脑胶质瘤MGMT、hMLH1和hMSH2基因启动子甲基化状态研究

目的：探讨脑胶质瘤患者O6-甲基鸟嘌呤-DNA甲基转移酶基因MGMT和错配修复基因hMLH1、hMSH2启动子CpG岛甲基化状态,及其在烷化剂化疗中的意义。方法：采用甲基化特异性PCR（MSP）方法检测39例脑胶质瘤和6例正常脑组织MGMT、hMLH1和hMSH2基因启动子区的甲基化状态,免疫组化方法测定蛋白表达。结果：脑胶质瘤患者组织MGMT、hMLH1和hMSH2基因启动子区甲基化发生率分别为46.2%、10.3%和20.5%,3种基因启动子未甲基化模式与其对应蛋白表达模式相似,并与患者性别、年龄、病理类型和病理分级无明显相关性。回顾性分析患者资料,显示39例脑胶质瘤患者中,MGMT基因甲基化的患者生存期显著高于MGMT基因未甲基化患者（P〈0.05,Log-rank检验）。结论：MGMT及错配修复基因甲基化是脑胶质瘤发生过程中常见的分子事件,可能与肿瘤的发生有关;检测MGMT、hMLH1和hMSH2基因启动子甲基化状态,在判断脑胶质瘤患者预后和预测烷化剂化疗耐药性中可能具有重要意义。     

子宫内膜异位症中hMLH1、hMSH2基因启动子区的甲基化研究

目的：探讨错配修复基因1（hMLH1）、错配修复基因2（hMSH2）启动子区甲基化与子宫内膜异位症的关系。方法：采用甲基化特异性PCR方法检测23例子宫内膜异位症组织和20例正常子宫内膜中hMLH1、hMSH2基因启动子区的甲基化状态。结果：hMLH1基因在子宫内膜异位症中的甲基化率为39％（9/23）,在正常子宫内膜组织中的甲基化率为5％（1／20）,两者比较,差异有统计学意义（P〈0．05）;hMSH2基因在子宫内膜异位症中的甲基化率为8％（2／23）,在正常子宫内膜组织中的甲基化率为5％（1／20）,两者比较,无明显差异（P〉0．05）。结论：hMLH1基因启动子区甲基化可能与子宫内膜异位症发病有关;hMSH2基因启动子区甲基化与子宫内膜异位症之间不存在显著联系。     

脑胶质瘤组织中p16基因启动子区CpG岛甲基化检测及其临床意义研究

目的：研究脑胶质瘤中p16基因启动子区甲基化情况及其临床意义。方法：用甲基化特异性PCR技术检测42例脑胶质瘤组织和癌旁正常脑组织中p16基因启动子甲基化,并分析该基因启动子甲基化与临床病理特征之间的关系。结果：脑胶质瘤组织中p16基因异常甲基化率（38.27%）显著高于癌旁正常脑组织中p16基因的异常甲基化率（8.8%,P=0.000）。发生甲基化的肿瘤组织或者正常脑组织中p16基因mRNA和蛋白表达显著降低。此外,p16基因异常甲基化和肿瘤病理分级有相关性（P=0.007）,而与患者性别、年龄及肿瘤类型等临床特征无关（P=0.669,0.869和0.944）。结论：p16基因启动子区CpG岛高甲基化与p16表达下调相关,推测p16启动子区CpG岛高甲基化是导致p16基因在脑胶质瘤中表达下调的重要因素,有望成为脑胶质瘤早期辅助诊断的分子标志物之一。     

MGMT 甲基化影响替莫唑胺对胶质母细胞瘤疗效的研究

目的:探讨MGMT甲基化如何影响替莫唑胺对胶质母细胞瘤的治疗效果。方法:选取41个胶质母细胞瘤患者(根据相同替莫唑胺化疗方案治疗下临床结局的不同分为两组)的肿瘤组织,采用甲基化特异性聚合酶链反应分析胶质瘤组织中MGMT基因启动子区过甲基化状态,同时采用免疫组织化学法分析胶质瘤组织中MGMT蛋白表达情况。结果:临床结局不佳组中,以MGMT蛋白表达阳性的肿瘤为主(72.2%),而在结局相对良好组中,MGMT蛋白表达的阳性率仅为39.1%;在MGMT蛋白表达阳性的22例胶质母细胞瘤组织中,7例MGMT启动子甲基化,阳性率为31.8%,在MGMGT蛋白表达阴性的19例中,14例MGMT启动子甲基化,阳性率为73.7%(P<0.05)。结论:MGMT基因启动子区的甲基化状态与MGMT蛋白的表达相关。MGMT基因启动子过甲基化,MGMT蛋白表达较低;MGMT基因启动子去甲基化,MGMT蛋白表达较高。MGMT启动子过甲基化通过抑制MGMT基因的表达而增加替莫唑胺的疗效。     

异柠檬酸脱氢酶-1 突变及MGMT 甲基化在脑胶质瘤的诊断及治疗研究进展

异柠檬酸脱氢酶-1(Isocitrate dehydrogenase-1,IDH1)突变是脑胶质瘤中基因突变所导致的代谢物生成异常,突变型胶质瘤与普通型胶质瘤具有不同的分子生物学特征,并常与患者良好预后相关。MGMT(O6-methylguanine-DNA methyltransferase)启动子甲基化能够降低胶质瘤中DNA修复蛋白MGMT的表达,提高患者对化疗药物烷化剂治疗的敏感性。IDH1突变和MGMT启动子甲基化在胶质瘤的生长过程中存在关联,二者的联合检测对胶质瘤的预后及治疗方式具有一定的提示作用,在胶质瘤的分子病理学诊断方面具有很大的应用前景。目前IDH1突变和MGMT启动子甲基化的检测已逐渐应用于临床,但其诊断方式及治疗指导意义有待进一步研究探讨。本文就IDH1基因突变及MGMT启动子甲基化在胶质瘤诊断及治疗方面的研究进展进行综述。     

RASSF1A和Runx3基因启动子区甲基化在胃癌组织中的表达及意义

目的：检测胃癌组织中RASSFlA和Runx3基因启动子区甲基化状态,探讨二者与胃癌发生发展的关系。方法：采用甲基化特异性PCR（MSP）技术检测57例胃癌组织和相应癌旁组织及30例正常胃黏膜组织中RASSFlA和Runx3基因启动子区甲基化状态。结果：RASSFlA和Runx3甲基化在正常组未见表达。胃癌组RASSFlA基因甲基化率为64．9％（37／57）,明显高于癌旁组的7．0％（4／57）,差异有统计学意义（P〈0．05）,胃癌组Runx3基因甲基化率为49．1％（28／57）,明显高于癌旁组5．3％（3／57）,差异有统计学意义（P〈O．05）。胃癌组RASSFlA和Runx3基因甲基化率为68．4％（39／57）,明显高于癌旁组的8．8％（5／57）,差异有统计学意义（P〈0．05）。结论：RASSFlA和Runx3基因启动子区高甲基化与胃癌的发生密切相关,有望为胃癌的早期诊治提供理论依据。     

人脑胶质瘤细胞中GDNF 基因启动子1 区甲基化状态的研究

目的:观察GDNF启动子1区在人脑胶质瘤细胞中的甲基化修饰状态,以期探讨其对于GDNF在胶质瘤中高表达的影响。方法:基因测序检测10例胶质瘤与5例正常脑组织中GDNF基因序列,比较其基因是否有突变发生;重亚硫酸盐修饰后基因测序检测20例胶质瘤(10例低级别和10例高级别)与5例正常脑组织中GDNF启动子1区甲基化修饰状态。结果:GDNF启动子1区基因在胶质瘤中没有发生突变;GDNF启动子1区甲基化修饰在正常脑组织、低级别、高级别中发生率分别为72.25%、86.25%、86.75%。在胶质瘤中的甲基化修饰水平比正常脑组织明显增高(P<0.05),而高低级别之间无显著性差异。结论:在胶质瘤细胞中,GDNF启动子1区发生了高甲基化修饰,这种修饰很可能会影响GDNF基因的表达。     

MGMT基因启动子甲基化与膀胱尿路上皮癌生物学行为关系的研究

目的 检测膀胱尿路上皮癌组织中O^6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）基因启动子甲基化状态,探讨MGMT甲基化与其蛋白表达水平以及肿瘤生物学行为之间的关系.方法 应用免疫组织化学SP法和甲基化特异性PCR（MSP）分别检测60例膀胱尿路上皮癌及15例正常膀胱黏膜组织中MGMT蛋白表达情况和MGMT基因启动子甲基化状态.结果 膀胱癌组织中MGMT蛋白阳性表达率为35.0 %（21/60）,低于正常膀胱组织（86.7 %,13/15,P〈0.01）.膀胱癌组织中MGMT甲基化阳性率为45.0 %（27/60）,明显高于正常膀胱组织（0.0 %,0/15,P〈0.01）;MGMT启动子甲基化与其蛋白表达呈负相关（r = -0.453,P〈0.01）;并且高级别膀胱癌中MGMT甲基化阳性率（70.6 %,12/17）要比低级别膀胱癌高（34.9 %,15/43）,（P〈0.05）,而MGMT甲基化与膀胱癌临床分期无明显关系.结论 MGMT启动子甲基化可能参与了膀胱尿路上皮癌的发生和肿瘤分化,MGMT启动子甲基化有望成为预判膀胱癌预后的重要标记.     

hMLH1和hMSH2种系突变携带者累积患癌风险研究

目的：探讨遗传性非息肉病性结直肠癌（HNPCC）家系中错配修复基因hMLH1和hMSH2种系突变携带者发生HNPCC相关恶性肿瘤的累积风险度。方法：通过随访14个HNPCC家系中222例hMLH1或hMSH2种系突变携带者与非携带者,应用SPSS14.0统计软件包分析种系突变携带者在不同年龄点发生HNPCC相关恶性肿瘤的累积风险度及两种基因种系突变累积患癌风险的差异。结果：hMLH1或hMSH2种系突变携带者肿瘤发生率为63.8%（60/94）,非突变携带者肿瘤发生率为0.8%（1/128）,种系突变携带者发生恶性肿瘤的相对危险度为非突变携带者的317.6倍;种系突变携带者发生各种HNPCC相关恶性肿瘤的累积风险度随年龄的增加逐渐增大,在60岁时发生各种HNPCC相关恶性肿瘤、结直肠癌、胃癌等的平均累积风险度分别为92.4%、81.3%、29.6%,40岁以前发生胃癌的平均风险度较低（6.1%）;hMLH1与hMSH2种系突变携带者发生各种HNPCC相关恶性肿瘤、结直肠癌、胃癌等累积风险度的差异无统计学意义（均为P＆gt;0.05）。结论：hMLH1或hMSH2种系突变携带者为HNPCC家系中患癌高危人群,发生HNPCC相关恶性肿瘤的风险度随年龄的增加而增大,最常发生恶性肿瘤的部位为胃和结直肠;hMLH1与hMSH2种系突变携带者发生各种HNPCC相关恶性肿瘤的累积风险度无明显差异。     

hMLH1基因启动子CpG岛甲基化致MSI与肿瘤的关系

通过人类错配修复基因( hMLHl)启动子CpG岛甲基化与微卫星不稳定性(MSI)的分析,探讨癌症发病的机制.错配修复基因hMLH1启动子CpG岛甲基化是hMLH1基因失活的重要机制,而hMLH1的表达失活则可导致MSI的产生,促进癌症的发生.根据一系列研究得出结论,在肿瘤组织中hMLH1基因启动子CpG岛甲基化和微卫星不稳定(MSI)有显著相关性,并在癌症早期发生、发展过程中起重要作用.因此临床检测hMLH1基因启动子CpG岛甲基化及微卫星不稳定可能成为癌症鉴别诊断、评价预后、指导化疗的分子标志物之一.     

The global DNA methylation surrogate LINE-1 methylation is correlated with MGMT promoter methylation and is a better prognostic factor for glioma

Gliomas are the most frequently occurring primary brain tumor in the central nervous system of adults. Glioblastoma multiformes (GBMs, WHO grade 4) have a dismal prognosis despite the use of the alkylating agent, temozolomide (TMZ), and even low grade gliomas (LGGs, WHO grade 2) eventually transform to malignant secondary GBMs. Although GBM patients benefit from promoter hypermethylation of the O(6)-methylguanine-DNA methyltransferase (MGMT) that is the main determinant of resistance to TMZ, recent studies suggested that MGMT promoter methylation is of prognostic as well as predictive significance for the efficacy of TMZ. Glioma-CpG island methylator phenotype (G-CIMP) in the global genome was shown to be a significant predictor of improved survival in patients with GBM. Collectively, we hypothesized that MGMT promoter methylation might reflect global DNA methylation. Additionally in LGGs, the significance of MGMT promoter methylation is still undetermined. In the current study, we aimed to determine the correlation between clinical, genetic, and epigenetic profiles including LINE-1 and different cancer-related genes and the clinical outcome in newly diagnosed 57 LGG and 54 GBM patients. Here, we demonstrated that (1) IDH1/2 mutation is closely correlated with MGMT promoter methylation and 1p/19q codeletion in LGGs, (2) LINE-1 methylation levels in primary and secondary GBMs are lower than those in LGGs and normal brain tissues, (3) LINE-1 methylation is proportional to MGMT promoter methylation in gliomas, and (4) higher LINE-1 methylation is a favorable prognostic factor in primary GBMs, even compared to MGMT promoter methylation. As a global DNA methylation marker, LINE-1 may be a promising marker in gliomas.     

The essential role of histone H3 Lys9 di-methylation and MeCP2 binding in MGMT silencing with poor DNA methylation of the promoter CpG island

Silencing of the O (6)-methylguanine-DNA methyltransferase (MGMT) gene, a key to DNA repair, is involved in carcinogenesis. Recent studies have focused on DNA hypermethylation of the promoter CpG island. However, cases showing silencing with DNA hypomethylation certainly exist, and the mechanism involved is not elucidated. To clarify this mechanism, we examined the dynamics of DNA methylation, histone acetylation, histone methylation, and binding of methyl-CpG binding proteins at the MGMT promoter region using four MGMT negative cell lines with various extents of DNA methylation. Histone H3K9 di-methylation (H3me2K9), not tri-methylation, and MeCP2 binding were commonly seen in all MGMT negative cell lines regardless of DNA methylation status. 5Aza-dC, but not TSA, restored gene expression, accompanied by a decrease in H3me2K9 and MeCP2 binding. In SaOS2 cells with the most hypomethylated CpG island, 5Aza-dC decreased H3me2K9 and MeCP2 binding with no effect on DNA methylation or histone acetylation. H3me2K9 and DNA methylation were restricted to in and around the island, indicating that epigenetic modification at the promoter CpG island is critical. We conclude that H3me2K9 and MeCP2 binding are common and more essential for MGMT silencing than DNA hypermethylation or histone deacetylation. The epigenetic mechanism leading to silent heterochromatin at the promoter CpG island may be the same in different types of cancer irrespective of the extent of DNA methylation.     

Expression and promoter methylation status of hMLH1, MGMT,APC, and CDH1 genes in patients with colon adenocarcinoma

Colorectal cancer (CRC) is the third most common cancer in men and the second in women worldwide. CRC development is the result of genetic and epigenetic alterations accumulation in the epithelial cells of colon mucosa. In the present study, DNA methylation, an epigenetic event, was evaluated in tumoral and matching normal epithelium in a cohort of 61 CRC patients. The results confirmed and expanded knowledge for the tumor suppressor genes hMLH1, MGMT, APC, and CDH1. Promoter methylation was observed for all the examined genes in different percentage. A total of 71% and 10% of the examined cases were found to be methylated in two or more and in all genes, respectively. mRNA and protein levels were also evaluated. Promoter methylation of hMLH1, MGMT, APC, and CDH1 genes was present at the early stages of tumor’s formation and it could also be detected in the normal mucosa. Correlations of the methylated genes with patient’s age and tumor’s clinicopathological characteristics were also observed. Our findings suggest that DNA methylation is a useful marker for tumor progression monitoring and that promoter methylation in certain genes is associated with more advanced tumor stage, poor differentiation, and metastasis.