DNA甲基化及其在肿瘤中的作用

DNA甲基化 (methylatioon)就是加上甲基 (CH3- )基团的碱基修饰过程 ,基因甲基化的过程就是基因表达受到抑制的过程。DNA甲基化与对抗核酶的抗性、细胞防御机制、基因激活与印记、细胞的分化与发育 ,以及衰老和癌变相关。癌基因低甲基化、抑癌基因高甲基化、错配修复基因高甲基化、基因启动子DNA甲基化以及DNA甲基转移酶活性改变等都与肿瘤的发生密切相关。     

DNA甲基化及其在肿瘤中的作用

DNA甲基化（methylation)就是加上甲基（CH3－）基团的碱基修饰过程，基因甲基化的过程就是基因表达受到抑制的过程。DNA甲基化与对抗核酶的抗性、细胞防御机制、基因激活与印记、细胞的分化与发育，以及衰老和癌变相关。癌基因低基化、抑癌基因高甲基化、错配修复基因高甲基化、基因启动子DNA甲基化以及DNA甲基转移酶活性改变等都与肿瘤的发生密切相关。     

DNA甲基化与肿瘤相关性研究进展

DNA甲基化主要指DNA 5'胞嘧啶甲基化,引起基因表达异常。近几年研究发现,在癌细胞中,肿瘤抑制基因甲基化的发生率与肿瘤抑制基因突变或缺失发生的概率大致相等,均可导致肿瘤抑制基因功能的丧失[1]。在许多组织来源的肿瘤中,尤其是造血系统来源的肿瘤,例如骨髓增生异常综合征,基因的异常高甲基化可以导致其进展为白血病[2]。DNA甲基化与基因的缺失、突变等遗传学改变不同,甲基化的DNA核苷酸序列未发生改变,仅通过个别碱基的修饰来影响基因转录,是可逆的。可通过人为的干预拮抗,诱导失活的基因表达。一些DNA甲基化抑制剂,例如5-氮杂胞苷和5-氮杂脱氧胞苷可以使失活的肿瘤抑制基因恢复其正常功能,为白血病和其他相关疾病的治疗提供了新的手段。1 DNA甲基化DNA甲基化,即在DNA甲基转移酶的作用下,将S2腺苷酰甲硫氨酸(SAM)分子上的甲基转移到DNA分子中胞嘧啶残基的第五位碳原子上,有两种DNA甲基化酶,即维持甲基转移酶(DNM T 1)和重新甲基化酶(DNM T 3a,DNM T 3b)。维持甲基化酶的作用是识别子代DNA双链中亲代单链上已甲基化的CpG位点,催化互补单链的胞嘧啶(C)发生甲基化。重新甲基化作用是使非甲...     

甲基化CpG结合蛋白(MeCPs)在肿瘤表遗传学研究中的进展

肿瘤是严重威胁人类健康的疾病之一，近年来大量的研究表明：肿瘤的发生不但存在遗传学上的改变，而且存在表遗传学的改变。表遗传学的改变主要包括DNA甲基化和组蛋白修饰。在哺乳动物中，DNA甲基化是指在DNA甲基化转移酶催化下，以孓腺苷甲硫氨酸（SAM）为甲基化供体，将甲基转移到特定碱基的过程。在人类，DNA甲基化主要发生在CpG二核苷酸上，形成5-甲基胞嘧啶（5mC），以甲基CpG（mCpG）形式出现。研究表明：DNA甲基化与基因表达沉默密切相关。     

DNA甲基化与肿瘤的侵袭和转移

肿瘤的侵袭和转移是肿瘤的重要特性，也是肿瘤致死的主要原因。近年来对肿瘤侵袭和转移的机制作了大量的研究，发现DNA的甲基化在肿瘤侵袭和转移中发挥重要的作用。DNA甲基化是表观遗传学分子机制之一，参与基因表达的调控。在肿瘤侵袭和转移过程中，DNA甲基化可以通过调节肿瘤转移相关基因的表达从而影响肿瘤的侵袭和转移。     

DNA甲基化异常与皮肤肿瘤

DNA甲基化（DNA methylation）异常主要指基因组中CpG岛发生甲基化修饰,引起基因表达异常。近几年的研究表明,DNA甲基化异常与肿瘤的发生关系密切。其主要的致癌机制为：抑癌基因启动子区被异常甲基化导致基因表达沉默进而导致肿瘤的发生;细胞周期调控基因甲基化异常致使细胞调控周期紊乱使得肿瘤细胞增殖过速;肿瘤侵袭相关基因甲基化异常致使肿瘤扩散转移以及DNA损伤修复基因异常甲基化使得肿瘤的发生机率增加。     

DNA甲基化与基因沉默及肿瘤

DNA甲基化在胚胎发育及肿瘤演变过程中都扮演了重要角色,相关方面的研究日益受到关注。现对近年来DNA甲基化的分子学基础及其与基因沉默和肿瘤关系的研究进行综述。     

DNA及RNA甲基化检测在肿瘤诊断中的研究进展

在恶性肿瘤的发生及发展过程中,遗传异常以及基因表观遗传学的改变起到了重要的作用。同遗传学异常一样,表观遗传的改变也是一种可反映肿瘤发生早期的分子机制。DNA甲基化作为表观遗传学的一种重要方式,在控制基因的表达、维持基因组稳定性、细胞分化和胚胎发育中起着重要的作用;RNA甲基化则主要包括后转录过程对真核基因表达的调控。因此,检测肿瘤细胞中这2种甲基化的异常成为早期诊断肿瘤的重要手段。本文就DNA及RNA甲基化与肿瘤的关系以及这2种甲基化检测方法做一综述。     

DNA甲基化

DNA甲基化是指甲基基团与DNA的CpG二核苷酸的胞嘧啶核苷共价结合，它对哺乳动物基因及胚胎的正常发育具有重要意义，但也给基因带来额外负担。肿瘤细胞在整体低甲基化同时伴某些特殊部位的起甲基化使原来正常的甲基化成份频繁破坏，甲基化发生在肿瘤抑制基因的启动子区时将抑制剂基因从而使细胞正常生长失控或导致突变。DNA甲基化在DNA修复和基因稳定性方面都有作用，DNA甲基转移酶家族的发现在甲基化研究领域开辟了一个新时期。     

抑癌因子DNA高甲基化与肿瘤

肿瘤DNA甲基化的异常包括基因组整体甲基化水平降低或和某些基因启动子区域甲基化水平异常升高。前者可导致原癌基因活化及基因组不稳定等,而基因启动子区域发生异常高甲基化则可导致基因转录沉默,使重要基因如抑癌基因等低表达或不表达及增加点突变,并通过影响多条细胞信号转导通路来干预细胞周期和细胞凋亡等,从而影响肿瘤的发生发展及预后和治疗。了解抑癌因子DNA高甲基化与肿瘤的研究现状,可望为揭示肿瘤发生机制提供新的思路,为肿瘤诊断和治疗提供新的标记物和靶点。     

骨髓增生异常综合征的表观遗传改变

表观遗传改变是指不涉及DNA序列变化但影响细胞基因表达的一些可遗传的改变,主要包括DNA启动子的甲基化和组蛋白的各种共价修饰。表观遗传改变与肿瘤的发生密切相关,在多种表观遗传相关分子共同参与下,组蛋白氨基酸残基去乙酰化或甲基化,抑癌基因启动子甲基化,使各种转录因子无法与DNA结合而导致抑癌基因失活。目前已知,骨髓增生异常综合征(MDS)存在多种抑癌基因的表观遗传改变,也存在着表观遗传调节蛋白PcG的表达异常,这些都与MDS的进展和预后有着密切的关系,提示了它们可作为新的疾病标志物应用于临床。本文就MDS的存在的表观遗传改变分子机制、抑癌基因失活和PcG蛋白异常作一综述。     

DNA甲基化调控的MicroRNA在乳腺癌中的研究进展

微小核糖核酸(miRNA)是一类内源性非编码小RNA, 长约18~25个核苷酸, 其通过与特定靶mRNA结合来抑制mRNA翻译过程, 从而在转录后水平调节靶基因的表达水平, 与恶性肿瘤的发生、发展、侵袭以及转移密切相关。前期大量研究发现, DNA甲基化可能是调控乳腺癌中miRNA表达异常的机制, 包括癌基因miRNA低甲基化激活和抑癌基因miRNA高甲基化失活, 从而导致miRNA调控的下游靶基因表达异常, 参与乳腺癌的发生发展。因此, 本文主要就DNA甲基化调控的miRNA在乳腺癌中的研究进展作一综述。      

Epigenomic reactivation screening to identify genes silenced by DNA hypermethylation in human cancer

Promoter DNA hypermethylation is the key epigenetic mechanism by which tumor suppressor genes are inactivated in human cancer. The discovery of the importance of this mechanism has spurred the development of a number of approaches to identify genes silenced by DNA hypermethylation in cancer. One of the most useful of these approaches is an epigenomic reactivation screening strategy that combines treatment of cancer cells in vitro with DNA methyltransferase and/or histone deacetylase inhibitors, followed by global gene expression analysis using microarrays, to identify upregulated genes. This approach is most effective when complemented by microarray analyses to identify genes repressed in primary tumors. Epigenomic reactivation screening has a number of key advantages over direct analysis of cancer-associated DNA methylation changes; most notably, it directly identifies genes in which epigenetic changes lead to altered gene expression. An increasing number of studies have utilized epigenomic reactivation screening to discover novel tumor suppressor genes in cancer. The results of some of the most recent studies are highlighted in this review.     

Comparison of biological effects of non-nucleoside DNA methylation inhibitors versus 5-aza-2'-deoxycytidine

DNA cytosine methylation plays a considerable role in normal development, gene regulation, and carcinogenesis. Hypermethylation of the promoters of some tumor suppressor genes and the associated silencing of these genes often occur in certain cancer types. The reversal of this process by DNA methylation inhibitors is a promising new strategy for cancer therapy. In addition to the four well-characterized nucleoside analogue methylation inhibitors, 5-azacytidine, 5-aza-2'-deoxycytidine (5-Aza-CdR), 5-fluoro-2'-deoxycytidine, and zebularine, there is a growing list of non-nucleoside inhibitors. However, a systemic study comparing these potential demethylating agents has not been done. In this study, we examined three non-nucleoside demethylating agents, (-)-epigallocatechin-3-gallate, hydralazine, and procainamide, and compared their effects and potencies with 5-Aza-CdR, the most potent DNA methylation inhibitor. We found that 5-Aza-CdR is far more effective in DNA methylation inhibition as well as in reactivating genes, compared with non-nucleoside inhibitors.     

Mechanistic and prognostic significance of aberrant methylation in the molecular pathogenesis of human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide, accounting for an estimated 600,000 deaths annually. Aberrant methylation, consisting of DNA hypomethylation and/or promoter gene CpG hypermethylation, is implicated in the development of a variety of solid tumors, including HCC. We analyzed the global levels of DNA methylation as well as the methylation status of 105 putative tumor suppressor genes and found that the extent of genome-wide hypomethylation and CpG hypermethylation correlates with biological features and clinical outcome of HCC patients. We identified activation of Ras and downstream Ras effectors (ERK, AKT, and RAL) due to epigenetic silencing of inhibitors of the Ras pathway in all HCC. Further, selective inactivation of SPRY1 and -2, DAB2, and SOCS4 and -5 genes and inhibitors of angiogenesis (BNIP3, BNIP3L, IGFBP3, and EGLN2) was associated with poor prognosis. Importantly, several epigenetically silenced putative tumor suppressor genes found in HCC were also inactivated in the nontumorous liver. Our results assign both therapeutic and chemopreventive significance to methylation patterns in human HCC and open the possibility of using molecular targets, including those identified in this study, to effectively inhibit HCC development and progression.     

非小细胞肺癌血清8个抑癌基因启动子CpG岛甲基化的联合检测及意义

目的：探讨非小细胞肺癌患者血清中8个抑癌基因启动子C pG岛甲基化的联合检测在诊断非小细胞肺癌患者中的意义,以期提高非小细胞肺癌早期无创检出率,提升治愈率。方法选取62例非小细胞肺癌患者设为肺癌组,选取30例肺部良性疾病患者作为对照组,选取16例健康者作为健康组,取3个组研究对象的血清采取癌基因测序法检测8种基因启动子区域甲基化情况,比较3个组研究对象的上述指标,分析上述指标的变化和3个组研究对象不同临床状态的相关性。结果肺组结肠腺瘤性息肉病基因（A PC ）、钙粘蛋白13（CD H 13）、死亡相关蛋白激酶基因（DAPK）、人类O6-甲基鸟嘌呤-DNA-甲基转移酶（MGMT）、RAS相关区域家族 F2（RASSF2）、人类Runt相关转录因子3（RUNX3）、RAS相关区域家族1A （RASSF1A）和TMS1/ASC基因甲基化检出率均明显高于对照组,组间比较差异具有统计学意义（P＜0．05）,非小细胞肺癌患者血清中抑癌基因8个指标联合检测敏感性达到93．54％,特异性达到90．3％,敏感性明显高于所有单项检测和4个指标联合检测。结论 A PC、CD H 13等8个抑癌基因异常甲基化状态的联合检测可以明显提高非小细胞肺癌患者抑癌基因甲基化的检出率。该8个基因异常甲基化有望成为非小细胞肺癌辅助诊断和预后判断的分子标记,并有可能成为非小细胞肺癌治疗的新靶点,从而提高临床对非小细胞肺癌患者的诊治水平。     

Simultaneous analysis of the methylation profile of 26 cancer related regions in invasive breast carcinomas by MS-MLPA and drMS-MLPA

Genetic and epigenetic events play a critical role in the tumorigenic process of breast cancer. The more genes are studied, the more accurate the epigenetic “signature” can be established. The aim of our work has been to apply the technique Methylation-Specific Multiplex Ligation dependent Probe Amplification (MS-MLPA) to study the methylation profile of 26 cancer related gene regions in breast cancers. Secondly, we aimed to establish if the epigenetic “signature” could serve to detect circulating tumor DNA (ctDNA) in breast cancer patients. The MS-MLPA was successfully setup and allowed to establish which regions were preferentially associated with the tumor process. The analysis permitted also to detect significant concurrent methylation between some genes. The detection of ctDNA could be performed by a “double-round” MS-MLPA (drMS-MLPA) approach and nested-Methyl Specific PCR (Nested-MSP). This development is an important novelty and served to detect a small amount of tumor DNA shaded into the blood stream of breast cancer patients. We conclude that MS-MLPA is an excellent assay to analyze the methylation profile of a tumor. The 82 studied samples presented a specific methylation “mark”. These studies serve to enhance the knowledge of the role of epigenetic alterations in breast tumors and can contribute to the development of personalized diagnosis, surveillance and treatment strategies.     

Antimitogenic and chemosensitizing effects of the methylation inhibitor zebularine in ovarian cancer

Deoxycytosine methylation within CpG islands of tumor suppressor genes plays a prominent role in the development and progression of drug-resistant ovarian cancer. Consequently, epigenetic therapies directed toward tumor suppressor demethylation/reexpression could potentially reverse malignant phenotypes and chemosensitize recalcitrant tumors. In this report, we examined the demethylating agent zebularine [1-(beta-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one], in comparison with the well-known methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC), for its ability to inhibit ovarian cancer cell proliferation and to demethylate and induce tumor suppressor genes. Zebularine exerted significant (>5-aza-dC) antiproliferative effects against the ovarian cancer cell lines Hey, A2780, and the cisplatin-resistant A2780/CP in a dose-dependent manner (65% versus 35% inhibition at 48 hours, zebularine versus 5-aza-dC). Moreover, 48-hour treatment with 0.2 mmol/L zebularine significantly induced demethylation of the tumor suppressors ras-associated domain family 1A and human MutL homologue-1. RASSF1A gene reexpression was also observed, as was reexpression of two other tumor suppressors, ARHI and BLU, although levels differed from those induced by 5-aza-dC. Global analyses of DNA methylation revealed similar overall demethylation (2.5- to 3-fold) by 5-aza-dC and zebularine as determined by methyl acceptance assay. However, differences in demethylation of individual loci were observed as determined by differential methylation hybridization. Finally, we found that zebularine could resensitize the drug-resistant cell line A2780/CP to cisplatin, with a 16-fold reduction in the IC50 of that conventional agent. In summary, zebularine seems to be a promising clinical candidate, singly or combined with conventional regimens, for the therapy of drug-resistant ovarian cancer.     

Cancer Genes Hypermethylated in Human Embryonic Stem Cells

Developmental genes are silenced in embryonic stem cells by a bivalent histone-based chromatin mark. It has been proposed that this mark also confers a predisposition to aberrant DNA promoter hypermethylation of tumor suppressor genes (TSGs) in cancer. We report here that silencing of a significant proportion of these TSGs in human embryonic and adult stem cells is associated with promoter DNA hypermethylation. Our results indicate a role for DNA methylation in the control of gene expression in human stem cells and suggest that, for genes repressed by promoter hypermethylation in stem cells in vivo, the aberrant process in cancer could be understood as a defect in establishing an unmethylated promoter during differentiation, rather than as an anomalous process of de novo hypermethylation.