肺癌细胞株APC基因启动子甲基化对其转录的影响

背景与目的:抑癌基因家族性腺瘤样结肠息肉病易感基因(adenomatous polyposis coli,APC)启动子区的高甲基化在很多肿瘤中被发现,与这些肿瘤的发生发展相关.本实验室在肺癌患者肿瘤组织中检测到APC甲基化率达47%,为了研究其在肺癌细胞株中的甲基化情况,并进一步了解甲基化对其转录的影响,本研究检测了3株肺癌细胞的抑癌基因APC启动子甲基化状态及其对该基因转录水平的影响.方法:提取3株肺癌细胞株(肺腺癌细胞株SPC-A1、小细胞肺癌细胞株NCI-H446、大细胞肺癌细胞株NCI-H460)的DNA,以经转甲基处理和未做处理的脐带血DNA为阳性、阴性对照,亚硫酸氢盐化学修饰后,用甲基化特异性基因扩增(methylation specific-polymerase chain reaction,MSP)和甲基化基因芯片对APC基因启动子1A CpG岛甲基化进行研究,并用实时荧光定量PCR(real time-polymerase chain reaction)技术,以Sybr-GreenⅠ为荧光染料,β-actin基因为内参照,检测mRNA转录;对甲基化阳性的NCI-H460细胞,分别用1、5、10、15 μmol/L的5'-杂氮-2'-脱氧胞嘧啶(5-aza-2-deoxycytidine,5-aza-dC)试剂进行脱甲基化,提取RNA,荧光定量检测其转录变化.结果:SPC-A1和NCI-H446细胞APC甲基化阴性,NCI-H460细胞APC甲基化阳性;甲基化芯片检测NCI-H460细胞在APC启动子1A 5个CpG位点均存在甲基化(687、707、714、719、726),SPC-A1和NCI-H446甲基化阴性,荧光定量结果NCI-H460的APC转录较SPC-A1和NCI-H446有明显的下降,仅为二者平均的30.04%;经5-aza-dC脱甲基化作用后,NCI-H460细胞的APC表达增加了约5～10倍,其中10 μmol/L浓度作用下,APC表达增加最多.结论:肺癌细胞株NCI-H460中存在APC基因高甲基化,5-aza-dC脱甲基化试剂可以激活其转录.     

乳腺癌患者外周血浆中游离的肿瘤相关DNA检测及其临床价值

目的探讨外周血浆中游离DNA变异在乳腺癌早期诊断、疗效评估和复发监控中应用的可行性。方法采用甲基化特异性PCR(MSP)方法,检测84例乳腺癌患者肿瘤组织、癌旁正常腺体组织及外周血浆中游离的肿瘤相关DNA钙黏素E(E-cadherin)和APC基因启动子甲基化畸变状况,选择10例乳腺良性疾病患者的血浆作为正常对照。结果乳腺癌肿瘤组织E-cadherin和APC基因启动子甲基化畸变频率分别为52.4%和45.2%,相应外周血浆中相同的DNA变异阳性检出率分别为33.3%和31.0%。外周血浆中DNA甲基化变异与肿瘤组织的甲基化畸变状况显著相关(E- cadherin,P<0.001;APC,P=0.002)。血浆DNA E-cadherin和APC基因甲基化畸变检测的灵敏度分别为63.6%和63.2%,特异度分别为100%和95.7%。肿瘤组织及外周血浆中游离DNA甲基化畸变与临床分期、病理类型、肿块大小及受体状况无相关性(P>0.05)。癌旁正常腺体组织及健康对照血浆中均未检测到E-cadherin和APC基因甲基化变异。结论在受检乳腺癌患者中,约1/3外周血浆中可检测到与原发肿瘤相同的E-cadherin和(或)APC基因甲基化畸变,与临床分期无相关性。在乳腺癌早期诊断、疗效评估和复发监控中,检测外周血浆中游离的肿瘤相关DNA变异有一定的可行性。     

DNA修复酶基因MGMT启动子区异常甲基化与食管癌的关系

背景与目的： 分析食管癌组织中MGMT启动子区CpG岛甲基化状态和食管癌发病风险的关系,探讨MGMT基因启动子的异常甲基化在食管癌筛查及早期诊断中的意义。 材料与方法： 对江苏淮安的91例新发食管癌病例的癌组织、癌旁组织及外周血血浆样本提取DNA,应用甲基化特异性PCR(MSP)分析MGMT启动子区CpG岛的甲基化状态;对食管癌组织和癌旁组织提取总RNA,采用SYBR GREEN I 实时荧光定量逆转录-聚合酶链反应(RT-PCR)测定MGMT的mRNA水平。 结果： MGMT启动子区CpG岛异常甲基化与食管癌发病风险增高有关联 (OR=7.750, 95％CI=2.736～21.955);MGMT启动子区CpG岛甲基化状态与MGMT mRNA水平无显著相关;血浆循环DNA中MGMT启动子区CpG岛甲基化与癌组织中MGMT启动子区CpG岛甲基化相关(P<0.01),血浆循环DNA中MGMT启动子区CpG岛甲基化检出率与癌组织中MGMT启动子区CpG岛甲基化检出率中度相关(Kappa=0.603, P<0.01)。 结论： MGMT基因的启动子区CpG岛的异常甲基化与食管癌发病风险增加有关;检测血浆循环DNA中MGMT基因启动子的异常甲基化,可为食管癌的筛查、早期诊断提供有价值的信息。     

血浆 MAL 基因甲基化检测对于肺癌早期诊断的意义

目的：研究肺癌患者、肺结核患者及健康人群 T 淋巴细胞相关蛋白（T －lymphocyte maturation －asso-ciated protein,MAL）基因启动子区的甲基化状态,进一步探讨血浆 MAL 基因甲基化检测对于肺癌早期诊断及鉴别肺癌与肺结核疾病的意义。方法：采用甲基化特异性 PCR（methylation special PCR,MSP）方法,检测75例肺癌组织标本、癌旁组织标本和对应的血浆标本及58例肺结核组织标本及对应的血浆标本的 MAL 基因启动子区甲基化状态;同时检测30例正常人群的血浆标本 MAL 甲基化状态作为对照。结果：肺癌组织和癌旁组织中 MAL 基因甲基化发生率分别为78．7％（59／75）和2．7％（2／75）,差别具有统计学意义（P ＜0．001）;肺癌和肺结核组织中 MAL 基因甲基化发生率分别为78．7％（59／75）和3．4％（2／58）,差别具有统计学意义（P＜0．001）;肺癌患者与肺结核患者对应的血浆中 MAL 基因甲基化发生率分别73．3％（55／75）和1．7％（1／58）,差别也具有统计学意义（P ＜0．001）。正常健康人群的血浆标本中未发现 MAL 基因甲基化改变;肺癌组和肺结核组分别与健康人群组比较,肺癌组明显高于健康人群（P ＜0．001）,而肺结核组患者与健康人群比较无明显差异（P ＝0．47）。组织标本和血浆标本的检出率具有一致性（P ＞0．05）。根据病理类型、TNM分期、性别、吸烟史等对肺癌患者进行分组,各组之间均无明显差异（P ＞0．05）。结论：MSP 法检测血浆 MAL 基因甲基化状态,可以作为一种有潜力的肺癌早期诊断方法,对于鉴别肺癌和肺结核也有一定的帮助。     

腺瘤性结肠息肉病基因甲基化在肝细胞癌分子诊断中的价值

目的:建立甲基化敏感性限制性内切酶-定量PCR(methylation-sensitive restriction enzyme-quantitative PCR,MSRE-qPCR)方法,并应用该方法探讨血浆腺瘤性结肠息肉病(adenomatous polyposis coli,APC)基因甲基化检测在肝细胞癌(hepatocellular carcinoma,HCC)诊断中的应用价值。方法:用HhaⅠ消化DNA样品,qPCR技术评估酶切效率,建立优化的MSRE-qPCR方法。用MSRE-qPCR法检测45例肝组织(20例HCC及其相应匹配的非癌组织和5例正常肝组织)中APC甲基化水平;应用亚硫酸氢盐测序PCR(bisulfi te sequencing PCR,BSP)对MSRE-qPCR检测结果进行验证,并与甲基化特异性PCR(methylation-specifi c PCR,MSP)检测方法比较。运用MSRE-qPCR技术检测72例HCC、37例肝良性病变和41例健康志愿者血浆标本的APC甲基化状态。结果:建立的MSRE-qPCR方法可定量检测低至1%的APC甲基化片段。MSRE-qPCR和MSP检测结果均显示,HCC组织中APC基因发生高频率甲基化。MSRE-qPCR检测结果经BSP验证准确无误,且与MSP检测结果具有较好的一致性(Kappa=0.955,P<0.0001)。HCC患者血浆APC甲基化水平高于肝良性病变及健康志愿者(P<0.0001)。血浆APC甲基化分析与血清甲胎蛋白(alpha-fetoprotein,AFP)联合检测可提高HCC诊断效率。结论:MSRE-qPCR可定量检测APC甲基化水平。血浆APC甲基化分析对于HCC的非侵入性诊断具有重要价值。     

乳腺癌患者外周血清中APC基因启动子甲基化异常的检测及其意义

背景与目的:检测肿瘤患者外周血中肿瘤相关标志物是当前肿瘤研究的热点之一,恶性肿瘤患者外周血中存在游离的肿瘤相关DNA已引起肿瘤学界的极大关注,人们曾在多种肿瘤患者血清中发现与原发肿瘤相同的DNA变异.本研究以APC(adenomatous polyposis coli)基因启动子甲基化作为肿瘤标志物,探讨乳腺癌患者外周血清中游离的肿瘤相关DNA与肿瘤组织及临床病理参数的相关性.方法:采用甲基化特异性PCR(methylation specific-PCR,MSP)方法,分别检测84例乳腺癌组织、癌旁正常腺体组织及外周血清中游离DNA APC基因启动子甲基化状况.结果:84例乳腺癌组织APC基因启动子甲基化频率为45.2%(38/84),相应外周血清中同样DNA变异阳性检出率为31.0%(26/84).外周血清中DNA甲基化变异与肿瘤组织的甲基化状况显著相关(r=0.977,P=0.002).检测外周血清中APC基因甲基化的敏感性为68.4%,特异性为97.8%.肿瘤组织及外周血清中游离DNA甲基化异常与临床分期、病理类型、肿块大小及受体状况无相关性(P＞0.05).肿瘤组织未检测到甲基化患者的血清中及健康人血清中均未检测到该基因甲基化变异.结论:乳腺癌患者外周血清中肿瘤相关DNA甲基化与肿瘤组织中相同基因的变异显著相关.     

肝癌患者血浆RASSF1A与APC基因甲基化诊断和预后价值分析

目的:探讨肝癌患者血浆中抑癌基因RASSF1A与APC启动子区域甲基化与诊断及生存期预后的关系。方法:收集102例肝癌、80例肝硬化及100名健康人血浆,提取DNA,采用甲基化特异性聚合酶链反应法检测RASSF1A与APC基因启动子区域甲基化状态,随访其生存期并分析基因甲基化状态与肝癌诊断及预后的关系。结果:RASSF1A基因启动子区域甲基化阳性率在肝癌和肝硬化血浆中分别为51.96%(53/102)和15.00%(12/80),差异有统计学意义,χ2=26.677,P=0.000;APC基因启动子区域甲基化阳性率在肝癌和肝硬化血浆中分别为47.06%(48/102)和22.50%(18/80),差异有统计学意义,χ2=11.700,P=0.001。102例肝癌患者中,44例(43.14%)AFP检测为阳性;58例AFP阴性的患者中,RASSF1A基因和APC基因启动子区域甲基化的阳性率分别为53.45%(31/58)和46.55%(27/58)。RASSF1A基因启动子区域甲基化阳性组与阴性组生存期比较,差异有统计学意义,P=0.011;而APC基因阳性组与阴性组生存期比较,差异无统计学意义,P=0.175。Cox多因素分析结果显示,AFP、临床分期、治疗方式及门静脉瘤栓有无为肝癌独立预后因子,RASSF1A和APC基因同时甲基化是肝癌的独立预后因子,P=0.019。结论:RASSF1A与APC基因启动子区域甲基化对肝癌、特别是AFP阴性肝癌的诊断有帮助,RASSF1A和APC基因启动子区域同时甲基化可以作为评价肝癌生存期预后的独立预后因子。     

肺癌患者外周血血浆中p16基因异常甲基化的检测

目的 分析肺癌患者外周血血浆中p16基因启动子区异常甲基化发生情况,探讨血浆中p16基因异常改变作为肺癌临床辅助诊断分子生物学标志物的可能性。方法 利用半巢式甲基化特异性PCR技术,检测了137例肺癌患者血浆和112例相对应肿瘤组织DNA p16基因的异常甲基化情况。结果 在血浆和肿瘤组织中的p16基因甲基化检出率分别为75．2％和80．4％;其中鳞癌、腺癌、腺鳞癌和小细胞肺癌患者血浆标本的阳性率分别为77．9％,65．1％,75．1％和91．7％。血浆DNAp16基因甲基化仅在肿瘤组织存在同样甲基化的病例中被检出。血浆和肿瘤组织中,p16基因的甲基化异常改变与肿瘤的分期、分型无明显相关性。结论 分析血浆DNA的p16基因异常甲基化有可能成为辅助肺癌诊断的有效方法之一。     

用尿沉淀细胞DNA甲基化状态的分析检测膀胱癌

目的：确定尿沉淀细胞DNA中的13个肿瘤相关基因启动子的甲基化谱式分析在膀胱癌诊断中的价值。方法：用定性甲基化特异性（methylation specific polymerase chain reaction，MSP）的方法，对92例临床确诊的膀胱癌患者、23例非肿瘤性尿路疾病患者、6例脑外科患者、7例健康志愿者检测了尿沉淀细胞DNA中肿瘤相关基因启动子的甲基化状态。结果：在临床确诊的92例膀胱癌患者中被检测的13个基因的高甲基化状态出现频率显著高于23例非肿瘤性尿路疾病患者，差异有统计学意义（P〈0．05）。而6例脑外科患者和7例正常健康人的尿沉淀细胞DNA中，上述基因均为去甲基化状态。若以任一个基因高甲基化为膀胱癌的指征，88．0％（81／92例）的膀胱癌可被检出。结论：MSP法分析尿沉淀细胞DNA中肿瘤相关基因启动子的甲基化状态可有效地检出膀胱癌。     

非小细胞肺癌中ASC基因启动子甲基化的意义

目的：探讨非小细胞肺癌（NSCLC）中ASC基因启动子甲基化与患者临床特征之间的关系以及去甲基化试剂对肺癌细胞系中ASC基因甲基化状态的逆转。方法：应用甲基化特异性PCR（MSP）检测48例NSCLC患者肿瘤组织和3株肺癌细胞系中ASC基因的甲基化状态，RT—PCR检测去甲基化试剂诱导肺癌细胞系中甲基化ASC基因的重新表达。结果：NSCLC肿瘤组织和相对应的正常肺组中ASC基因启动子甲基化的发生率分别为49．7％和8-3％，二者之间有显著性差异（P〈0．01）。重度吸烟患者的肿瘤组织中ASC基因启动子甲基化的发生率明显增高（P〈0．05），早期的NSCLC（T1）中ASC基因启动子甲基化也有相对较高的发生率（P〈0．05）。去甲基化试剂5-氮-2’-脱氧胞苷（5-aza-2＇-deoxycytidine）可使甲基化的ASC基因启动子发生逆转而获得重新表达。结论：ASC基因启动子的甲基化可能与吸烟引起的非小细胞肺癌有关．并可能是非小细胞肺癌发展过程中的一个早期事件，甲基化的ASC基因可以成为NSCLC治疗的靶点。     

Quantitative adenomatous polyposis coli promoter methylation analysis in tumor tissue, serum, and plasma DNA of patients with lung cancer.

The serum of cancer patients often harbors increased free DNA levels, which can potentially be used for cancer detection. Because genetic and epigenetic alterations of the adenomatous polyposis coli (APC) gene are common events in gastrointestinal tumor development, we sought to investigate the frequency and level of aberrant APC promoter methylation in primary tumors and paired preoperative serum or plasma samples of lung cancer patients by semiquantitative methylation-specific fluorogenic real-time PCR. We detected methylation of APC in 95 of 99 (96%) primary lung cancer tissues. Forty-two of 89 (47%) available serum and/or plasma samples from these cases carried detectable amounts of methylated APC promoter DNA. In contrast, no methylated APC promoter DNA was detected in serum samples from 50 healthy controls. A highly elevated APC methylation level in lung cancer tissue was the only independent factor predicting inferior survival in this cohort (P = 0.015). APC methylation analysis appears to be promising as a prognostic factor in primary lung cancer and as a noninvasive tumor marker in plasma and/or serum DNA.     

Methylation assay for the diagnosis of lung cancer on bronchial aspirates: a cohort study.

PURPOSE: Recent studies have detected aberrant promoter methylation of adenomatous polyposis coli promoter 1 A (APC), cyclin-dependent kinase inhibitor-2A (p16(INK4a)), retinoic acid receptor beta2, and RAS association domain family protein 1 (RASSF1A) in bronchial aspirates and suggested their use as biomarkers for lung cancer diagnostics. The purpose of this study was to validate these candidate marker genes in a retrospective cohort study. EXPERIMENTAL DESIGN: Bronchial aspirates collected from a cohort comprising 247 patients with suspected lung cancer were investigated retrospectively regarding aberrant promoter methylation using a quantitative methylation-specific real-time PCR (QMSP). RESULTS: Eighty-nine patients were diagnosed with primary lung cancer, 102 had benign lung disease, and 56 showed miscellaneous other conditions. A panel consisting of APC, p16(INK4a), and RASSF1A emerged as useful combination. This panel detected aberrant methylation in bronchial aspirates of 22 of 35 (63%) and 21 of 44 (44%) centrally and peripherally located primary lung cancers, respectively. Bronchial aspirates also showed aberrant methylation in 5 of 7 (71%) patients with a recurrent lung cancer and in 8 of 30 (27%) cases without tumor recurrence. In contrast, only 1 of 102 patients with benign lung disease displayed a (false) positive test result. Rarely, aberrant methylation was found in patients with other malignancies (3 of 16). The QMSP assay correctly confirmed lung cancer in 8 of 12 (67%) cases with an ambiguous cytology. Moreover, it disclosed 9 of 26 (35%) of peripheral tumors lacking simultaneous cytologic or histologic diagnosis of malignancy. CONCLUSIONS: Our findings suggest that the QMSP assay could be applied as a reflex test in cases of suspected lung cancer that defy a definite diagnosis by conventional methods. Thus, the assay could be a useful diagnostic adjunct especially regarding peripheral tumors.     

Quantitative methylation-specific polymerase chain reaction gene patterns in urine sediment distinguish prostate cancer patients from control subjects.

PURPOSE: Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of prostate cancers and is a promising marker for cancer detection. We sought to develop a test for prostate cancer based on a quantitative methylation-specific polymerase chain reaction (QMSP) of multiple genes in urine sediment DNA. PATIENTS AND METHODS: We tested urine sediment DNA for aberrant methylation of nine gene promoters (p16INK4a, p14(ARF), MGMT, GSTP1, RARbeta2, CDH1 [E-cadherin], TIMP3, Rassf1A, and APC) from 52 patients with prostate cancer and 21 matched primary tumors by quantitative fluorogenic real-time polymerase chain reaction. We also analyzed urine sediments from 91 age-matched individuals without any history of genitourinary malignancy as controls. RESULTS: Promoter hypermethylation of at least one of the genes studied was detected in urine samples from all 52 prostate cancer patients. Urine samples from the 91 controls without evidence of genitourinary cancer revealed no methylation of the p16, ARF, MGMT, and GSTP1 gene promoters, whereas methylation of RARbeta2, TIMP3, CDH1, Rassf1A, and APC was detected at low levels. CONCLUSION: Overall, methylation found in urine samples matched the methylation status in the primary tumor. A combination of only four genes (p16, ARF, MGMT, and GSTP1) would theoretically allow us to detect 87% of prostate cancers with 100% specificity. Our data support further development of the noninvasive QMSP assay in urine DNA for early detection and surveillance of prostate cancer.     

肝细胞癌中APC基因启动子甲基化及蛋白表达的研究

目的：探讨肝细胞癌中APC基因启动子甲基化状态及蛋白表达的临床意义。方法：应用甲基化特异性聚合酶链反应（MSP）和免疫组织化学方法检测61例HCC及相应的癌旁肝组织中APC基因启动子甲基化状态和蛋白表达水平,分析甲基化与临床资料及蛋白表达的关系。结果：癌组织及癌旁组织中APC基因启动子甲基化阳性率分别为26.23%和11.47%（P〈0.05）。癌与癌旁组织APC蛋白表达无统计学差异（P〉0.05）。APC基因启动子甲基化与临床分期、门脉癌栓、术后复发、肝外转移、肿瘤大小、肿瘤分化、肿瘤个数及血清AFP值无关。APC基因启动子甲基化与蛋白表达无相关性。结论：APC启动子区甲基化可能参与了肝癌的发生,但在HCC的发展中的作用仍需进一步研究。     

PAX1基因甲基化特异性定量PCR检测在宫颈癌筛查中的意义

目的 探讨定量测定配对盒家族基因1（PAX1）甲基化水平在宫颈癌筛查中的意义。方法 收集正常宫颈组织20例、宫颈上皮内瘤变Ⅰ（CINⅠ）组织15例、CINⅡ组织16例、CINⅢ组织19例和宫颈癌组织22例。采用甲基化特异性实时定量聚合酶链反应（QMSP）定量检测不同宫颈组织中PAX1甲基化水平,甲基化特异性PCR（MS-PCR）检测宫颈癌组织PAX1基因区域甲基化情况,并同时行第2代杂交捕获 人乳头瘤病毒 DNA（HC2-HPV-DNA）检测。采用受试者工作特征曲线（ROC）比较两者对宫颈癌的诊断效率。甲基化率通过甲基化指数（PMR）进行指标量化。结果 宫颈癌组织的PMR为（75.27±30.61）％,显著高于CIN Ⅲ的（12.90±10.80）％和其他良性病变及正常组织（P＜0.001）。QMSP 检测PAX1甲基化的ROC曲线下面积（AUC）、灵敏度和特异度分别为0.98、100.0％和84.3％,优于HC2-HPV-DNA检测的0.82、100.0％和52.5％,差异有统计学意义（P＜0.001）。MS-PCR检测结果显示,在癌组织原发灶、转移性癌组织、毗邻原发灶的正常组织和距原发灶边缘＞3cm的正常组织中,PAX1的甲基化率分别为95.0％（19／20）、100.0％（4／4）、70.0％（14／20）和35.0％（7／20）,组间比较差异有统计学意义（P＜0.001）。结论 QMSP方法定量检测PAX1的甲基化具有极高的灵敏度,且其特异度超过目前常用的HC2-HPV-DNA检测方法,在临床筛查和宫颈癌的早期诊断中可能具有潜在的应用价值。     

DNA methylation alterations in the pancreatic juice of patients with suspected pancreatic disease

Molecular markers of pancreatic neoplasia could aid in the evaluation of visible pancreatic lesions and indicate neoplasia invisible to imaging. We evaluated methylation-specific PCR (MSP) assays that detect aberrantly methylated DNA for their use as markers of pancreatic neoplasia. Methylation analysis was done on pancreatic juice collected endoscopically or surgically from 155 individuals with suspected pancreatic disease: 56 patients had pancreatic ductal adenocarcinoma, 17 had intraductal papillary mucinous neoplasms, 26 had symptomatic chronic pancreatitis, 12 controls lacked evidence of pancreatic disease, and 44 were asymptomatic individuals at increased risk of developing familial pancreatic cancer undergoing screening for pancreatic neoplasia. Pancreatic juice DNA was analyzed for promoter methylation using conventional MSP assays for 17 genes. For six genes, pancreatic juice methylation was quantified using real-time quantitative MSP (QMSP; Cyclin D2, FOXE1, NPTX2, ppENK, p16, and TFPI2). Quantifying pancreatic juice methylation using QMSP with a cutoff of >1% methylated DNA could better predict pancreatic cancer than detecting methylation using conventional MSP. In the endoscopic group, 9 of 11 patients with pancreatic cancer, but none of 64 individuals without neoplasia had > or =1% methylation for two or more of the best five QMSP assays (82% sensitivity and 100% specificity; P < 0.0001). The prevalence of pancreatic juice methylation in patients with chronic pancreatitis was less than in patients with pancreatic cancer but higher than in controls and similar to high-risk individuals. The detection and quantification of aberrantly methylated DNA in pancreatic juice is a promising approach to the diagnosis of pancreatic cancer.