巢式甲基化特异性PCR检测不同类型组织标本中WIF-1基因启动子异常甲基化

目的:采用一种高灵敏度的DNA甲基化分析方法,即巢式甲基化特异性PCR法(nested-MSP,nMSP),检测外科手术切除新鲜组织、石蜡包埋组织及纤维支气管镜活检组织中WIF-1基因启动子的异常甲基化状态。方法:将基因组DNA变性成为单链,用亚硫酸氢盐修饰单链DNA,所有未甲基化的胞嘧啶被转变为尿嘧啶,而甲基化的胞嘧啶则不变。设计针对甲基化和非甲基化等位基因的特异引物,进行巢式PCR扩增,最后经凝胶电泳检测目的片段。结果:在3种类型的原发性非小细胞肺癌标本中都检测出了WIF-1基因启动子的异常甲基化。结论:巢式甲基化特异性PCR是一种灵敏度高、特异性强的甲基化检测方法,可广泛应用于不同类型标本基因启动子甲基化的分析。     

巢式甲基化特异性PCR检测肝细胞癌患者血清中RASSF1A和CDH13基因启动子区甲基化

为了检测肝细胞癌患者血清中RASSF1A和CDH13基因启动子的甲基化状态,收集肝细胞癌患者及健康对照者的血清标本,采用巢式甲基化特异性PCR(nMSP)法检测RASSF1A和CDH13基因启动子区甲基化状态.结果肝细胞癌患者血清样品中RASSF1A和CDH13基因启动子区甲基化率为53.12%和31.25%,68.75%的患者血清可以检测到异常甲基化,正常对照血清中未检测到RASSF1A和CDH13基因启动子区甲基化,RASSF1A和CDH13基因甲基化与患者的临床病理资料无明显相关性(P>0.05);表明nMSP法检测血清中RASSF1A和CDH13基因启动子区甲基化具有较高的敏感性,可为肝细胞癌的筛查、早期诊断和预后判断提供有价值的信息.     

动脉粥样硬化病人LDL受体基因启动子序列的高甲基化改变

探讨动脉粥样硬化（Atherosclerosis,AS）病人LDLR基因启动子区12个CpG二核苷酸结构甲基化的改变。改进并联合应用巢式、Touchdown—PCR,甲基化敏感单链构象分析法（methylationsensitive—single—stand conformation analysis,MS—SSCA）,对61例AS病人及28例健康对照者的LDLR基因启动子区CpG岛的甲基化状态进行检测。在AS患者中发现三种不同的甲基化模式,即完全甲基化、部分甲基化和非甲基化。61例AS患者中有2例完全甲基化,13例部分甲基化,其余为非甲基化．甲基化率为24．59％;28例正常对照者中仅1例发生部分甲基化,甲基化率为3．57％。两组甲基化率差异有显著性（P〈0．05）。AS病人LDLR基因启动子区甲基化程度增高,在LDLR基因调控区的高甲基化修饰可能参与了AS的发病。同时,改进并联合应用的巢式PCR、Touchdown—PCR．MS—SSCA法降低了对DNA模板严格度的要求,提高了灵敏度和特异性,在检测基因甲基化中具有方便、经济、效率高的优点,有良好的应用前景。     

白血病患者骨髓中RASSFlA基因启动子甲基化状态及其临床意义

目的：通过检测各类型白血病骨髓中RASSFlA基因启动子区甲基化水平,探讨其对白血病分型的临床检测意义。方法：抽选93例不同类型白血病患者（观察组）予以甲基化特异性PCP（MSP）方法进行骨髓RASSFlA基因甲基化状态检测,研究不同类型白血病甲基化状态差异,同期抽选93例非白血病者为对照研究（对照组）。结果：观察组中有13例（13．98％）检测到RASSFlA基因甲基化,而对照组中RASSFlA基因甲基化率为0％,比较差异显著（P〈0．05）。不同类型白血病RASSFlA甲基化率比较：淋巴系显著高于髓系（P〈O．05）,急性与慢性白血病比较差异无显著性（P〉0．05）。结论：白血病骨髓中MSP法检测存在RASSFlA甲基化;而RASSFlA基因在淋巴系白血病中的甲基化概率明显增高,因此,对RASSFlA进行甲基化检测有可能作为白血病临床诊断分型的生物学指标之一。     

脑胶质瘤患者组织和血清MGMT、hMLH1和hMSH2基因启动子甲基化状态检测

目的探讨脑胶质瘤患者组织和血清中MGMT、hMLH1和hMSH2基因启动子CpG岛甲基化发生率及相关性。方法甲基化特异性PCR（MSP）检测39例脑胶质瘤组织样本及32例预处理的脑胶质瘤血清样本中MGMT、hMLH1和hMSH2基因启动子区的甲基化状态。结果脑胶质瘤组织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％,肿瘤组织中至少有一种基因甲基化的发生率为64．1％（25／39）;在脑胶质瘤患者外周循环血液中检测到了相关基因甲基化系列,并且与组织中基因甲基化发生率明显相关。结论MGMT、hMLH1和hMSH2基因启动子甲基化是脑胶质瘤发生过程中常见的分子事件,血清中相关基因DNA甲基化检测有可能为脑胶质瘤诊断和个体化化疗提供一种稳定的无创性检测指标。     

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。     

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。     

卵巢上皮癌中ING4基因启动子的甲基化状态及其临床意义

目的：探讨卵巢上皮癌中ING4基因启动子的甲基化状态及其临床意义。方法：收集2005年7月至2012年6月哈尔滨医科大学附属第一医院行全面分期手术并经病理检查确诊的150例卵巢上皮癌组织标本,并以同期因子宫肌瘤或子宫腺肌症行子宫全切除术或次全切除术并经病理检查确诊为正常卵巢组织的150例标本作为对照组。采用甲基化特异性PCR（MSP）技术检测卵巢上皮癌组织与正常卵巢组织中ING4基因启动子的甲基化状态,蛋白印迹法检测1NG4蛋白的表达,并分析ING4基因启动子的甲基化状态与卵巢上皮癌临床病例特征的关系。结果：卵巢上皮癌组织中ING4基因启动子的甲基化阳性率为42．7％（64／150）,明显高于正常卵巢组织（4％,6／150）,差异有统计学意义（P〈0．05）。ING4基因启动子甲基化阳性的卵巢上皮癌组织中ING4蛋白表达阴性或弱阳性;1NG4基因启动子甲基化阴性的卵巢上皮癌和正常卵巢组织中ING4蛋白表达阳性;在64例1NG4基因启动子甲基化的卵巢上皮癌组织中,ING4蛋白表达强度与ING4基因启动子的甲基化程度呈负相关（r=-0．435,P〈0．05）。卵巢上皮癌组织中,1NG4基因甲基化的阳性率随着手术病理分期和组织学分级的增加而增加（P〈0．05）;卵巢透明细胞癌（55．6％,10／18）和卵巢子宫内膜样癌（59-3％,16／27）中ING4基因甲基化的阳性率显著高于浆液性囊腺癌（33．9％,20／59）和粘液性囊腺癌（39．1％,18／46）（P〈0．05）;ING4基因启动子的甲基化状态与患者的年龄、有无腹水及淋巴结转移均无显著相关性（P〉0．05）。结论：ING4基因启动子的甲基化可能促进了其在卵巢上皮癌组织中的表达失活,进而促进了卵巢上皮癌的生长和分化。     

DLC-1基因在MCF-7人乳腺癌细胞系中低表达的机制探究

目的：探究DLC-1基因在MCF-7人乳腺癌细胞系中低表达的机制。方法：应用甲基化特异性PCR（MSP）检测人乳腺癌细胞MCF-7的DLC-1基因甲基化状态,不同浓度的5-氮杂-2＇-脱氧胞嘧啶（5-Aza-CdR）处理人乳腺癌细胞MCF-7,RT-PCR及Real-time PCR定量检测用药前后细胞中DLC-1基因mRNA表达水平变化。结果：DLC-1基因启动子区CpG岛呈甲基化状态,经过5-Aza-CdR处理后,DLC-1基因启动子区呈去甲基化状态,并且其mRNA恢复表达。结论：抑癌基因DLC-1 CpG岛甲基化是导致该基因低表达的原因之一,5-Aza-CdR能逆转DLC-1基因甲基化状态。     

Highly sensitive determination of the methylated p16 gene in cancer patients by microchip electrophoresis

The p16 tumor suppressor gene is inactivated by promoter region hypermethylation in many types of tumor. Recent studies showed that aberrant methylation of the p16 gene is an early event in many tumors, especially in lung cancer, and may constitute a new biomarker for early detection and monitoring of prevention trials. We detected tumor-associated aberrant hypermethylation of the p16 gene in plasma and tissue DNA from 153 specimens using a modified semi-nested methylation-specific PCR (MSP) combining plastic microchip electrophoresis or slab gel electrophoresis, respectively. Specimens were from 79 lung cancer patients, 15 abdominal tumor patients, 30 positive controls and 30 negative controls. The results showed that the positive rate obtained by microchip electrophoresis was more than 26.6% higher and the same specificity was kept when compared with slab gel electrophoresis. The microchip electrophoresis can rapidly and accurately analyze the PCR products of methylated DNA and obviously improve the positive rate of diagnosis of cancer patients when compared with gel electrophoresis. This method with the high assay sensitivity might be used for detection of methylation of p16 gene and even to facilitate early diagnosis of cancer patients.     

p16INK4A and CDH13 hypermethylation in tumor and serum of non-small cell lung cancer patients

Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the etiopathogenesis of lung cancer and is a promising tool for cancer detection. In the present study, promoter hypermethylation of p16INK4A and CDH13 genes was investigated in tumor tissue and in matched serum from 61 patients with histologically confirmed non-small cell lung cancer. Using a fluorescence-based method of methylation-specific PCR (F-MSP), methylation of p16INK4A and CDH13 was detected in 79% and 66% of tumors, respectively, and was not significantly related to conventional clinicopathological characteristics of patients or tumors. Methylation of both genes was observed in 52% of tumors and of at least one gene in 92% of lesions. In matched serum, hypermethylation of p16INK4A and CDH13 was observed in 26% and 23% of patients, respectively, but as they were not associated, the methylation of at least one gene was detected in 39% of patients. In conclusion, the frequency of p16INK4A or CDH13 hypermethylation in patient serum, together with evidence of their early occurrence in lung cancerogenesis and the total lack of methylation in serum from healthy individuals, offer a promising tool for non invasive early detection of lung cancer.     

宫颈癌患者血浆和组织中FHIT基因5′端CpG岛甲基化状态的研究

为了检测宫颈癌患者血浆和组织中FHIT基因5′端CpG岛甲基化状态, 以找到无创伤性诊断宫颈癌的新指标, 选取151例宫颈癌患者的血浆和30例患者的宫颈癌组织为研究对象,用MSP的方法检测FHIT基因5′端CpG岛甲基化状态, 并对MSP产物进行克隆和测序。结果在宫颈癌患者血浆和组织中, FHIT基因5′端CpG岛甲基化率为30.46%和53.33%, 血浆和组织的总体符合率为80%。而对照中均未检测到甲基化状态。随着患者临床分期和组织学分级的增加, FHIT基因甲基化的检出率也在逐渐的增加。表明宫颈癌患者的血浆和肿瘤组织中FHIT基因5′端CpG岛甲基化的发生是高频事件, 使用FHIT基因作为标记可以对宫颈癌患者进行无创伤诊断和预后的评估。     

宫颈癌患者血浆和组织中FHIT基因5′端CpG岛甲基化状态的研究

为了检测宫颈癌患者血浆和组织中FHIT基因5′端CpG岛甲基化状态, 以找到无创伤性诊断宫颈癌的新指标, 选取151例宫颈癌患者的血浆和30例患者的宫颈癌组织为研究对象,用MSP的方法检测FHIT基因5′端CpG岛甲基化状态, 并对MSP产物进行克隆和测序。结果在宫颈癌患者血浆和组织中, FHIT基因5′端CpG岛甲基化率为30.46%和53.33%, 血浆和组织的总体符合率为80%。而对照中均未检测到甲基化状态。随着患者临床分期和组织学分级的增加, FHIT基因甲基化的检出率也在逐渐的增加。表明宫颈癌患者的血浆和肿瘤组织中FHIT基因5′端CpG岛甲基化的发生是高频事件, 使用FHIT基因作为标记可以对宫颈癌患者进行无创伤诊断和预后的评估。     

Quantitative detection of methylated SOCS-1, a tumor suppressor gene, by a modified protocol of quantitative real time methylation-specific PCR using SYBR green and its use in early gastric cancer detection

Although methylation-specific PCR (MSP) is a sensitive technique in the detection of DNA hypermethylation, it is not quantitative. Here we described a modified PCR protocol to quantify methylated SOCS-1 gene by real time MSP using SYBR green, which involves an additional PCR step after the 72 degrees C extension step. This modified protocol is also useful in the quantitative detection of methylated SOCS-1 gene in serum samples of gastric cancer patients.     

Wnt signaling activation and WIF-1 silencing in nasopharyngeal cancer cell lines

Aberrant activation of Wingless-type (Wnt) signaling pathway plays a critical role in oncogenesis of various human cancers. Wnt inhibitory factor-1 (WIF-1) is a secreted antagonist of Wnt signaling and acts through direct binding to Wnt in the extracellular space. Recently, we reported Wnt signaling in various human malignancies. In addition, we identified in lung cancer that WIF-1 is silenced due to promoter hypermethylation. In this study, we found constitutive activation of Wnt signaling and WIF-1 silencing in nasopharyngeal carcinoma (NPC) cell lines. Furthermore, by utilizing methylation-specific PCR and sequence analysis, we demonstrated that frequent hypermethylation of the WIF-1 promoter correlates with WIF-1 silencing in NPC cell lines. Our results indicate that aberrant Wnt signaling is a common event in NPC carcinogenesis linked with WIF-1 silencing in at least cell lines. Strategies targeting these molecules should be potentially promising in treating NPC.     

Investigation of tumor-derived extracellular DNA in blood of cancer patients by methylation-specific PCR

The frequency of APC, RASSF1A, RARbeta, CDH1 and CDH13 gene promoter methylation in samples of DNA isolated from breast and lung patient plasma was studied in order to develop the noninvasive tumor-specific DNA detection method. Methylation of at least one of genes was detected in extracellular DNA from most of the cancer blood specimens. The results obtained indicate that promoter hypermethylation of a number of marker genes represents a promising serum marker for early breast and lung cancer detection.