DNA甲基化与胃癌的相关性

在胃癌的发展过程中常发现肿瘤相关基因的异常甲基化存在,其中包括全基因组的低甲基化和某些抑癌基因的高甲基化.另外,DNA甲基化水平似乎还与胃癌的预后密切相关.近年来为揭示DNA甲基化与胃癌的相关性进行了大量研究.     

人胃癌总基因DNA与c—myc,c—Ha—ras基因甲基化的改变

DNA甲基化（DNAmethylation）在调节基因表达及维持细胞正常分化中起重要作用[1]。各种恶性肿瘤的形成过程中或多或少地有DNA甲基化的改变，包括总基因组DNA甲基化水平降低和各种癌基因特定位点的DNA甲基化模式的改变[2，3]。国外有较多的动物实验和细胞培养的报道，国内有过胃癌c－Ha－ras基因甲基化变化的分析[4]．但都未有较系统的人胃癌细胞总基因组DNA与特定位点DNA甲基化改变的对比研究．我们将胃癌（Gastriccancer，C）区、癌旁（Paracancer，P）区、外周正常（Normal，N）区粘膜组织细胞DNA标本分为两份，同时作甲基化…     

肿瘤相关基因高甲基化与胃癌早期诊断的研究进展

近年的研究表明在肿瘤发生过程中 ,除了DNA序列改变外 ,Epigenetics(表遗传学或拟遗传学 )在肿瘤形成过程中也具有重要作用。表遗传学是指在细胞分裂过程中进行 ,不改变相关基因的DNA序列而研究相关基因表达的变化[1] 。其中DNA甲基化是研究得最多、也是最深入的 1种表遗传学表达机制。通过对胃癌与DNA甲基化和去甲基化、DNA甲基化和染色质结构的关系的研究[2 ] ,揭示了DNA甲基化在胃癌形成过程中的重要作用 ,目前正成为研究胃癌发生的热点领域。本文拟就近年来正常细胞DNA甲基化、CpG岛甲基化、胃癌相关基因的高甲基化研究及其在…     

对顺铂耐药胃癌细胞的DNA甲基化表型标志物初筛

目的:寻找和鉴定对顺铂耐药胃癌细胞的异常甲基化基因。方法:我们拟胃癌细胞和耐药细胞为研究对象,采用DNA甲基化芯片,对比分析两种细胞DNA甲基化表达谱差异,结合甲基化特异性PCR（MS-PCR）技术验证获得的异常甲基化基因。结果:利用DNA甲基化谱芯片对胃癌细胞和耐药细胞两个细胞系的基因组DNA甲基化谱进行分析,发现1 095个甲基化差异位点;并筛选出36个高甲基化,14个低甲基化修饰基因。然后通过MS-PCR方法对这50个基因的甲基化修饰在细胞水平上又进行了验证分析,最终筛选出与甲基化谱芯片结果一致的 14种基因,包括11种高甲基化和3种低甲基化修饰基因。结论:胃癌癌细胞发生顺铂耐药时,细胞基因组存在广泛的DNA甲基化修饰改变。     

胃癌中微小RNA与DNA甲基化调控的关系

 微小RNA（miRNA）是一类长度约22个核苷酸的非编码单链小分子RNA,在各种生理病理过程中发挥了重要作用,其表达失调和肿瘤的发生发展有密切联系。DNA甲基化在人类基因组中属于表观遗传学调控的范畴,无论DNA的超甲基化或低甲基化都与胃癌等多种肿瘤的发生发展有关。     

DNA甲基化和E-cadherin基因甲基化与胃癌关系研究进展

目的：总结DNA甲基化和E-cadherin基因甲基化与胃癌关系的研究现状。方法：应用PubMed及万方数据知识服务平台检索系统,以“胃癌、DNA甲基化、Eu钙黏蛋白基因和CpG岛”等为关键词,检索1979-01-2013-05的相关文献,共检索到英文文献2093篇,中文文献672篇。纳入标准：1）关键词包括胃癌及DNA甲基化;2）文中包括E-钙黏蛋白基因启动子区CpG岛甲基化。剔除标准：1）非E-钙黏蛋白基因启动子区CpG岛的甲基化;2）关键词有E-钙黏蛋白但文中无甲基化描述。符合纳入标准的中文文献56篇,英文文献43篇,根据剔除标准剔除中文文献46篇,英文文献23篇,最后纳入分析30篇文献。结果：DNA甲基化在基因表达、调控过程中发挥重要作用,其功能紊乱在恶性肿瘤形成过程中具有重要作用,与胃癌的发生密切相关。E-cadherin基因能抑制肿瘤细胞的浸润和转移,E-cadherin基因启动子区CpG岛甲基化是胃肠道肿瘤E-cadherin基因失活的主要原因,可引起E-cadherin表达下降或缺失,与胃癌的发生、浸润、转移和预后密切相关。结论：DNA甲基化和E-cadherin基因甲基化与胃癌的形成、浸润和转移等关系密切,但其要成为胃癌防治的靶点仍需进一步研究。     

胃癌患者外周血与胃癌组织中ERCC1基因甲基化的关系及其意义

背景与目的：胃癌的发生基于基因和表观遗传学机制,表观遗传学的改变在胃癌的发展中起到重要作用。DNA甲基化是目前研究最多、最为深入的一种表观遗传学表达机制。DNA甲基化是一个可逆性过程。核苷酸切除修复交叉互补基因1(excision repair cross-complementing gene 1,ERCC1)是一种DNA损伤修复基因。本研究检测胃癌患者外周血与胃癌组织中ERCC1基因启动子CpG岛甲基化状态,探讨两者的关系及其意义。方法：采用甲基化特异性PCR技术,检测30例胃癌患者外周血、胃癌组织中ERCC1基因启动子CpG岛甲基化状态。结果：胃癌组织中ERCC1基因启动子CpG岛甲基化率为76.7%(23/30),外周血中ERCC1基因启动子CpG岛甲基化率为63.3%(19/30),差异无统计学意义。结论：胃癌患者外周血中的ERCC1基因启动子CpG岛甲基化率与胃癌组织中相似,检测胃癌患者外周血中的ERCC1基因启动子CpG岛甲基化状态为治疗胃癌提供一个简便、快捷、可靠的途径,同时也为以ERCC1基因启动子CpG岛甲基化作为靶点治疗胃癌提供了可靠的理论依据。     

原发胃癌中p15基因异常及启动子区甲基化状态

检测胃癌组织中p15基因异常及启动子甲基化状态。方法：p15基因及启动子区域，用PCR-SSCP、MSP（甲基化特异的PCR）法和测序等方法对100例胃癌患者的癌组织和癌旁组织进行检测。结果：9％的病例具有p15基因启动子区的高甲基化，p15基因发现两例DNA序列改变。结论：p15基因在胃癌中起一定作用且p15基因启动子区域高甲基化是胃癌中p15基因失活的重要原因。     

HPLC检测胃癌DNA甲基化的初步研究

DNA甲基化与基因功能密切相关。在肿瘤和转化细胞中，总基因组DNA和某些看家基因的甲基化水平下降。我们以前曾以甲基化酶温育、3H－S-腺苦甲硫氨酸（3HAM）掺入法研究过人胃癌总基因组DNA甲基化情况，现在我们又以高效液相色谱法（HPLC）检测其总基因组DNA中5一甲基胞嚼陡（'"C）的含量，以与前种方法的结果比较，进一步明确人胃癌DNA甲基化水平的降低情况。1材料与方法1．1对象1994年3月～7月间本院进展期胃癌手术标本中已行'H－SAM掺入法检测DNA甲基化者的癌（C）区、癌旁（P）区和外周远端对照组织（N）区粘膜标本21例。1…     

胃癌细胞系和胃癌组织中EphA7基因的高甲基化及其临床意义

背景与目的：启动子区CpG岛高甲基化是导致基因转录水平下调的重要表观遗传学机制,我们前期的研究发现EphA7基因在部分胃癌中表达下调.本研究探讨EphA7基因下调的机制及其临床意义.方法：检测6株胃癌细胞及62例胃癌标本中EphA7基因甲基化状态.采用同位素掺入方法对胃癌细胞系的EphA7基因表达进行半定量RT-PCR测定;利用亚硫酸氢钠处理DNA后,进行DNA测序和甲基化特异性PCR检测.结果：对胃癌细胞系亚硫酸氢钠修饰后DNA测序发现,在EphA7基因启动子区内CpG岛存在超甲基化现象,利用甲基化特异性PCR检测胃癌标本证实,在部分胃癌组织中存在高甲基化.高甲基化与胃癌细胞的分化程度有关（P=0.03）.结论：高甲基化是导致该基因下调的机制之一.EphA7基因在胃癌的发生过程中可能发挥一定作用.     

LINE-1 hypomethylation in gastric cancer, detected by bisulfite pyrosequencing, is associated with poor prognosis

Background

Genome-wide DNA hypomethylation plays an important role in genomic instability and carcinogenesis. DNA methylation in the long interspersed nucleotide element-1, L1 (LINE-1) repetitive element is a good indicator of the global DNA methylation level. In some types of human neoplasms, LINE-1 methylation level is attracting interest as a predictive marker for patient prognosis. However, the prognostic significance of LINE-1 hypomethylation in gastric cancer remains unclear.

Methods

Using 203 resected gastric cancer specimens, we quantified LINE-1 methylation using bisulfite-pyrosequencing technology. A Cox proportional hazards model was used to calculate the hazard ratio (HR), adjusted for the clinical and pathological variables.

Results

Gastric cancers showed significantly lower LINE-1 methylation levels compared to matched normal gastric mucosa (p < 0.0001; n = 74). Tumoral LINE-1 methylation range was 11.6–97.5 on a 0–100 scale (n = 203; mean 71.4, median 74.4, standard deviation 12.9). LINE-1 hypomethylation was significantly associated with shorter overall survival [log-rank p = 0.029; univariate HR 2.01, 95 % confidence interval (CI) 1.09–3.99, p = 0.023; stage-matched HR 1.88, 95 % CI 1.02–3.74, p = 0.041; multivariate HR 1.98, 95 % CI 1.04–4.04, p = 0.036]. No significant effect modification was observed by any of the covariates in survival analysis (all p interaction >0.25).

Conclusions

LINE-1 hypomethylation in gastric cancer is associated with shorter survival, suggesting that it has potential for use as a prognostic biomarker.     

Discovery of aberrant expression of R-RAS by cancer-linked DNA hypomethylation in gastric cancer using microarrays

Although hypomethylation was the originally identified epigenetic change in cancer, it was overlooked for many years in preference to hypermethylation. Recently, gene activation by cancer-linked hypomethylation has been rediscovered. However, in gastric cancer, genome-wide screening of the activated genes has not been found. By using microarrays, we identified 1,383 gene candidates reactivated in at least one cell line of eight gastric cancer cell lines after treatment with 5-aza-2'deoxycytidine and trichostatin A. Of the 1,383 genes, 159 genes, including oncogenes ELK1, FRAT2, R-RAS, RHOB, and RHO6, were further selected as gene candidates that are silenced by DNA methylation in normal stomach mucosa but are activated by DNA demethylation in a subset of gastric cancers. Next, we showed that demethylation of specific CpG sites within the first intron of R-RAS causes activation in more than half of gastric cancers. Introduction of siRNA into R-RAS-expressing cells resulted in the disappearance of the adhered cells, suggesting that functional blocking of the R-RAS-signaling pathway has great potential for gastric cancer therapy. Our extensive gene list provides other candidates for this class of oncogene.     

Promoter methylation of cyclin D2 gene in gastric carcinoma

Methylation of CpG island in the promoter has been recognized as an important mechanism for regulation of gene expression. Although considerable work has been done on the epigenetic control of tumor suppressor genes, little is known about the potential role of promoter CpG demethylation in the activation of oncogenes. The cyclin D2 gene is overexpressed in a subset of gastric carcinoma. To determine whether hypomethylation of cyclin D2 is involved in stomach carcinogenesis, we studied methylation of CpG islands in the cyclin D2 gene by methylation-specific PCR in 34 gastric carcinoma specimens, 21 corresponding non-neoplastic mucosae, and 8 gastric carcinoma cell lines. We also measured levels of cyclin D2 mRNA in 23 of the gastric carcinoma cases and in the gastric carcinoma cell lines. Hypomethylation of the cyclin D2 promoter was found in 24 (71%) of the 34 tumor tissues and in 6 (29%) of the 21 corresponding non-neoplastic mucosa, the incidence being significantly different (p=0.002; Fisher's exact test). Moreover, hypomethylation of cyclin D2 was more common in stage III and IV tumors than in stage I and II tumors (p=00.014; Fisher's exact test). All of three cell lines with promoter hypomethylation expressed detectable levels of cyclin D2 mRNA. Treatment of cyclin D2-negative cells lines harboring promoter hypermethylation with demethylating agent, 5-Aza-2'-deoxycytidine, led to a reactivation of cyclin D2 expression. These results suggest that DNA hypomethylation is a mechanism underlying the increased expression of cyclin D2 in cancer cells and that demethylation of cyclin D2 may be involved in development and progression of gastric carcinoma.     

Effects of Helicobacter pylori eradication on methylation status of E-cadherin gene in noncancerous stomach.

PURPOSE: Promoter hypermethylation of E-cadherin plays an important role on gastric cancer development. Whereas E-cadherin methylation was frequently detected in the stomach of Helicobacter pylori-infected individuals, we tested whether eradication of H. pylori alters the methylation status of the noncancerous gastric epithelium. EXPERIMENTAL DESIGN: Endoscopic biopsies were taken from the antrum and corpus of H. pylori-infected subjects without gastric cancer. Presence of methylated E-cadherin sequences in the gastric specimens was detected by methylation-specific PCR. Bisulfite DNA sequencing was done to determine the topographical distribution and changes in methylation profiles with H. pylori eradication. RESULTS: Among the 28 H. pylori-infected subjects (median age, 44.5 years), 15 (53.6%) had E-cadherin methylation detected in stomach at baseline. Discordant methylation patterns between the antrum and corpus were noted in six patients. One year after successful H. pylori eradication, there was a significant reduction in the methylation density of the promoter region and exon 1 of the E-cadherin gene as detected by bisulfite DNA sequencing (P < 0.001). CONCLUSION: Promoter methylation in E-cadherin was frequently detected in the stomach of H. pylori-infected individuals. Eradication of H. pylori might possibly reduce the methylation density in E-cadherin gene and the chance of subsequent neoplastic transformation.     

DNA甲基化在肿瘤中的研究进展

DNA甲基化是调节真核生物基因表达的重要方式之一.DNA甲基化在正常细胞癌变过程中发挥重要的作用.在多种肿瘤类型中,控制细胞周期、增殖、凋亡、转移、耐药性以及细胞内信号传导的相关基因存在异常甲基化.     

Distinctive pattern of LINE-1 methylation level in normal tissues and the association with carcinogenesis

Genome-wide losses of DNA methylation have been regarded as a common epigenetic event in malignancies and may play crucial roles in carcinogenesis. Limited information is available on the global methylation status in normal tissues and other cancer types beyond colonic carcinoma. Here we applied the combined bisulfite restriction analysis PCR to evaluate the methylation status of LINE-1 repetitive sequences in genomic DNA derived from microdissected samples from several human normal and neoplastic tissues. We found that methylation of LINE-1 in leukocytes was independent of age and gender. In contrast, normal tissues from different organs showed tissue-specific levels of methylated LINE-1. Globally, most carcinomas including breast, colon, lung, head and neck, bladder, esophagus, liver, prostate, and stomach, revealed a greater percentage of hypomethylation than their normal tissue counterparts. Furthermore, DNA derived from sera of patients with carcinoma displayed more LINE-1 hypomethylation than those of noncarcinoma individuals. Finally, in a colonic carcinogenesis model, we detected significantly greater hypomethylation in carcinoma than those of dysplastic polyp and histological normal colonic epithelium. Thus, the methylation status is a unique feature of a specific tissue type and the global hypomethylation is a common epigenetic process in cancer, which may progressively evolve during multistage carcinogenesis.     

Methylenetetrahydrofolate reductase 677C/T gene polymorphism, gastric cancer susceptibility and genomic DNA hypomethylation in an at-risk Italian population

We performed a case-control study to examine the relationship between MTHFR C677T gene polymorphism (MTHFR677C/T) and gastric cancer susceptibility in at-risk populations in central Italy. To explore genomic DNA hypomethylation as a potential etiologic mechanism, this phenomenon was evaluated in carriers of the MTHFR677T/T genotype and carriers of the wild-type MTHFR677C/C genotype. Lymphocyte genomic DNA from 162 gastric cancer patients and 164 controls was used for MTHFR677C/T genotyping. Unconditional regression analysis with ORs and 95% CIs was used to investigate the association of the polymorphism with disease. Genomic DNA methylation status by an established enzymatic assay that measures the DNA accepting capacity of methyl groups (inversely related to endogenous methylation) was assessed in a random sample of 40 carriers of the wild-type MTHFR677C/C genotype and 40 carriers of the MTHFR677T/T genotype. The global allelic distribution was in Hardy-Weinberg equilibrium. The MTHFR677T allele was significantly associated with gastric cancer risk with an OR of 2.49 (95% CI 1.48-4.20) in heterozygous MTHFR677C/T carriers and an OR of 2.85 (95% CI 1.52-5.35) in homozygous MTHFR677T/T carriers. This risk association was retained in subgroup analyses by tumor histotype and location. Genomic DNA hypomethylation status in MTHFR677T/T carriers was significantly higher than in subjects with wild-type MTHF677C/C genotype (p = 0.012). In the studied population, MTHFR677T played the role of a moderate-penetrance gastric cancer susceptibility allele. Possession of the MTHFR677T/T genotype was significantly associated with genomic DNA hypomethylation. These findings deserve further investigation in the context of novel strategies for gastric cancer prevention.     

DNA demethylation and cancer: therapeutic implications

The epigenome, which is comprised of chromatin and its associated proteins and the patterns of covalent modification of DNA by methylation, sets up and maintains gene expression programs. A hallmark of cancer is a paradoxical aberration of DNA methylation patterns, a global loss of DNA methylation, that coexists with regional hypermethylation of certain genes. The hypermethylation of tumor-suppressor genes has attracted significant attention recently and DNA methylation inhibitors are being tested as potential anticancer agents. However, emerging data suggests that hypomethylation plays a role in activating genes required for metastasis and invasion. It is proposed here that hypermethylation and hypomethylation in cancer are independent processes, which target different programs at different stages in tumorigenesis. Understanding the relative roles of hypomethylation and hypermethylation in cancer has clear implications on the therapeutic use of agents targeting the DNA methylation machinery, which are discussed in this review.