Runx3基因甲基化及Runx3蛋白表达在结肠癌发生中的意义

目的:评价Runx3 基因启动子区CpG 岛甲基化状态及Runx3 蛋白表达在结肠癌发生中的作用及临床意义.方法:应用DNA 甲基化特异性PCR(MSP) 技术检测结肠癌、结肠腺瘤、正常结肠黏膜Runx3 基因CpG 岛甲基化状态,应用免疫组织化学发法检测Runx3 蛋白的表达.结果:MSP 方法检测发现Runx3 基...     

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。     

胃癌患者p16基因启动子甲基化检测及其临床意义

目的 探讨p16基因启动子CpG岛甲基化在胃癌发生、发展中的作用.方法 应用特异性甲基化PCR(MSP)检测58例胃癌及癌旁组织和30例正常胃黏膜组织中p16基因启动子CpG岛的甲基化状态,分析其与胃癌临床病理参数的关系.结果26例(44.8％)胃癌组织、5例(8.6％)癌旁组织也检测出甲基化的存在,30例正常胃黏膜组织标本中均未检测出p16基因甲基化的存在(P ＜0.05);p16基因甲基化与胃癌淋巴结转密切相关(P＜0.05).结论 p16基因启动子CpG岛甲基化可促进胃癌的发生、发展;此基因的异常甲基化可作为胃癌早期诊断的敏感指标.     

p16基因高甲基化在胃癌发展中的作用

目的:通过检测胃癌、癌前病变和正常对照组中p16基因启动子区CpG岛甲基化水平及其表达,并结合临床病理资料,分析他们在胃癌发生、发展中的作用.方法:用甲基化特异性聚合酶链反应(methylation-specific PCR,MSP)检测41例胃癌组织、40例癌前病变组织和38例正常对照组织中p16基因启动子5′CpG岛甲基化;应用免疫组化检测基因的蛋白表达.结果:胃癌组织中p16基因甲基化阳性率为56.1%(23/41),癌前病变组织中为17.5%(7/40),而正常对照组织中为2.6%(1/38),前组与后两组之间的差异有显著性(P<0.05).胃癌组织中p16基因表达阳性率为51.2%,癌前病变组织中为90.0%,正常对照组织中为100.0%,前组与后两组之间的差异有显著性(P<0.05).低分化型胃癌组织中的p16基因甲基化阳性率明显高于高分化型(81.3%vs 40.0%,P<0.05).有淋巴结转移的胃癌组织中,p16基因甲基化阳性率与无转移组的差异有显著性(81.0%vs 30.0%,P<0.05).浸润深达浆膜层的胃癌组织中,甲基化阳性率与未达浆膜层组无统计学差异(60.0%vs 52.4%,P>0.05).胃癌组织中p16基因甲基化阳性组的蛋白表达阳性率显著低于甲基化阴性组(26.1%vs 83.3%,P<0.01).结论:胃癌组织中存在有p16基因启动子5′CpG岛高甲基化,并导致其基因表达率显著低于正常对照及癌前病变组织.p16基因的高甲基化与胃癌分化程度、淋巴结转移相关.p16基因甲基化的发生,从正常、癌前病变到胃癌有逐渐增加的趋势,提示其基因CpG岛高甲基化有可能作为诊断早期胃癌的一项较为敏感的指标.     

胃癌RASSF1A基因启动子CpG岛甲基化与DNMT1表达的意义

目的探讨RAS相关区域家族1A（RASSF1A）基因启动子CpG岛甲基化和DNA甲基转移酶1（DN-MT1）的表达与胃癌发生的关系。方法运用甲基化特异性聚合酶链反应和免疫组织化学方法检测胃癌癌旁正常组织和癌组织RASSF1A基因启动子CpG岛甲基化发生率及DNMT1的表达情况。结果癌旁正常组织中RASSF1A基因启动子CpG岛甲基化发生率、DNMT1阳性表达率显著低于相应癌组织（P均〈0.01）。RASSF1A启动子CpG岛甲基化患者DNMT1阳性表达率与非甲基化患者比较无统计学差异（P〉0.05）。结论RASSF1A基因启动子区CpG岛甲基化和DNMT1的高表达可能与胃癌发生有一定关系。     

SOCS-3基因在结肠癌中的表达及其甲基化测定

目的通过对SOCS-3基因在结肠癌和癌旁组织中的表达及其启动子甲基化状态的测定,探讨其与结肠癌发生、发展和转移的关系。方法收集40例结肠癌患者的肿瘤标本、20例癌旁组织及10例正常结肠组织,运用甲基化特异性PCR测定SOCS-3基因CpG岛甲基化状态,同时运用实时定量PCR分析SOCS-3基因在结肠癌组织中的表达水平。结果40例结肠癌组织中有34例(85%)存在SOCS-3基因CpG岛的异常甲基化,癌旁组织中有2例(10%),而正常结肠组织中未检测到SOCS-3基因CpG岛的异常甲基化;结肠癌组织中SOCS-3基因CpG岛甲基化组与无甲基化组相比,其SOCS-3基因的相对表达量明显减少(P0.05),表明SOCS-3基因CpG岛甲基化可导致SOCS-3基因表达降低。SOCS-3基因CpG岛甲基化与性别、年龄无关(P0.05),而与肿瘤病理分级、TNM分期有关(P0.05)。结论结肠癌中存在SOCS-3基因CpG岛异常甲基化,且CpG岛的甲基化导致其基因表达降低。SOCS-3基因CpG岛甲基化可能参与了结肠癌的发生、发展和转移。     

CpG岛甲基子表型阳性的散发性大肠癌的临床病理特征

目的 探讨CpG岛甲基子表型阳性的散发性大肠癌的临床病理特征.方法 采用甲基化特异性PCR方法对71例散发性大肠癌患者行p14、hMLH1、p16、MGMT和MINT1共5个基因启动子甲基化检测,分析CpG岛甲基子表型阳性的散发性大肠癌的临床病理特征.结果 71例散发性大肠癌患者中共检出CpG岛甲基子表型阳性者15例,阳性率为21.1%.CpG岛甲基子表型阳性的散发性大肠癌中右半结肠癌（40.0%比12.5%,P＜0.05）、低分化癌（46.7%比14.3%,P＜0.05）、淋巴结转移（86.7%比48.2%,x2=7.112,P＜0.05）、Dukes C期或D期（86.7%比50.0%,x2=6.519,P＜0.05）所占比例均显著高于CpG岛甲基子表型阴性者.结论 CpG岛甲基子表型阳性的散发性大肠癌具有右半结肠癌多发、低分化多见、常有淋巴结转移和分期较晚的特点.     

肼苯哒嗪对LOVO结肠癌细胞株p16基因的影响

目的 探讨DNA甲基化抑制剂肼苯哒嗪对LOVO结肠癌细胞株p16抑癌基因的转录调控作用及对细胞株生长增殖的生物学影响,寻找治疗肿瘤的新靶点.方法 用不同浓度的肼苯哒嗪作用结肠癌细胞株,四唑盐(MTT)比色法观察肼苯哒嗪对细胞生长的影响,逆转录聚合酶链反应(RT-PCR)检测对LOVO结肠癌细胞p16基因mRNA表达的影响;MSP法测LOVO结肠癌细胞启动子区CpG岛甲基化情况.结果 肼苯哒嗪能抑制LOVO结肠癌细胞的生长,且此效应与浓度相关(P<0.05);肼苯哒嗪能使LO-VO结肠癌细胞中甲基化的p16基因去甲基化重新表达;浓度越高的肼苯哒嗪作用后的LOVO结肠癌细胞p16基因mRNA表达越强.结论 肼苯哒嗪能抑制LOVO结肠癌细胞的生长,并且使甲基化失活的p16基因重新表达.     

MGMT、hMLH1和hMSH2基因启动子甲基化状态对脑胶质瘤预后的影响

目的探讨脑胶质瘤DNA修复基因O6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）和错配修复基因（MMR）（hMLH1、hMSH2）启动子甲基化状态及其对患者预后和烷化剂化疗敏感性的影响。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,免疫组化方法测定其蛋白表达。绘制Kaplan-merier生存曲线。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%,而正常脑组织相应基因启动子区未发生甲基化;三种基因启动子未甲基化模式与其对应蛋白表达模式相似。MGMT基因甲基化的脑胶质瘤患者存活率显著高于未甲基化者（P〈0.05）;MMR基因甲基化患者中MGMT基因甲基化与未甲基化者的生存期无统计学差异（P〉0.05）。结论hMLH1、hMSH2及MGMT甲基化是脑胶质瘤发生过程中常见的分子事件;联合检测MGMT、hMLH1和hM-SH2基因启动子甲基化状态可判断脑胶质瘤患者的预后及其对烷化剂化疗的敏感性。     

胃癌患者TIMP3基因启动子区CpG岛甲基化状态及蛋白表达

目的探讨胃癌组织中TIMP3基因启动子区CpG岛甲基化及蛋白的表达与胃癌生物学行为的关系,并分析TIMP3基因启动子区CpG岛甲基化与其蛋白表达的相关性。方法采用甲基化特异性PCR(MSP)技术和免疫组织化学SP法检测45例患者的胃癌组织、正常胃黏膜组织中TIMP3基因启动子区CpG岛甲基化情况及蛋白的表达。结果 TIMP3基因启动子区CpG岛在胃癌及正常组织中的甲基化阳性率分别为31.1%和0(P<0.05);TIMP3蛋白在胃癌及正常组织中的阳性表达率分别为46.7%和90.5%(P<0.05);TIMP3基因启动子区CpG岛甲基化及蛋白的表达分别与胃癌的分化程度、淋巴结转移有关(P<0.05)。TIMP3基因启动子甲基化与蛋白表达呈明显的负相关(P<0.05)。结论 TIMP3基因启动子区CpG岛的异常甲基化是引起蛋白表达缺失的主要原因,TIMP3基因启动子区CpG岛的异常甲基化与胃癌的发生发展有关。     

Extensive but hemiallelic methylation of the hMLH1 promoter region in early-onset sporadic colon cancers with microsatellite instability.

BACKGROUND AND AIMS: Methylation of the hMLH1 promoter region is frequently observed in microsatellite instability (MSI)-positive sporadic colorectal carcinomas. We studied hMLH1 promoter methylation in peripheral blood lymphocytes of 87 index patients representing 29 cases of hereditary nonpolyposis colorectal cancers (HNPCCs), 28 cases of atypical HNPCCs, and 30 sporadic cases of the development of early-onset colorectal carcinomas or multiple primary cancers. METHODS: Methylation of the hMLH1 promoter region was analyzed by Na-bisulfite polymerase chain reaction/single-strand conformation polymorphism analysis or methylation-specific polymerase chain reaction. MSI, allelic status of the hMLH1 locus, and loss of hMLH1 protein expression were examined in cases for which tumor tissues were available. RESULTS: Extensive methylation of the hMLH1 promoter was detected in peripheral blood lymphocytes of 4 of 30 patients with sporadic early-onset colon cancer, among whom multiple primary cancers (1 colon and 1 endometrial cancer) developed in 2 cases. This methylation was not detected in analyses of HNPCC or atypical HNPCC groups or healthy control subjects. MSI was positive, and extensive methylation was detected in both cancers (colon and endometrial cancer) and normal tissues (colon, gastric mucosa, endometrium, and bone marrow) in all of the examined cases (3 of 3). Analysis of a polymorphic site in the hMLH1 promoter in 2 informative cases showed that methylation was hemiallelic. In 1 case, the unmethylated allele was lost in the colon cancer but not in the metachronous endometrial cancer. CONCLUSIONS: Constitutive, hemiallelic methylation of the hMLH1 promoter region was shown to be associated with carcinogenesis in sporadic, early-onset MSI-positive colon cancers.     

Extensive methylation of hMLH1 promoter region predominates in proximal colon cancer with microsatellite instability.

BACKGROUND & AIMS: Methylation of the hMLH1 promoter region has been suggested to cause microsatellite instability (MSI) in sporadic colorectal carcinoma (CRC). We studied the methylation profile in a wide region of the hMLH1 promoter and compared with the hMLH1 protein expression and MSI status in 88 cases of sporadic CRC. METHODS: Na-bisulfite treatment and polymerase chain reaction single-strand conformation polymorphism analysis was performed using 5 sets of polymerase chain reaction primers spanning the promoter region of the hMLH1 to examine methylation status. Results were compared with immunostaining using anti-hMLH1 monoclonal antibody and MSI status of the tumor samples. RESULTS: Methylation status was classified as full or partial methylation. Full methylation indicates the methylation of all CpG sites in the examined regions. Methylation of the hMLH1 promoter was observed in 88.9% (16 of 18) of CRCs showing high frequency MSI (MSI-H), among which 89% (14 of 16) had full methylation with reduced hMLH1 protein expression. All cases showing full methylation were proximal colon tumors with MSI-H. In cases with partial methylation, only the upstream region of the hMLH1 promoter was methylated. Partial methylation was also shown in 33.3% (6 of 18) of the normal mucosa of MSI-H cases. Frequencies of methylation were significantly correlated with female gender (P = 0.0009) and aging (P = 0.007). CONCLUSIONS: Full methylation of the hMLH1 promoter region and subsequent gene inactivation may play a crucial role in the carcinogenesis of MSI-H CRCs in the proximal colon. Methylation upstream of the hMLH1 promoter appears to be an early event in the carcinogenesis of MSI-H tumors.     

多基因甲基化在胰腺癌发病机制中的作用

目的:检测基NRASSF1A,p16,SOCS-1和 hMLH1在胰腺癌中甲基化的作用．方法:采用饱和氯化钠法提取肿瘤组织(n= 21)和瘤旁组织(n=21)DNA．采用甲基化特异 PCR对上述基因甲基化状态进行分析．结果:在癌旁组织中,RASSF1A,p16,SOCS-1 和hMLH1甲基化频率分别为36．4％,13．6％, 13．6％和4．5％;在胰腺癌中分别为59．1％, 40．9％,31．8％和18．2％．P16甲基化频率在两种组织间具有显著性差异(x2=4．13,P<0．05)． 45．5％胰腺癌被检出存在2个或2个以上基因甲基化,明显高于癌旁组织(9．1％,x2=7．33, P<0．01)．31．8％胰腺癌没有检出任何一种基因甲基化．结论:多基因甲基化是部分胰腺癌发展过程中一种早期事件,在这部分胰腺癌的发病中起重要作用．     

MS-HRM在遗传性非息肉性大肠癌筛查中的应用

目的探讨甲基化敏感性高分辨率熔解曲线分析(MS-HRM)在遗传性非息肉性大肠癌(HNPCC)筛查中的应用价值。方法采用实时荧光定量PCR技术检测miR-195在41例散发性大肠癌(SCRC)和9例HNPCC患者癌组织及对应癌旁正常组织(距离癌组织5 cm)中的表达水平,随后使用MS-HRM检测miR-195启动子区域CpG岛甲基化情况。结果 miR-195在HNPCC癌组织中的表达量为1.20±1.48,甲基化比例为55.56%(5/9);miR-195在SCRC组织中的表达量为0.76±1.06,甲基化比例为58.54%(24/41),差异无统计学意义(P0.05);miR-195在肠癌组织及癌旁正常组织中的平均表达水平分别为0.837±1.145和2.236±2.468,差异具有统计学意义(P0.05)。结论 miR-195在SCRC和HNPCC癌组织中的表达有无差异尚待进一步研究。miR-195可能有助于抑制肠癌发展,并且因其甲基化而参于大肠癌的发病机制。     

结肠癌中心和边缘组织增殖能力的差异及相关基因的表达

目的探索结肠癌组织不同区域肿瘤细胞是否存在增殖能力的差异，且这种区域性差异和多重抑痛基因p16及其可变读码框架pl4基因的表达情况及其甲基化状态的相关性。方法免疫组化法测定癌块不同区域增殖蛋自Ki67和P16、P14的表达差异，激光显微切割技术结合甲基化特异性PCR、巢式逆转录PCR观察同一癌块不同区域癌细胞p16及p14基因甲基化状态的改变及其mRNA的表达情况。结果在42份癌组织标本的中心，增殖蛋自Ki67均呈强阳性表达，而P16和P14蛋白表达缺失；在癌组织标本的边缘．有13份Ki67表达阳性（30．1％）。不同区域癌细胞的增殖能力差异有统计学意义（P〈0．05）．且与年龄、性别、Dukes分期、p14的表达无关，但与p16的表达显著相关（X^2=25．37，P〈0．01）。且p16基因的甲基化状态和mRNA的表达也存在明冠的区域性差异（P〈0．05）。结论人结肠癌组织中存在肿瘤细胞增殖能力的区域性差异，在癌组织边缘肿瘤细胞侵袭力增强的同时增殖能力下降，癌边缘区P16基因去甲基化后的再表达可能是这一细胞事件的分子原因。     

Genetic instability and aberrant DNA methylation in chronic hepatitis and cirrhosis--A comprehensive study of loss of heterozygosity and microsatellite instability at 39 loci and DNA hypermethylation on 8 CpG islands in microdissected specimens from patients with hepatocellular carcinoma

A study was conducted to examine the significance of genetic instability and aberrant DNA methylation during hepatocarcinogenesis. Genomic DNA was extracted from 196 microdissected specimens of noncancerous liver tissue that showed no marked histologic findings or findings compatible with chronic hepatitis or cirrhosis, and 80 corresponding microdissected specimens of hepatocellular carcinoma (HCC) from 40 patients. Loss of heterozygosity (LOH) and microsatellite instability (MSI) were examined by polymerase chain reaction (PCR) using 39 microsatellite markers, and DNA methylation status on 8 CpG islands was examined by bisulfite-PCR. In noncancerous liver tissues, LOH, MSI, and DNA hypermethylation were found in 15 (38%), 6 (15%), and 33 (83%) of 40 cases, respectively. The incidence of DNA hypermethylation in histologically normal liver was similar to that in chronic hepatitis and cirrhosis, although neither LOH nor MSI was found in histologically normal liver. In cancerous tissues, LOH, MSI, and DNA hypermethylation were found in 39 (98%), 8 (20%), and 40 (100%) of 40 cases, respectively. CpG islands of the p16 gene and methylated in tumor 1, 2, 12, and 31 clones were frequently methylated in cancerous tissues, although neither the thrombospondin-1 nor the human Mut L homologue (hMLH1) gene was methylated. Absence of silencing of the hMLH1 gene by DNA hypermethylation is consistent with the low incidence of MSI in HCCs. The results of this study indicate that LOH and aberrant DNA methylation contribute to hepatocarcinogenesis; DNA hypermethylation in particular, which precedes or may even cause LOH, is as an early event during hepatocarcinogenesis.     

食管鳞癌组织p16基因调控区甲基化及其蛋白表达研究

目的探讨p16基因在食管癌变过程中表达缺失与其启动子区甲基化的关系。方法采用MSP免疫组化方法,检测环太行山地区45例食管鳞癌患者癌组织p16基因启动子区甲基化状态及蛋白表达情况。结果p16基因在癌组织中表达异常41例（91．1％）,间变组织中表达异常38例（84．4％）,发生纯合型甲基化的组织分别为33例（73．3％）（癌组织）和32例（71．1％）（间变组织）,而其周围正常组织26例（57．8％）均发生了p16启动子区的杂合型甲基化。p16基因纯合型甲基化与癌组织、间变组织、p16蛋白表达缺失相关（P〈0．05）。结论该地区食管癌组织p16基因在癌前病变中p16启动子区即发生了纯合型甲基化、食管癌变的早期事件。p16基因启动子区甲基化可单独影响p16蛋白的正常表达。     

胃癌组织中肿瘤相关基因甲基化及其表达与叶酸和代谢酶MTHFR基因多态性的关系

目的:探讨人胃癌组织中癌基c-myc,抑癌基因p16INK4A,p21WAF1,错配修复基NhMLH1和hM2SH2的甲基化状态及其表达与叶酸、MTHFR基因多态性的关系．方法:胃癌38例手术切除标本的癌区、癌旁和外周正常黏膜组织,运用FOL ACS:180自动化学发光系统测定叶酸含量,PCR-RFLP技术检测MTHFR基因677(C→T)和1298(A→C)两个常见多态,并分别以Real—time RT-PCR和甲基化特异性PCR (MSP)技术检测肿瘤相关基因的表达和甲基化状态．结果:c-myc表达升高,p16INK4A,hMLH1和hMSH2表达降低的胃癌黏膜组织其基因启动子区异常甲基化．p21WAF1,hMSH2表达降低, p16INK4A高甲基化者叶酸水平明显降低,c-myc低甲基化和表达升高者中均存在低叶酸水平．MTHFR 677CC基因型的胃癌黏膜组织p16INK4A甲基化升高且表达降低,而其余肿瘤相关基因的甲基化及其表达与MTHFR两个常见多态均无明显相关性．结论:DNA甲基化在胃癌的发生、发展中具有重要作用,叶酸水平和MTHFR基因多态性通过影响部分肿瘤相关基因的甲基化状态而调控其表达．     

Methylation and mutation analysis of p16 gene in gastric cancer

AIM: To study methylation, frequencies of homozygous deletion and mutation of p16 gene in gastric carcinoma.METHODS: The methylation pattern in exon i and exon 2 of p16 gene was studied with polymerase chain reaction (PCR), using methylation sensitive restriction endonuclease HpaⅡ and methylation insensitive restriction endonuclease MspI. PCR technique was used to detect homozygousdeletions of exon 1 and exon 2 of p16 gene and single strand conformation polymorphism (SSCP) technique was used to detect the mutation of the gene.RESULTS: Hypermethylation changes in exon 1 and exon 2 of p16 gene were observed in 25 % and 45 % of 20 gastric cancer tissues, respectively, while no methylation abnormality was found in normal tissues. The homozygous deletion frequency of exon i and exon 2 of p16 gene in 20 gastric cancer tissues was 20 % and 10 %, respectively. No mutation was found in exon i of p16 gene, while abnormal single strands were found in 2 (10 %) cases in exon 2 as detected by SSCP.CONCLUSION: The results suggest that hypermethylation and abnormality of p16 gene may play a key role in the progress of gastric cancer. Hypermethylation of exon 2 of p16 gene may have effects on the carcinogenesis of gastric mucosa and may be a later event.