1 protein levels were depleted after MMA. These data support the idea that MMA has demethylation and antimetastasis effects on lung cancer cells. This mechanism might be mediated by the interaction of MMA and DNMT1, leading to the depletion of the DNMT1 protein and the reversal of the metastasis phenotype in lung cancer cells.     

Epigenetic CpG Demethylation of the Promoter and Reactivation of the Expression of Neurog1 by Curcumin in Prostate LNCaP Cells

Curcumin (CUR), a major bioactive polyphenolic component from turmeric curry, Curcuma longa, has been shown to be a potent anti-cancer phytochemical with well-established anti-inflammatory and anti-oxidative stress effects. Chromatin remodeling-related epigenetic regulation has emerged as an important mechanism of carcinogenesis, chemoprevention, and chemotherapy. CUR has been found to inhibit histone acetyltransferase activity, and it was also postulated to be a potential DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitor. In this study, we show that when human prostate LNCaP cells were treated with CUR, it led to demethylation of the first 14 CpG sites of the CpG island of the Neurog1 gene and restored the expression of this cancer-related CpG-methylation epigenome marker gene. At the protein level, CUR treatment had limited effects on the expression of epigenetic modifying proteins MBD2, MeCP2, DNMT1, and DNMT3a. Using ChIP assay, CUR decreased MeCP2 binding to the promoter of Neurog1 dramatically. CUR treatment showed different effects on the protein expression of HDACs, increasing the expression of HDAC1, 4, 5, and 8 but decreasing HDAC3. However, the total HDAC activity was decreased upon CUR treatment. Further analysis of the tri-methylation of histone 3 at lysine 27 (H3K27me3) showed that CUR decreased the enrichment of H3K27me3 at the Neurog1 promoter region as well as at the global level. Taken together, our present study provides evidence on the CpG demethylation ability of CUR on Neurog1 while activating its expression, suggesting a potential epigenetic modifying role for this phytochemical compound in human prostate cancer cells.     

吸入氢气对脓毒症小鼠海马组织 DNA甲基化的影响

目的 评价吸入氢气对脓毒症小鼠海马组织的 DNA甲基化状态的影响。方法 54只健康雄性 C57BL/6小鼠采用随机数字表法分为 3组：假手术组（Sham组）、脓毒症组（Sepsis组）和氢气治疗组（Sepsis+H2组）,每组 18只。Sepsis组和 Sepsis+H2组采用盲肠结扎穿孔法（CLP）制备小鼠脓毒症模型,Sepsis+H2组小鼠于手术后 1 h和 6 h吸入用空气混合的 2%氢气 1 h,Sham组只开腹不进行盲肠结扎和穿孔。3组小鼠于假手术或 CLP后 1、3、7 d取小鼠海马组织,比色法测定全基因组 DNA甲基化水平;实时定量 PCR法检测 DNA甲基化转移酶（DNMTs,包括 DNMT1、DNMT3a和 DNMT3b）的 mRNA水平;Western blot法检测 DNMT1、DNMT3a和 DNMT3b蛋白表达水平。结果 与 Sham组比较,Sepsis组小鼠在建模后 1、3、7 d海马组织全基因组甲基化水平明显下降（P＜0.05）,DNMT1和 DNMT3a的 mRNA和蛋白表达水平升高,DNMT3b的 mRNA和蛋白表达水平降低（P＜0.05）;与 Sepsis组比较,Sepsis+H2组全基因组甲基化水平升高（P＜0.05）,DNMT1和 DNMT3a的 mRNA和蛋白表达水平下降,DNMT3b的 mRNA和蛋白表达水平升高（P＜0.05）。结论 吸入氢气可纠正脓毒症小鼠海马组织的 DNA甲基化紊乱状态,改善 DNA甲基化紊乱状态是氢气治疗脓毒症相关性脑病的重要机制之一。     

Effects of subchronic cadmium poisoning on DNA methylation in hens

Cadmium is a persistent pollutant that poses a threat to most biological organisms including birds. Although toxicity of cadmium is mainly linked to cancer, the mechanism of its carcinogenic activity remains poorly understood. Since DNA methylation is linked to cancer, we have examined the effect of cadmium on DNA methylation and DNMTs mRNA expression in hen liver and kidney. Sixty 50-day-old hyline-white hens were randomly allocated into 3 equal groups; a control group fed a basal diet, a low-dose group fed the basal diet spiked with 140 mg/kg CdCl₂, and a high-dose group fed the basal diet spiked with 210 mg/kg CdCl₂. After 60 days, liver and kidney samples were analysed for cadmium by FAAS, DNA methylation level by HPLC and DNMTs mRNA levels by semi-quantitative RT-PCR. The DNA methylation levels and the expressions of DNMT1 and DNMT3a mRNA in liver and kidney were significantly elevated by the cadmium treatment but there was no change in the expression of DNMT3b mRNA in the two tissues. The fact that cadmium increases DNA methylation and the expressions of DNMT1 and DNMT3a mRNA in liver and kidney suggests DNA methylation may be involved in the carcinogenic action of cadmium.     

Methylation of PARP-1 promoter involved in the regulation of nano-SiO2-induced decrease of PARP-1 mRNA expression

Nano silicon dioxide (nano-SiO₂) is becoming more and more widely applied in the fields of industry. The potential toxic effects of nano-SiO₂ and its hazard to human health are drawing more attention. The mRNA expression of poly(ADP-ribose) polymerases-1(PARP-1), a pivotal repair gene, has been decreased by nano-SiO₂ exposure. However, the effect of epigenetic modification on nano-SiO₂-induced low PARP-1 expression has not been reported. In this study, HaCaT cells with or without DNA methyltransferase 1(DNMT1) knock down were incubated with nano-SiO₂ and then further treated with DNMT inhibitor, 5-aza-2-deoxycytidine (DAC), which is a kind of key epigenetic modification reagents. Real-time Q-PCR and western blotting were used to examine the mRNA and protein expression of PARP-1. For promoter methylation status of PARP-1, methylation-specific PCR (MSP) and Bisulfite sequencing assay were performed. Results showed a dramatic decrease of PARP-1 expression on mRNA and protein level and a simultaneously obvious increase in the level of PARP-1 methylation in nano-SiO₂-treated cells compared to the control group. Further, the expression and promoter methylation of PARP-1 in HaCaT cells were restored following DNMT1 knock down, suggesting that the effects of PARP-1 promoter hypermethylation are mediated at least in part by DNMT1. Taken together, methylation of PARP-1 promoter might be involved in the regulation of nano-SiO₂-induced decrease of PARP-1 expression.     

Aberrant methylation accounts for cell adhesion-related gene silencing during 3-methylcholanthrene and diethylnitrosamine induced multistep rat lung carcinogenesis associated with overexpression of DNA methyltransferases 1 and 3a

To evaluate the significance of alterations in cell adhesion-related genes methylation during lung multistep carcinogenesis induced by the genotoxic carcinogens 3-methylcholanthrene (MCA) and diethylnitrosamine (DEN), tissue samples microdissected from MCA/DEN-induced rat lung carcinogenesis model were subjected to methylation-specific PCR to evaluate the DNA methylation status of CADM1, TIMP3, E-cadherin and N-cadherin. Immunohistochemistry was used to determine protein expression of CADM1, TIMP3, N-cadherin and the DNA methyltransferases (DNMTs) 1, 3a and 3b. E-cadherin hypermethylation was not detected in any tissue. CADM1, TIMP3 and N-cadherin hypermethylation was correlated with the loss of their protein expression during the progression of pathologic lesions. The prevalence of DNA methylation of at least one gene and the average number of methylated genes increased with the histological progression. DNMT1 and DNMT3a protein expression increased progressively during the stages of lung carcinogenesis, whereas DNMT3b overexpression was only found in several samples. Furthermore, DNMT1 protein expression levels were correlated with CADM1 methylation, and DNMT3a protein expression levels were correlated with CADM1, TIMP3 and N-cadherin methylation. The average number of methylated genes during carcinogenesis was significantly correlated with DNMT1 and DNMT3a protein expression levels. Moreover, mRNA expression of CADM1 significantly increased after treatment with DNMT inhibitor 5-aza-2′-deoxycytidine in CADM1-methylated primary tumor cell lines. Our findings suggest that an accumulation of hypermethylation accounts for cell adhesion-related gene silencing is associated with dynamic changes in the progression of MCA/DEN-induced rat lung carcinogenesis. We suggest that DNMT1 and DNMT3a protein overexpression may be responsible for this aberrant DNA methylation.     

Towards a pharmacology of DNA methylation.

DNA methylation plays an important role in controlling gene-expression programs. Increasing evidence indicates that the enzyme responsible for replicating the DNA methylation pattern, DNA methyltransferase 1 (DNMT1), has a role in cancer. In this article, it is suggested that DNMT1 is a multifunctional protein that has regulatory activities in addition to DNA methylation activity. These functions are assembled into one protein to ensure the coordinate replication of DNA and its methylation pattern. The regulatory activities of DNMT1 are proposed to be involved in cellular transformation and should, therefore, serve as the targets for novel anti-cancer agents.     

5-Aza-2′-deoxycytidine,a DNA methylation inhibitor,induces cytotoxicity,cell cycle dynamics and alters expression of DNA methyltransferase 1 and 3A in mouse hippocampus-derived neuronal HT22 cells

Epigenetic processes such as DNA methylation are essential for processes of gene expression in normal mammalian development. DNA methyltransferases (DNMT) are responsible for initiating and maintaining DNA methylation. It is known that 5-Aza-CdR, an inhibitor of DNMT induces cytotoxicity by reducing DNMT activity in various tumor cell lines. However, disturbances in neuronal DNA methylation may also play a role in altered brain functions. Thus, it was of interest to determine whether alterations in DNA methylation might be associated with neuronal functions by using 5-Aza-CdR, on mouse hippocampus-derived neuronal HT22 cell line. In particular, the aim of this study was to investigate the effects of 5-Aza-CdR on cell growth inhibition, cell cycle arrest, apoptosis as well as the expression levels of DNMT in HT22 cells. HT22 cells were incubated with 5 or 20 μmol/L 5-Aza-CdR for 24 h. Data showed that 5-Aza-CdR at both concentrations significantly inhibited proliferation of HT22 cells and exacerbated cytoplasmic vacuolization. Flow cytometry analysis demonstrated that 5-Aza-CdR treatment at both concentrations decreased early apoptosis but enhanced late apoptosis. Cell cycle analysis illustrated that 5-Aza-CdR treatment induced S phase arrest. Further, incubation with 5-Aza-CdR produced a down-regulation in expression of mRNA and protein DNMT1 and 3A but no marked changes were noted in DNMT 3B and p21 expression. In addition, DNMT1 activity was significantly decreased at both 5-Aza-CdR concentrations. Evidence indicates that 5-Aza-CdR induced cytotoxicity was associated with altered mRNA and protein expression of DNMT 1 and 3A associated with reduced DNMT1 activity in HT22 cells which might affect brain functions.     

The effect of thiopurine drugs on DNA methylation in relation to TPMT expression

The thiopurine drugs 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are well-established agents for the treatment of leukaemia but their main modes of action are controversial. Thiopurine methyltransferase (TPMT) metabolises thiopurine drugs and influences their cytotoxic activity. TPMT, like DNA methyltransferases (DNMTs), transfers methyl groups from S-adenosylmethionine (SAM) and generates S-adenosylhomocysteine (SAH). Since SAM levels are dependent on de novo purine synthesis (DNPS) and the metabolic products of 6-TG and 6-MP differ in their ability to inhibit DNPS, we postulated that 6-TG compared to 6-MP would have differential effects on changes in SAM and SAH levels and global DNA methylation, depending on TPMT status. To test this hypothesis, we used a human embryonic kidney cell line with inducible TPMT. Although changes in SAM and SAH levels occurred with each drug, decrease in global DNA methylation more closely reflected a decrease in DNMT activity. Inhibition was influenced by TPMT for 6-TG, but not 6-MP. The decrease in global methylation and DNMT activity with 6-MP, or with 6-TG when TPMT expression was low, were comparable to 5-aza-2′-deoxycytidine. However, this was not reflected in changes in methylation at the level of an individual marker gene (MAGE1A). The results suggest that a non-TPMT metabolised metabolite of 6-MP and 6-TG and the TPMT-metabolised 6-MP metabolite 6-methylthioguanosine 5’-monophosphate, contribute to a decrease in DNMT levels and global DNA methylation. As demethylating agents have shown promise in leukaemia treatment, inhibition of DNA methylation by the thiopurine drugs may contribute to their cytotoxic affects.     

异硫氰酸苯已酯诱导骨髓瘤U266细胞P16基因去甲基化

目的研究异硫氰酸苯己酯(PHI)对骨髓瘤U266细胞株P16基因的去甲基化作用并探讨其作用机制。方法应用半定量逆转录-聚合酶链反应检测U266细胞经过PHI处理后DNA甲基转移酶DNMT1、DNMT3a和DNMT3b基因的mRNA的表达变化,用甲基化特异性聚合酶链反应检测PHI作用前后U266细胞株P16基因甲基化状态的变化。结果使用不同浓度的PHI处理U266细胞72h后,U266细胞表达DNMT1、DNMT3a、DNMT3bmRNA的水平明显降低并呈浓度依赖性。以不同浓度的PHI处理U266细胞10d后,P16基因的甲基化状态被逐渐逆转。结论 PHI可能通过抑制DNA甲基转移酶的表达水平诱导P16基因产生去甲基化,使失活的抑癌基因重新激活,诱导瘤细胞凋亡。     

SOCS1 hypermethylation mediated by DNMT1 is associated with lipopolysaccharide-induced inflammatory cytokines in macrophages

Macrophages activation which releases the pro-inflammatory cytokines is an essential event in the process of inflammation. SOCS1 has been shown to act as a negative regulator of cytokine signals and plays a key role in the suppression of tissue injury and inflammatory diseases. DNA methylation mediated by specific DNA methyltransferases1 (DNMT1) which contributes to the epigenetic silencing of multiple genes. SOCS1 promoter hypermethylation is by far the best categorized epigenetic change in tumors. Our study with a view to investigate whether the loss of SOCS1 due to SOCS1 promoter methylation was involved in the course of inflammatory cytokines released from lipopolysaccharide (LPS)-stimulated macrophages. Here, we found that treatment of LPS-induced RAW264.7 macrophage cells with the DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-azadC) reduced aberrant promoter hypermethylation of SOCS1 and prevented the loss of the expression of SOCS1 in macrophages which secret inflammatory cytokines. Knockdown of DNMT1 gene not only attenuated the SOCS1 gene promoter methylation but also up-regulated the expression of SOCS1 in activated RAW264.7 cells. Furthermore, silencing of DNMT1 prevented the activation of JAK2/STAT3 pathway in LPS-induced RAW264.7 cells. These studies demonstrated that DNMT1-mediated SOCS1 hypermethylation caused the loss of SOCS1 expression results in negative regulation of activation of the JAK2/STAT3 pathway, and enhanced the release of LPS-induced pro-inflammatory cytokines such as TNF-α and IL-6 in macrophages.     

Analysis of aberrant methylation in DNA repair genes during malignant transformation of human bronchial epithelial cells induced by cadmium

Cadmium (Cd) and its compounds are well-known human carcinogens, but the mechanisms underlying the carcinogenesis are not entirely understood yet. Aberrant methylation was investigated in order to obtain insight into the DNA repair-related epigenetic mechanisms underlying CdCl(2)-induced malignant transformation of human bronchial epithelial cells (16HBE). Gene expression and DNA methylation were assessed in untreated control cells; 5th, 15th, and 35th passage of CdCl2-treated cells and tumorigenic cells (TCs) from nude mice by using high-performance liquid chromatography, real-time PCR, Western blot analysis, and methylation-specific PCR assay. During Cd-induced malignant transformation, global DNA methylation progressively increased and was associated with the overexpression of the DNA methyltransferase genes DNMT1 and DNMT3a but not DNMT3b. Expression of both the messenger RNA and proteins of the DNA repair genes (hMSH2, ERCC1, XRCC1, and hOGG1) progressively reduced and DNA damage increased with Cd-induced transformation. The promoter regions of hMSH2, ERCC1, XRCC1, and hOGG1 were heavily methylated in the 35th passage transformed cells and the TCs. The DNA demethylating agent 5-aza-2'-deoxycytidine could reverse the Cd-induced global DNA hypermethylation, DNMT hyperactivity, and the silencing of hMSH2, ERCC1, XRCC1, and hOGG1 in a time-dependent manner. The results indicate that DNMT1 and DNMT3a overexpression can result in global DNA hypermethylation and silencing of the hMSH2, ERCC1, XRCC1, and hOGG1 genes. They may partly explain the epigenetic mechanisms underlying the carcinogenesis due to Cd.     

Interleukin 1β and Prostaglandin E2 affect expression of DNA methylating and demethylating enzymes in human gingival fibroblasts

Periodontitis is a common chronic inflammatory condition that results in increased levels of inflammatory cytokines and inflammatory mediators. In addition to oral disease and tooth loss, it also causes low-grade systemic inflammation that contributes to development of systemic conditions including cardiovascular disease, pre-term birth, diabetes and cancer. Chronic inflammation is associated with epigenetic change, and it has been suggested that such changes can alter cell phenotypes in ways that contribute to both ongoing inflammation and development of associated pathologies. Here we show that exposure of human gingival fibroblasts to IL-1β increases expression of maintenance methyltransferase DNMT1 but decreases expression of de novo methyltransferase DNMT3a and the demethylating enzyme TET1, while exposure to PGE2 decreases expression of all three enzymes. IL-1β and PGE2 both affect global levels of DNA methylation and hydroxymethylation, as well as methylation of some specific CpG in inflammation-associated genes. The effects of IL-1β are independent of its ability to induce production of PGE2, and the effects of PGE2 on DNMT3a expression are mediated by the EP4 receptor. The finding that exposure of fibroblasts to IL-1β and PGE2 can result in altered expression of DNA methylating/demethylating enzymes and in changing patterns of DNA methylation suggests a mechanism through which inflammatory mediators might contribute to the increased risk of carcinogenesis associated with inflammation.     

Doxorubicin inhibits DNMT1, resulting in conditional apoptosis

Chemotherapy using DNA intercalators is one of the most successful approaches to cancer treatment. Although DNA intercalators are believed to inhibit DNA polymerases and topoisomerases, resulting in the induction of apoptosis in tumor cells, other factors potentially inhibited by the anthracycline antibiotics remain to be elucidated. Herein, we show that the enzymatic activity of DNMT1, the primary DNA methyltransferase in mammalian cells, is inhibited by DNA intercalators, such as doxorubicin, in an in vitro assay. Enzymatic analyses indicate that doxorubicin inhibits the catalytic activity of DNMT1 via DNA intercalation. We also found that apoptosis was induced in DNMT1(+/+) HCT116 cells by only a limited range of doxorubicin dose, meaning that apoptotic cell death is "conditional" with respect to the concentration of the DNA intercalating drug. It is noteworthy that conditional apoptosis is not observed in human colorectal cancer cells lacking DNMT1 but can be induced in DNMT1(-/-) cells by transfection of a plasmid expressing DNMT1. Our results suggest that DNMT1 is one of the major targets of doxorubicin resulting in drug-induced apoptosis in human cancer cells. We propose that expression levels of DNMT1 in tumor cells may affect the effectiveness of doxorubicin in chemotherapy.     

甲氨蝶呤治疗类风湿关节炎对Th17细胞甲基化的影响及其机制

目的： 探讨甲氨蝶呤片（MTX）治疗类风湿关节炎（RA）对Th17细胞DNA甲基化的影响及其机制。方法： 将2018年3月至2019年3月在某院住院治疗的67例使用MTX口服治疗的RA患者作为观察对象。采集患者治疗前后静脉血检测红细胞沉降率（ESR）、C反应蛋白（CRP）、血小板计数（PLT）及白细胞介素-17（IL-17），分离外周血单个核细胞（PBMC）测定Th17细胞比例，分析PBMC中维甲酸相关孤核受体-γt（ROR-γt）、DNA甲基转移酶1（DNMT1）、DNMT3a、DNMT3b基因mRNA表达水平，并检测ROR-γt基因甲基化程度，分别对患者Th17细胞比例与ESR、CRP、PLT及IL-17水平和ROR-γt基因甲基化程度与IL-17水平进行相关性分析。结果： 患者治疗前Th17细胞DNA甲基化程度低，ESR快，CRP及PLT水平高，Th17细胞比例及IL-17水平高，DNMT1基因mRNA表达水平低，ROR-γt基因mRNA表达水平高，ROR-γt基因甲基化比例低；治疗后ESR、CRP及PLT水平显著降低（P<0.05），Th17细胞比例及IL-17水平显著降低（P<0.001），DNMT1基因mRNA表达水平显著升高（P<0.001），ROR-γt基因mRNA表达水平显著降低（P<0.001），ROR-γt基因甲基化比例显著增加（P<0.05）；Th17细胞比例与IL-17水平呈正相关，ROR-γt基因甲基化程度与IL-17水平呈负相关（P<0.05）。结论： 甲氨蝶呤可通过提高类风湿关节炎患者Th17细胞ROR-γt基因甲基化程度抑制体内IL-17分泌改善和延缓疾病进程，同时此过程有DNMT1酶参与。     

Effects of long-term low-dose formaldehyde exposure on global genomic hypomethylation in 16HBE cells

Formaldehyde (FA), a volatile organic compound, is a ubiquitous air pollutant that is classified as ‘Carcinogenic to humans (Group 1)’ by IARC (2006). As a well-recognized human carcinogen, its carcinogenic mechanisms are still poorly understood. Previous studies have emphasized on genetic changes. However, little is known about the epigenetic mechanisms of FA exposure. In this study, We not only characterized the epigenomic response to long-term low-dose FA exposure in 16HBE cells, but also examined the expression of DNA methyltransferases (DNMTs) and the methyl-CpG-binding protein DNA-binding domain protein 2 (MBD2). Each week the 16HBE cells were treated with 10 μM FA for 24 h (h). After 24 weeks (W) of exposure to FA, the level of genomic DNA methylation gradually decreased in a time-related manner. Moreover, our results showed that FA exposure down-regulated the expression of DNMT3a and DNMT3b at both mRNA and protein level, and up-regulated the levels of DNMT1 and MBD2 at both mRNA and protein level. Our study indicated that long-term FA exposure could disrupt genomic DNA methylation, which may be one of the possible underlying carcinogenic mechanisms of FA.