RASSF1A基因在胶质瘤组织中甲基化研究及临床意义

目的：探讨胶质瘤组织及脑正常组织中抑癌基因RASSF1A启动子区甲基化程度及与临床特征的关系。方法：采用甲基化特异性聚合酶链反应（MS-PCR）方法检测46例脑胶质瘤（其中星形细胞瘤19例，室管膜瘤16例，胶质母细胞瘤11例）及6例脑正常组织中RASSF1A基因启动子区甲基化状态。并对RASSF1A基因启动子区甲基化发生情况与临床各因素之间的关系进行分析。结果：（1）46例脑胶质瘤组织DNA标本中RASSF1A基因启动子区甲基化发生率为65．2％（30／46）；6例脑正常组织中RASSF1A基因未发生甲基化；RASSF1A在胶质瘤和脑正常组织之间发生甲基化率比较差异有显著性（P=0.017）。在46例脑胶质瘤中RASSF1A有30例发生甲基化，其中低级别组14例（14／26），高级别组16例（16／20），两组之间甲基化率比较无明显差异（P〉0．05）。其中星形细胞瘤、室管膜瘤、胶质母细胞瘤中甲基化发生率分别为63．2％（12／19）、68、8％（11／16）、63．6％（7／11），各组之间甲基化发生率比较均无统计学意义（P=0．211）。（2）RASSFIA基因启动子区甲基化程度与脑胶质瘤病理分型、肿瘤大小之间无显著相关性。结论：RASSF1A在胶质瘤中有甲基化发生，在脑正常组织中未发生甲基化，检测胶质瘤中RASSF1A基因启动子区甲基化情况对临床判断肿瘤发生发展有指导意义。     

染色体3p区抑癌基因在非小细胞肺癌中的甲基化状况与临床意义

背景与目的 DNA甲基化是表观遗传学的一种调控机制,染色体3p区等位基因缺失是肺癌发生中较频繁和早期的事件之一。检测染色体3p区5个典型抑癌基因DLEC1、RASSF1A、hMLH1、RARβ和FHIT在非小细胞肺癌(non-small cell lung cancer,NSCLC)中的甲基化状况,分析其临床意义。方法取78例NSCLC患者术中癌组织及相应正常肺组织标本,采用甲基化特异性聚合酶链反应(methylation specific PCR,MSP)检测基因启动子区甲基化状况,RT-PCR和免疫组化检测DLEC1基因表达。结果 78例NSCLC组织中,DLEC1、RASSF1A、RARβ和hMLH1甲基化频率分别为41.03%、39.74%、30.77%和16.67%,与正常组织相比差异均具有统计学意义。FHIT基因在癌组织和正常组织均无甲基化。DLEC1甲基化与患者临床分期(P=0.011)和淋巴结转移相关(P=0.019),而RASSF1A、RARβ、hMLH1基因甲基化以及平均甲基化指数与临床病理特征无关联。56.41%(44/78)的NSCLC组织中发现DLEC1基因表达下调或缺失,且与启动子甲基化有关。结论 3p区抑癌基因甲基化是NSCLC发生中的重要分子事件,可能作为NSCLC早期诊断的潜在生物标记,新型抑癌基因DLEC1失活与启动子高甲基化有关。     

NSCLC患者血浆p16和MGMT基因甲基化检测及临床意义

目的：检测非小细胞肺癌（non—small cell lung cancer，NSCLC）患者外周血血浆中p16基因、O^6-甲基乌嘌呤-DNA甲基转移酶（O^6-methylguanine—DNA methyhransferase，MGMT）基因启动子的甲基化状态，探讨p16、MGMT基因启动子的异常甲基化在NSCLC筛查及早期诊断中的意义。方法：利用巢式甲基化特异性聚合酶链反应法检测NSCLC患者外周血血浆p16、MGMT基因启动子的甲基化状态。结果：65例NSCLC血浆样品中分别发现19例（29．23％）p16基因启动子异常甲基化和16例（24．62％）MGMT基因启动子异常甲基化，45例正常对照血浆组未检测到p16、MGMT基因启动子的异常甲基化（P〈0．05），血浆中两基因甲基化检出率与NSCLC的分型及临床分期无明显相关性（P〉0．05）。结论：利用巢式甲基化特异性PCR法检测外周血血浆中p16、MGMT基因启动子的甲基化，可为NSCLC的筛查、早期诊断及预后判断提供有价值的信息。     

卵巢上皮性恶性肿瘤组织RASSF1A基因启动子区甲基化的研究

目的 探讨RASSF1A基因启动子区异常甲基化与卵巢上皮性恶性肿瘤发生、发展的关系。方法 应用甲基化特异性PCR方法,检测80例卵巢上皮性恶性肿瘤组织RASSF1A基因启动子区异常甲基化。结果 80例卵巢上皮性恶性肿瘤组织中,RASSF1A基因启动子区甲基化的发生率为52．5％,而相应痛旁正常组织中,RASSF1A基因启动子区均未发生甲基化（P〈0．05）。浆液性癌、黏液性癌和内膜样癌中,RASSF1A基因启动子区甲基化的发生率分别为54．2％、52．4％和45．5％,差异尤统计学意义。临床Ⅰ期、Ⅱ期卵巢上皮性恶性肿瘤RASSF1A基因启动子区甲基化的发生率分别为21．4％和16．7％,明显低于临床Ⅲ期（66．7％）和Ⅳ期（77．8％）。高分化组和中分化组RASSFlA基因启动子区甲基化的发牛率分别为34．5％和35．0％,均低于低分化组（80．6％）。结论 卵巢上皮性恶性肿瘤组织中存在RASSF1A基因启动子区的异常甲基化,甲基化与卵巢上皮性恶性肿瘤的临床分期和组织学分级有关。     

上皮性卵巢癌组织中RASSF1A和BRCA1及p16基因异常甲基化检测及其临床意义的研究

目的：探讨RASSF1A、BRCA1和p16基因异常甲基化在上皮性卵巢癌发生及发展中的作用。方法：采用甲基化特异性PCR法检测63例上皮性卵巢癌组织和相应的41例盆腹腔转移灶及10例癌旁卵巢组织中RASSF1A、BRCA1和p16基因启动子区甲基化状态。结果：上皮性卵巢癌组织原发灶及转移灶中RASSF1A、BRCA1和p16基因启动子区甲基化发生率分别为49．2％、25．4％、20．6％及58．5％、26．8％、22．0％，均显著高于正常卵巢组织中的发生率，P值均〈0．05。RASSF1A基因启动子区异常甲基化的发生率在临床Ⅰ、Ⅱ期显著低于Ⅲ、Ⅳ期，Х^2=13．018，P〈0．0001；在高、中分化癌的发生率均显著低于低分化癌，Х^2=8．481，P=0．004；Х^2=8．195，P=0．004。结论：RASSF1A、BRCA1和p16基因异常甲基化与上皮性卵巢癌的发生及发展相关，RASSF1A基因异常甲基化与上皮性卵巢癌临床分期及分化程度相关。     

河南林州地区贲门癌和癌旁组织中p16基因启动子甲基化及蛋白表达

目的探讨贲门癌变过程中p16基因启动子区甲基化和p16蛋白表达变化特征和规律及其相互关系。方法采用甲基化特异PCR（MSP）及免疫组化方法,检测林州地区32例贲门癌患者癌组织、癌旁不典型增生组织和正常组织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缺失18例（56%）,不典型增生组织中表达缺失8例（73%）;26例（81%）癌组织、7例（64%）不典型增生组织和18例（67%）正常组织发生了p16基因启动子区的甲基化。贲门癌组织中p16基因甲基化与表达缺失一致率为56%。差异无统计学意义,P〉0.05。结论p16蛋白表达缺失可能是贲门癌变过程中的重要分子事件,p16基因启动子区甲基化可能是导致其蛋白表达缺失的机制之一。     

非小细胞肺癌组织中3p区抑癌基因启动子的甲基化

目的：探讨3p位点上3个抑癌基因RASSF1α、RAR-β和FHIT启动子甲基化在非小细胞肺癌（non-smallcelllungcancer．NSCLC）组织中的作用。方法：应用甲基化特异的PCR（methyl-specific PCR,MSP）和RTPCR方法分别测定53例NSCLC组织中RASSF1α、RAR-β和FHIT的甲基化率与转录水平。结果：NSCLC癌组织和癌旁组织中的甲基化率分别是FHIT为53％和8％,RAR-β为42％和6％,RASSF1α为57％和9％;FHIT、RASSF1α的甲基化率在吸烟指数〉400年支组高于≤400年支组（P值分别为0．004与0．008）;FHIT、RASSF1α的甲基化率Ⅱ期高于Ⅰ期（P值分别为0．016与0．008）,Ⅲ期高于Ⅱ期（P值分别为0．033与0．029）。结论：3P位点RASSF1α、RAR-β和FHIT基因甲基化的发生与NSCLC的病理生理密切相关,而启动子CpG岛甲基化是导致转录沉默的重要机制。     

MSP法检测胃癌组织中RASSF1A基因启动子区甲基化改变

目的：探讨抑癌基因RASSF1A启动子区CpG岛甲基化与胃癌及临床病理特征的关系。方法：采用甲基化特异性PCR（methylation—specific PCR,MSP）法检测60例胃癌组织及相应癌旁组织和30例对照组织中RASSF1A基因启动子区甲基化状态。结果：胃癌组织中RASSF1A基因启动子区CpG岛甲基化率为65．0％（39／60）,艋著高于癌旁组织6．7％（4／60）,及对照组0％（0／30）（P〈0．01）。胃癌组织中不同年龄、性别、分化程度及淋巴结转移与否的RASSF1A基因甲基化率的差异均无统计学意义。结论：胃癌中RASSF1A基因启动子区的高甲基化提示其与胃癌的发生密切相关,MSP法对RASSF1A基因启动子区甲基化的检测有望成为胃癌早期监测的重要方法。     

结直肠癌中 RASSF1A 启动子甲基化与Cyclin D1和P53表达的关系

目的： 检测结直肠癌组织中Ras相关区域家族1A（ras-association domain family 1A, RASSF1A )基因启动子甲基化以及Cyclin D1和P53蛋白的表达,分析它们与结直肠癌临床病理特征的关系。 方法： 收集2008年8月至2009年8月长海医院病理科37例结直肠癌组织和14例癌旁组织(癌灶边缘5 cm以外组织）标本。甲基化特异性PCR（methylation-specific PCR,MSP）检测结直肠癌组织中 RASSF1A 基因启动子的甲基化,免疫组织化学法检测结直肠癌组织中 Cyclin D1和P53蛋白的表达,分析 RASSF1A 启动子甲基化、Cyclin D1和P53表达的关联性以及三者与结直肠癌临床病理特征的相关性。 结果： 37例结直肠癌组织中 RASSF1A 启动子甲基化有23例（62.16%）,14例癌旁组织中 RASSF1A 启动子甲基化有12例（85.71%）;37例癌组织中Cyclin D1阳性14例（37.84%）、P53阳性15例（40.54%）,14例癌旁组织中Cyclin D1和p53表达均为阴性。直肠癌组织中 RASSF1A 启动子甲基化阳性率高于结肠癌（P<0.05）。结直肠癌患者年龄与Cyclin D1表达呈负相关（P<005）,淋巴结转移与P53表达呈正相关（P<0.05）。结直肠癌组织中 RASSF1A 启动子甲基化与Cyclin D1和P53的表达无相关性。 结论： RASSF1A 启动子甲基化、Cyclin D1和P53蛋白表达三者的联合检测对研究结直肠癌的发生、发展有一定意义。     

非小细胞肺癌RASSF1A启动子甲基化状态临床研究

目的 探讨非小细胞肺癌(NSCLC)中RASSF1A启动子的甲基化状态及临床意义.方法 采用甲基化特异的PCR(MSP)检测150例NSCLC、34例癌旁正常组织和20例肺部良性病变中RASSF1A启动子甲基化状态.结果 150例NSCLC中58例发现RASSF1A启动子存在甲基化(38.7%),34例癌旁正常组织和20例肺部良性病变无一例发现RASSF1A启动子甲基化.RASSF1A启动子甲基化与年龄、性别、吸烟指数、组织类型、TNM临床分期均无关(P＞0.05),与开始吸烟年龄、肿瘤分化程度相关(P＜0.05).结论 RASSF1A启动子甲基化状态可以作为NSCLC诊断的一个候选分子标志.     

Epigenetic inactivation of RASSF1A in lung and breast cancers and malignant phenotype suppression

BACKGROUND: The recently identified RASSF1 locus is located within a 120-kilobase region of chromosome 3p21.3 that frequently undergoes allele loss in lung and breast cancers. We explored the hypothesis that RASSF1 encodes a tumor suppressor gene for lung and breast cancers. METHODS: We assessed expression of two RASSF1 gene products, RASSF1A and RASSF1C, and the methylation status of their respective promoters in 27 non-small-cell lung cancer (NSCLC) cell lines, in 107 resected NSCLCs, in 47 small-cell lung cancer (SCLC) cell lines, in 22 breast cancer cell lines, in 39 resected breast cancers, in 104 nonmalignant lung samples, and in three breast and lung epithelial cultures. We also transfected a lung cancer cell line that lacks RASSF1A expression with vectors containing RASSF1A complementary DNA to determine whether exogenous expression of RASSF1A would affect in vitro growth and in vivo tumorigenicity of this cell line. All statistical tests were two-sided. RESULTS: RASSF1A messenger RNA was expressed in nonmalignant epithelial cultures but not in 100% of the SCLC, in 65% of the NSCLC, or in 60% of the breast cancer lines. By contrast, RASSF1C was expressed in all nonmalignant cell cultures and in nearly all cancer cell lines. RASSF1A promoter hypermethylation was detected in 100% of SCLC, in 63% of NSCLC, in 64% of breast cancer lines, in 30% of primary NSCLCs, and in 49% of primary breast tumors but in none of the nonmalignant lung tissues. RASSF1A promoter hypermethylation in resected NSCLCs was associated with impaired patient survival (P =.046). Exogenous expression of RASSF1A in a cell line lacking expression decreased in vitro colony formation and in vivo tumorigenicity. CONCLUSION: RASSF1A is a potential tumor suppressor gene that undergoes epigenetic inactivation in lung and breast cancers through hypermethylation of its promoter region.     

Detection of RASSF1A aberrant promoter hypermethylation in sputum from chronic smokers and ductal carcinoma in situ from breast cancer patients

The newly identified 3p21.3 tumour suppressor gene RASSF1A is methylated in the majority of primary lung tumours, lung tumour cell lines and in a variable percentage of breast tumours. To determine the extent of RASSF1A promoter hypermethylation in early lung tumorigenesis, we analysed sputum samples from lung cancer patients and from current and former smokers using a sensitive methylation-specific PCR (MSP) technique. We also analysed RASSF1A promoter region hypermethylation in trios of normal breast/invasive ductal breast carcinoma/ductal carcinoma in situ (DCIS) from breast cancer patients and DCIS without invasive cancer. We found that 50% of small cell lung cancer (SCLC) and 21% of non-small cell lung cancer (NSCLC) patients had RASSF1A methylation, while one of two former smokers and four of 13 current smokers demonstrated RASSF1A methylation in sputum. Furthermore, two of the four current smokers and one former smoker showing RASSF1A methylation in their sputum developed cancer within 12-14 months of bronchoscopy. In our breast cancer trios, RASSF1A promoter hypermethylation was detected in 65% of invasive cancers, in 42% of corresponding DCIS but in none of the normal breast samples. In addition, we found that three out of 10 DCIS without invasive breast cancer also underwent RASSF1A promoter hypermethylation. Our findings suggest that RASSF1A promoter region hypermethylation may be a useful molecular marker for early detection of lung cancer. Furthermore, since RASSF1A promoter hypermethylation was detected in ductal carcinoma in situ, inactivation of RASSF1A may be an early event in breast tumorigenesis.     

Tumor-specific methylation in bronchial lavage for the early detection of non-small-cell lung cancer.

PURPOSE: The aim of this study was to identify tumor-specific methylation in bronchial lavage for the early detection of non-small-cell lung cancer (NSCLC) by differentiating the age-related methylation from the tumor-specific methylation in NSCLC. PATIENTS AND METHODS: Eighty-five NSCLC patients and 127 cancer-free subjects participated in this study. Aberrant methylation at the promoters of the p16, Ras association domain family 1A (RASSF1A), fragile histidine triad (FHIT), H-cadherin, and retinoic acid receptor beta (RARbeta) genes were evaluated in the resected tumor tissues and bronchial lavage samples of NSCLC patients and in the bronchial lavage samples of cancer-free subjects by methylation-specific polymerase chain reaction. RESULTS: Of the 127 cancer-free samples, methylation was detected in 6% for p16, 13% for RARbeta, 3% for H-cadherin, 4% for RASSF1A, and 28% for FHIT. Hypermethylation of the p16, RARbeta, H-cadherin, and RASSF1A genes was not associated with patient age and smoking, whereas hypermethylation of the FHIT promoter occurred more frequently in older patients (P =.02) and was associated with exposure to tobacco smoke (P =.001). A strong correlation between age and smoking was found in patients with hypermethylation of the FHIT gene (r = 0.36; P =.03). A total of 68% of the bronchial lavage samples from the 85 NSCLC patients showed methylation of at least one of p16, RARbeta, H-cadherin, and RASSF1A genes. CONCLUSION: Our study suggests that tumor-specific methylation of the p16, RASSF1A, H-cadherin, and RARbeta genes may be a valuable biomarker for the early detection of NSCLC in bronchial lavage, and that the age-related methylation of FHIT gene in the normal bronchial epithelium is related to the exposure to tobacco smoke.     

Epigenetic inactivation of the RAS-effector gene RASSF2 in lung cancers

RASSF2, a member of the RAS association domain family 1 (RASSF1), is a candidate tumor suppressor gene (TSG) that is silenced by promoter hypermethylation in several human cancers. In this study, we examined the expression of RASSF2 mRNA and the promoter methylation status in lung cancer cell lines and in tumor samples of 106 primary non-small cell lung cancers (NSCLCs) by methylation-specific PCR. RASSF2 expression was absent in 26% of small cell lung cancers (SCLCs; n=27 lines) and 50% of NSCLCs (n=42 lines). Promoter methylation of RASSF2 was found in 18% of the SCLC cell lines (n=22) and 62% of the NSCLC cell lines (n=26), and the methylation status was tightly associated with the loss of RASSF2 expression. RASSF2 expression was restored by treatment with 5-aza-2-deoxycytidine and/or trichostatin-A in the NSCLC cell lines which were absent of the expression. RASSF2 methylation was found in 31% of primary NSCLC tumors, and methylation was more frequent in the specimens from non-smokers (18 of 40, 45%) than in the specimens from smokers (15 of 66, 23%, P=0.014). We also examined the association of RASSF2 methylation with mutations of KRAS and EGFR and with promoter hypermethylation of RASSF1A; however, we could not find a significant association between RASSF2 methylation and these genetic and epigenetic changes. Our results indicate that aberrant methylation of the RASSF2 gene with the subsequent loss of RASSF2 expression plays an important role in the pathogenesis of lung cancers.     

RASSF1A基因甲基化与非小细胞肺癌预后的相关性研究

背景与目的研究发现在很多肿瘤中均存在RASSF1A基因启动子区域高甲基化状态导致基因表达失活的现象,本研究就RASSF1A基因启动子的甲基化状态与非小细胞肺癌预后的关系进行探讨。方法采用甲基化特异的PCR检测150例非小细胞肺癌和20例肺部良性病变RASSF1A启动子甲基化状态。结果150例非小细胞肺癌中58例发现RASSF1A启动子存在甲基化(58/158,38.7%),20例肺部良性病变中无一例发现RASSF1A启动子甲基化。存在RASSF1A启动子高甲基化的病例预后较未发现RASSF1A甲基化的病例差(P=0.004),Cox回归分析显示RASSF1A启动子的甲基化状态是非小细胞肺癌术后的一个预后相关因素(RR=1.584,95%CI:1.040-2.411,P=0.032)。结论MSP法检测RASSF1A启动子甲基化状态可以作为非小细胞肺癌术后的一个预后评价指标。