Hypermethylated promoter region of DR3, the death receptor 3 gene, in rheumatoid arthritis synovial cells

OBJECTIVE: To examine the promoter activity and protein expression of the death receptor 3 gene DR3, a member of the apoptosis-inducing Fas gene family, with particular reference to the methylation status of its promoter region in rheumatoid arthritis (RA). METHODS: Genomic DNA was prepared from peripheral blood mononuclear cells obtained from healthy individuals and from patients with RA and synovial cells obtained from patients with RA and osteoarthritis. The methylation status of the DR3 promoter was analyzed by bisulfite genomic sequencing and methylation-specific polymerase chain reaction techniques. Gene promoter activity and protein expression were examined using the luciferase reporter and Western blotting techniques. RESULTS: The promoter region of the DR3 gene contained many CpG motifs, including one CpG island that was specifically hypermethylated in synovial cells from patients with RA. Promoter assays showed that the promoter CpG island was essential for the transactivation of the DR3 gene and that forced hypermethylation of the CpG island with the bacterial methylase Sss I in vitro resulted in inhibition of the DR3 gene expression. Furthermore, the expression of DR-3 protein was down-modulated in association with methylation of the promoter CpG island in RA synovial cells. CONCLUSION: The CpG island in the DR3 gene promoter was specifically methylated to down-modulate the expression of DR-3 protein in rheumatoid synovial cells, which may provide resistance to apoptosis in RA synovial cells.     

Age- and sex-related analysis of methylation of 5′-upstream sequences of <Emphasis Type="Italic">Fmr-1</Emphasis> gene in mouse brain and modulation by sex steroid hormones

Fmr-1 gene is implicated in synaptic plasticity and thereby learning, memory and cognition, and methylation of Fmr-1 gene is necessary for memory development that is an age-dependent phenomenon. Aging in general has been reported to affect methylation of gene, however, nothing is known on the age dependent variation in methylation of Fmr-1 gene. Using the brain tissues from male and female mice of various age groups and sex steroid hormones (testosterone or 17beta-estradiol) as modulators, restriction enzymes Hpa II and Msp I and Southern blotting technique, we studied methylation of 5'-upstream sequences of Fmr-1 gene. Our data reveal that the methylation of the 5'-upstream sequences that include CpG islands in promoter and 5'-untraslated region (5'-UTR) gradually increases due to advancing age in both the sexes. 17beta-estradiol lowers the methylation significantly in the brain of mouse of both male and female mouse in age-dependent manner where as testosterone does not affect it appreciably. The alteration in the methylation may be attributed to altered DNA methyl transferase (DNMT) activity as the age increases from young to old, and the 17beta-estradiol may down regulate the DNMT activity in both the age and sex groups whereas the testosterone may not have similar effect on DNMT. Down regulation of methylation of Fmr-1 CpG island and/or 5'-UTR by 17beta-estradiol might lead to derepression of Fmr-1 gene especially in old age. This finding on Fmr-1 methylation is novel and it might have implications in understanding fragile X related disorders and age-dependent alteration in LTP and LTD.     

启动子区域甲基化对肺腺癌GPC5表达的调控作用及意义

目的 研究启动子区域CpG岛的甲基化对肺腺癌细胞GPC5表达的调节作用.方法 在线预测GPC5启动子区域CpG岛的分布,合成甲基化特异性聚合酶链反应(Methylation specific polymerase chain reaction,MSP)引物,甲基化酶特异性抑制剂5-氮杂-2'-脱氧胞嘧啶核苷(5-AZA)处理肺腺癌细胞系A549、SPC-A1及永生化支气管上皮细胞HBE,检测处理前后GPC5表达的变化.同时用MSP方法检测肺腺癌组织标本中GPC5基因启动子区域CpG岛甲基化情况,以探讨其可能的临床意义.结果 GPC5在正常的支气管上皮中不表达,而在肺腺癌细胞系中均有明显表达.5-AZA处理后,GPC5在A549及SPC-A1中的表达均上调,提示启动子区域CpG岛的甲基化参与了GPC5基因表达的调节.但肺腺癌组织标本中GPC5基因启动子区域CpG岛呈现低甲基化状态,提示这种低甲基化状态参与了肺腺癌细胞中GPC5表达的上调.结论 GPC5基因启动子区域的甲基化参与了其表达的调控,可能是导致肺腺癌中GPC5上调的一个重要因素.     

核转录因子E2相关因子2启动子CpG岛甲基化对其蛋白表达及COPD的影响

核转录因子E2相关因子2 (nuclear factor erythroid-2-related factor 2,Nrf2)在人体氧化与抗氧化平衡过程中起了重要的作用.氧化与抗氧化失衡是COPD的重要发病机制之一.近来有研究发现 Nrf2启动子CpG岛甲基化能够影响 Nrf2蛋白的表达,从而影响抗氧化防御过程.明确 Nrf2启动子CpG岛甲基化与 Nrf2蛋白表达及COPD的关系,对今后临床上治疗 COPD寻找新途径具有重要意义.     

EMs患者在位子宫内膜hMLH1蛋白表达变化及其基因启动子CpG岛甲基化观察

目的观察子宫内膜异位症（EM s）患者在位子宫内膜组织中hMLH1蛋白的表达变化及hMLH1基因启动子CpG岛甲基化情况。方法采用免疫组化法分别检测52例（Ⅰ、Ⅱ、Ⅲ、Ⅳ期分别为9、16、12、15例）EM s患者及30例健康志原者（对照组）在位子宫内膜中的hMLH1,采用甲基化特异性聚合酶链反应（MSP）法检测两组在位子宫内膜hMLH1启动子CpG岛甲基化情况。分离hMLH1启动子CpG岛甲基化异常者在位内膜中的腺细胞和间质细胞,采用MSP志愿分别检测两种细胞hMLH1启动子CpG岛甲基化情况。结果 EM s组中hMLH1蛋白表达减弱共6例,其中Ⅰ、Ⅱ、Ⅲ、Ⅳ期分别为0、0、1、5例,其余样本hMLH1蛋白表达均呈阳性;对照组30例无hMLH1蛋白表达减弱者,hMLH1蛋白表达均表现为阳性。EM s组中hMLH1启动子CpG岛部分甲基化3例,其中Ⅰ、Ⅱ、Ⅲ、Ⅳ期分别为0、0、0、3例,余均表现为非甲基化;对照组30例均表现为hMLH1启动子CpG岛非甲基化。EM s组Ⅳ期患者与Ⅰ、Ⅱ、Ⅲ期患者及对照组相比,P均〈0.05。3例hMLH1启动子CpG岛表现为部分甲基化者子宫内膜组织中hMLH1蛋白表达均减弱,其腺细胞中hMLH1启动子区表现为半甲基化,间质细胞hMLH1启动子区未见明显甲基化条带。结论 EM s患者在位子宫内膜组织中存在hMLH1表达减弱及hMLH1启动子CpG岛部分甲基化,主要集中在腺细胞,hMLH1异常与严重EM s的发病有关。     

Acquisition of aberrant DNA methylation is associated with frailty in the very old: findings from the Newcastle 85+ Study

Frailty is a major health problem in older people and, as the population ages, identification of its underlying biological mechanisms will be increasingly important. DNA methylation patterns within genomic DNA change during ageing and alterations in DNA methylation, particularly at gene promoter regions, can lead to altered gene expression. However the importance of altered DNA methylation in frailty is largely unknown. Using cross-sectional data from the Newcastle 85+ Study (all participants aged 85 years) frailty was operationalized by the Fried model. DNA methylation levels were assessed by highly quantitative pyrosequencing at the gene promoter associated CpG islands from a panel of five age-related methylation marker loci and at LINE-1 repetitive elements (as a surrogate for genome-wide methylation). While genome-wide methylation (as assessed at LINE-1 elements) showed no association with frailty status, there was a clear association between CpG island methylation and frailty. When compared to participants with CpG island methylation levels in the combined middle two (referent) quartiles, those in the lowest quartile had significantly decreased odds of frailty [odds ratio 0.47 (95 % CI 0.26–0.85); n = 321, p = 0.013]. Overall this study suggests a potential role for age-related changes in CpG island methylation in the development of frailty.     

hMLH1基因高甲基化在胃癌发生、发展中的作用

基因启动子区CpG岛甲基化使许多基因失活，从而导致恶性肿瘤的发生和发展。目的：检测hMLHl基因启动子区CpG岛甲基化水平，探讨其胃癌发生、发展中的作用。方法：以甲基化特异性聚合酶链反应（MSP）检测41例胃癌、40例癌前病变和38例对照组织中hMLHl基因启动子区CpG岛甲基化状态，并分析其与胃癌患者临床病理特征的关系。结果：胃癌组织中hMLHl基因启动子区CpG岛甲基化阳性率为34．1％，显著高于癌前病变组的5．0％和对照组的0％（P〈0．05）。hMLHl基因甲基化阳性率与胃癌患者的年龄和肿瘤浸润深度有关（阳性率分别为46．4％对7．7％和55．0％对14.3％，P〈0．05），与性别、肿瘤分化程度和淋巴结转移无关（阳性率分别为34．8％对33．3％、28．0％对43．8％和38．1％对30．0％）。结论：胃癌组织中存在hMLHl基因启动子区CpG岛高甲基化，可能与胃癌的发生、发展有关．且可能在老年胃癌患者的肿瘤发生过程中起重要作用。     

Hepatitis B viral DNA is methylated in liver tissues

The mechanisms that regulate hepatitis B virus (HBV) replication within the liver are poorly understood. Given that methylation of CpG islands regulates gene expression in human tissues, we sought to identify CpG islands in HBV-DNA and to determine if they are methylated in human tissues. In silico analysis demonstrated three CpG islands in HBV genotype A sequences, two of which were of particular interest because of their proximity to the HBV surface gene start codon (island 1) and to the enhancer 1/X gene promoter region (island 2). Human sera with intact virions that were largely unmethylated were used to transfect HepG2 cells and HBV-DNA became partially methylated at both islands 1 and 2 by day 6 following exposure of HepG2 to virus. Examination of three additional human sera and 10 liver tissues showed no methylation in sera but tissues showed methylation of island 1 in six of 10 cases and of island 2 in five of 10 cases. The cell line Hep3B, with integrated HBV, showed complete methylation of island 1 but no methylation of island 2. In conclusion, HBV-DNA can be methylated in human tissues and methylation may play an important role in regulation of HBV gene expression.     

Hedgehog通路中Ptchl基因在人胃癌中高甲基化状态分析

目的 研究Hedgehog通路中Ptchl基因表达及其甲基化状态在胃癌发生中的变化.方法 分别抽提10例胃癌组织及其癌旁＞3 cm组织和胃癌细胞株AGS的总RNA和基因组DNA.实时定量逆转录多聚酶链反应(QRT-PCR)检测Ptch基因的mRNA表达.应用软件分析Ptchl基因5'调控序列的CpG岛状态,亚硫酸氢盐测序PCR(BSP)分析甲基化水平.结果 对Ptchl mRNAla转录体转录起始位点(计为0点)上游-3950 bp和下游+2050 bp进行CpG岛分析,发现存在两个CpG岛,第1个为-1139 bp～+860 bp,第2个为+875 bp～+1692 bp.以第1个CpG岛的-870 bp～+229 bp区段内的19个CpG位点的BSP测序结果显示,胃癌细胞株AGS全部发生甲基化,胃癌组织中甲基化程度为16%～100%,平均64%±32%,癌旁组织甲基化程度为0%～42%,平均13%±14%,两组问差异有统计学意义(P＜0.05).Spearman秩相关分析发现,Ptchl基因甲基化同其表达呈负相关(r=-0.558,P=0.011).结论 Ptchl基因高甲基化参与胃癌的发生,可能为胃癌新的肿瘤标志物.     

CpG island methylation of the hTERT promoter is associated with lower telomerase activity in B-cell lymphocytic leukemia

OBJECTIVE: Expression of the catalytic subunit of the telomerase enzyme hTERT is essential for prolonging the replicative lifespan and is the rate-limiting step in cellular immortalization and carcinogenesis. Because hTERT expression is positively correlated with telomerase activity, its regulation is suggested as the major determinant of enzymatic activity. The hTERT promoter region contains two CpG islands, which are known to be target sites for de novo DNA methylation. To elucidate the impact of this epigenetic mechanism on telomerase activity, we analyzed the degree of hTERT promoter methylation in 30 patients with B-cell chronic lymphocytic leukemia. MATERIALS AND METHODS: hTERT promoter methylation was assessed using a methylation-specific competitive polymerase chain reaction assay. The assay is based on digestion of genomic DNA with a methylation-sensitive restriction enzyme before amplification with an internal standard. RESULTS: Patients exhibiting high telomerase activity showed significantly less methylation of the hTERT promoter core domain than patients with low enzyme activity. In addition, telomerase activity was significantly associated with telomere length and overall survival. CONCLUSIONS: Our data show that the degree of CpG island methylation of the hTERT promoter exhibits an impact on telomerase activity in a subgroup of patients with B-cell chronic lymphocytic leukemia and therefore is assumed to play a role in regulating hTERT gene expression in these patients.     

p16基因启动子区甲基化在人嗜铬细胞瘤和副神经节瘤中的变化

目的 检测嗜铬细胞瘤(PHEO)和副神经节瘤(PGL)中p16基因突变和启动子区DNA甲基化改变,分析其与患者临床特征之间的关系.方法收集34例(PHEO 20例、PGL14例)组织标本及患者临床资料,通过甲基化特异性PCR(MSP)测定p16基因启动子区甲基化状态,DNA测序检测基因序列以及RT-PCR方法测定其mRNA表达.结果 (1)34例肿瘤组织中未发现p16基因纯合缺失及点突变;(2)35.3%(12/34)的患者存在p16基因高甲基化,p16基因甲基化阳性标本中,PHEO和PGL分别占25%和75%,两者差异有统计学意义(P=0.005);p16基因甲基化在恶性、单发肿瘤、发病年龄早的亚组中有增高趋势(P＞0.05);(3)甲基化与非甲基化肿瘤组织之间p16 mRNA表达无统计学差异;不同特点的肿瘤中其mRNA表达亦无统计学差异,但恶性肿瘤p16 mRNA表达与良性肿瘤相比有下降的趋势(0.83±0.65对1.12±0.81,P=0.278).结论人PHEO和PGL中,p16基因纯合缺失和突变并不常见,但p16基因启动子区甲基化是其失活的主要形式.