血浆Ras相关区域家族蛋白1A基因甲基化在肝细胞癌分子诊断中的价值

目的:建立一种可检测微量DNA标本中DNA甲基化的甲基化敏感性限制性内切酶-定量PCR(methylation-sensitive restriction enzymes-based quantitative PCR,MSRE-qPCR)方法,并运用该技术探讨血浆Ras相关区域家族蛋白1A(Ras association domain family1A,RASSF1A)基因甲基化检测在肝细胞癌(hepatocellutar carcinoma,HCC)非侵入性诊断中的价值。方法:用MSRE HhaⅠ消化DNA样品,再用qPCR技术分析酶切结果,建立检测RASSF1A基因甲基化的MSRE-qPCR方法。以45例肝组织(20对HCC患者手术切除肿瘤标本及匹配非癌组织和5例正常肝)为材料,测试该方法的应用价值;运用亚硫酸氢盐测序PCR(bisulf ite sequencing PCR,BSP)技术进行进一步验证,并与甲基化特异性PCR(methylation specif ic PCR,MSP)方法相比较。再运用该技术检测150例血浆标本(包括72例HCC患者、37例肝硬化或慢性肝炎等良性病变患者和41例健康对照)的RASSF1A基因甲基化状态,并分析其与HCC患者临床病理参数的关系。结果:MSRE-qPCR法可定量检测低至1%以下的RASSF1A甲基化片段。20例HCC组织中有14例(70%)发生RASSF1A高甲基化,对应非癌组织中RASSF1A甲基化阳性率为25%,而5例正常肝组织均为阴性。MSRE-qPCR结果经BSP验证无误,且与MSP检测结果具有较好的一致性。HCC患者血浆RASSF1A甲基化阳性率(47/72,65.3%)显著高于健康对照(1/41,2.4%)和肝良性病变组(3/37,8.1%),差异均有统计学意义(P<0.0001)。联合检测血浆RASSF1A甲基化与血清AFP可显著提高HCC诊断效率。结论:建立的MSRE-qPCR方法要求样本少、操作简便、成本低廉,可定量检测RASSF1A基因甲基化水平。血浆RASSF1A甲基化分析对于HCC的非侵入性诊断具有重要价值。     

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.     

MSRE-gPCR技术分析多基因DNA甲基化对肝细胞癌的诊断价值

背景与目的:DNA甲基化是潜在的肿瘤标志物.本研究运用自行建立的甲基化敏感性限制性内切酶一定量PCR(methylation-sensitive restriction enzymes-based quantitative PCR,MSRE-gPCR)方法检测肝细胞癌(hepatocellutar carcinoma,HCC)组织中多个基因的DNA甲基化状态,筛选可用于HCC诊断的DNA甲基化标志物组合.方法:以47对HCC患者癌组织和癌旁组织、8例正常人肝组织为材料,运用MSRE-gPCR方法检测这102例肝组织中APC、GSTPI、RASSFIA、p16、SFRP1、RUNX3、Hint1、SOCS1和HIC-1基因的启动子甲基化状态.结果:APC、GSTPI、RASSF1A、p16、SFRP1、RUNX3基因在肿瘤组织中的甲基化率分别为70.2%、70.2%、63.8%、29.8%、44.7%和36.2%,均显著高于对应癌旁组织(P均＜0.05);而Hintl、SOCSI和HIC-1基因甲基化水平在HCC肿瘤和癌旁组织间无显著差异.联合检测APC、GSTP1、RASSF1A和SFRP1 4个靶点,可检出所有HCC病例.这些基因的甲基化水平与患者肿瘤大小、分化、包膜及HBV感染等临床病理参数均无显著相关性.结论:联合检测APC、GSTPI、RASSFIA和SFRPI的DNA甲基化状态对于HCC风险评估和分子诊断具有重要价值.     

肺癌患者血浆中APC基因启动子甲基化定量检测研究

背景与目的：APC基因编码的蛋白参与信号转导途径,大量研究证实APC启动子区的高甲基化在肿瘤的发生发展中起重要作用。本研究建立血浆APC基因实时荧光定量甲基化特异性基因扩增（methylation—specific PCR,MSP）检测方法,并对临床肺癌患者血浆进行检测,以确定该方法在肺癌诊断中的应用价值。方法：将APC基因启动子甲基化阳性肺癌细胞株NCI—H460细胞用有限稀释法获取单个集落形成的细胞,以经典的酚一氯仿法提取细胞DNA,并用MSP对APC基因甲基化进行验证,紫外分光光度计定量并以10倍稀释的浓度梯度依次投入到200μL健康人血浆中．得到模拟肺癌患者血浆,利用磁珠核酸提取方法从模拟肺癌及肺癌患者、肺部良性疾病患者和健康对照者血浆中提取DNA,对血浆DNA模板进行亚硫酸氢盐化学修饰;以模拟血浆样品作为标准品,采用外标准曲线法对各种血浆样品中APC甲基化进行定量分析。结果：所建立的实时荧光定量MSP检测方法的线性范围为1．5×10^2-1．5×10^5拷贝／mL。用该方法检测甲基化肿瘤细胞的DNA其最低检测限为1．5×10^2拷贝／mL。78例肺癌患者有40例组织中检测出APC基因甲基化阳性．其中的19例（47．5％）肺癌患者血浆中APC基因启动子甲基化阳性,APC甲基化浓度为1．67×10^2-6．78×10^3拷贝／mL,中位浓度为1．60×10^3拷贝／mL。38例组织阴性的肺癌患者、31例肺部良性疾病患者和23例健康者的血浆APC基因启动子甲基化均为阴性。结论：实时荧光定量MSP检测血浆APC基因甲基化在肺癌诊断方面具有潜在应用价值。     

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。     

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。     

定量PCR检测肝细胞癌患者血浆循环DNA及其临床意义

背景与目的:血循环DNA是一种新的肿瘤诊断及预后判断的标志物.本研究运用定量PCR技术检测肝细胞癌(hepatocellular carcinoma,HCC)患者血浆循环DNA含量并探讨其诊断价值.方法:收集72例HCC患者术前血浆样本,37例肝良性病变(肝硬化以及慢性肝炎)和41例健康志愿者的血浆样本,纯化血浆循环DNA,采用实时定量PCR技术对血浆DNA水平进行检测.应用接受者操作特性(receiver-operating characteristics,ROC)曲线分析血浆循环DNA在HCC诊断中的价值.结果:HCC中位血浆循环DNA浓度(173 ng/mL)显著高于健康对照(9 ng/mL)和肝良性病变组(46 ng/mL)(P<0.001);其ROC曲线下面积(area under the ROC curve,AUC)分别为0.949和0.874.而HCC血浆DNA浓度也显著高于肝硬化及慢性肝炎患者(P=0.001),AUC为0.703.以18.2 ng/mL作为诊断HCC的临界值,其诊断特异度为90.2%,敏感度达90.3%;与血清AFP联合检测可提高HCC诊断效率,AUC上升至0.974,其诊断特异度和敏感度分别为95.1%和94.4%.伴肝内播散或脉管癌栓HCC患者的血浆DNA浓度(261 ng/mL)明显高于不伴肝内播散灶或脉管痛栓患者(142 ng/mL,P=0.035).结论:定量PCR技术可精确定量血浆循环DNA浓度;血浆DNA分析对于HCC诊断,预测肿瘤转移潜能具有重要价值.     

慢性乙型肝炎病毒感染与p16INK4A基因启动子甲基化关系的研究

目的探讨慢性乙型肝炎病毒(HBV)感染对p16INK4A基因启动子甲基化的影响及其在HBV相关肝细胞癌(HCC)形成中的作用。方法选取23例HBV相关HCC癌及癌旁组织、25例慢性乙型肝炎肝穿刺组织,采用甲基化特异性PCR(MSP)检测p16INK4A基因启动子的甲基化状态;免疫组化EnVision二步法测定肝组织内HBsAg、HBcAg、HBeAg和HBx蛋白的表达;荧光实时定量PCR检测肝组织HBV DNA含量;PCR扩增和直接测序检测HBV x基因变异。结果癌组织p16INK4A基因启动子甲基化阳性率(47.83%)明显高于癌旁组织(17.39%),慢性乙型肝炎患者p16INK4A基因启动子甲基化阳性率(36.00%)与癌组织、癌旁组织差异无统计学意义。在癌旁组织和慢性乙型肝炎, p16INK4A基因启动子甲基化者HBx蛋白表达中位数分别为3.000和0.250,明显高于非甲基化者(0.500和0.000),但在癌组织中,HBx蛋白的表达与p16INK4A基因启动子甲基化状态无关。HBsAg、HBcAg、组织HBV DNA含量和x基因突变均与p16INK4A基因启动子甲基化状态无关。结论在癌前病变中,p16INK4A基因启动子甲基化与HBx蛋白高表达有关,HBx蛋白可能通过诱导p16INK4A基因启动子甲基化而使该抑癌基因失活,在HBV相关HCC形成中起重要作用。     

原发性肝癌(HCC)TACE治疗前后p16、AFP、sVEGF的动态变化

目的:探讨HCC患者经肝动脉灌注化疗栓塞术(TACE)治疗前、后p16基因甲基化、AFP和sVEGF变化的临床意义。方法:采用甲基化特异性PCR(MSP)法检测56例HCC患者血清p16基因甲基化率,ELISA法检测AFP和sVEGF。结果:HCC患者血清p16基因甲基化检出率为69.6%(39/56),AFP阳性检出率为80.4%(45/56),sVEGF阳性检出率为73.2%(41/56)。结论:血清DNA中p16基因甲基化检测可作为HCC分子诊断标志,三者联合检测对肝癌诊断有实用价值。     

肝细胞癌血浆LINE-1启动子区甲基化水平的研究

目的 探讨肝细胞癌（HCC）患者血浆中长散布核元件-1（LINE-1）基因启动子区CpG位点甲基化水平变化的临床意义。方法 应用亚硫酸氢盐测序 PCR（BSP）检测33例HCC患者（HCC组）、18例肝硬化患者（肝硬化组）及16例健康志愿者（正常组）的外周血血浆中LINE 1启动子区的CpG位点甲基化程度。结果 HCC组的LINE-1启动子区多个CpG位点甲基化水平及整体甲基化水平下降;进一步通过ROC曲线分析发现CpG位点1、位点7、位点8的甲基化水平对诊断HCC的能力较优,其敏感度和特异度分别为80.0％和78.8％、81.8％和71.4％、82.9％和75.8％。结论 LINE-1基因启动子区甲基化水平在HCC中明显下降,其中CpG位点1、位点7、位点8的甲基化水平可能对HCC诊断有意义。     

血浆GSTP1和SFRP1基因甲基化分析在肝细胞癌早期诊断中的价值

背景与目的:DNA甲基化是一种新的肿瘤诊断及预后判断的标志物.本研究运用自行建立的甲基化敏感性限制性内切酶一定量PCR(methylation-sensitive restriction enzymes-based quantitative PCR,MSRE-qPCR)方法检测血浆GSTP1和SFRP1基因的DNA甲基...     

宫颈癌APC基因启动子甲基化与其临床病理特征的关系

背景与目的：腺瘤性结肠息肉病（adenomatous polyposis coli,APC@基因是Wnt信号传导通路中重要的抑癌基因,其启动子区CpG岛高甲基化引起的基因失活可导致Wnt信号传导通路异常,与多种肿瘤有关。APC基因启动子甲基化与宫颈癌的关系研究较少。本研究检测宫颈癌标本中APC基因启动子甲基化状态,并分析其与临床病理特征及高危型人乳头瘤病毒（HPV@感染的关系,探讨APC基因启动子甲基化与宫颈癌的关系。方法：运用甲基化特异性PCR（MSP@检测95例宫颈癌标本及对照组20例正常宫颈组织的APC基因启动子甲基化状态,并分析APC基因甲基化状态与不同临床病理特征及高危型HPV感染之间的联系。结果：宫颈癌组APC基因甲基化率明显高于正常对照组（分别为56.8%,10%,P〈0.01@。宫颈癌APC基因甲基化检测和高危型HPV DNA检测结果有较好的一致性（Kappa=0.348,P〈0.001@。腺癌组甲基化率较鳞癌组高（分别为74.1%,50.0%,P〈0.05@。APC甲基化阳性率随肿块增大及淋巴结转移而增高（P〈0.05@。不同年龄、FIGO临床分期、WHO病理分级、肿瘤侵犯深度和APC基因甲基化未见明显关系（P〉0.05@。结论：宫颈癌APC基因启动子甲基化与高危型HPV具有良好的一致性。APC基因启动子高甲基化为频发事件,并与不同宫颈癌病理类型相关,可作为宫颈癌诊断及分型的生物学标志物。     

APC promoter hypermethylation contributes to the loss of APC expression in colorectal cancers with allelic loss on 5q

INTRODUCTION: Germ-line mutations of the APC gene are associated with familial adenomatous polyposis, and somatic mutations occur frequently in sporadic colorectal cancer. However, to abrogate APC function, both alleles must be inactivated. Recently, it has been demonstrated that epigenetic modification of the APC promoter influences APC silencing. Here we examined the influence of APC methylation on APC expression in tumors with and without LOH at the APC locus. MATERIAL AND METHODS: 137 sporadic colorectal cancer specimens were investigated for LOH at the 5q locus. The methylation status of the APC promoter was determined by methylation-specific PCR. APC expression was performed by immunohistochemistry. RESULTS: Expression was reduced or lost in 110 of 137 (80%) tumors and LOH at 5q was found in 13 of 132 (10%) tumors. There was no difference in 5q LOH between tumors with or without intact APC expression. Vice versa, there was no difference in the APC expression in tumors with 5q LOH. Aberrant APC promoter methylation was detected in 33 of 118 (28%) tumors investigated. Of the tumors with 5q LOH for which methylation data were available, 4 of 11 (36%) were methylated versus 28 of 105 (27%) of those without LOH. No difference in methylation was observed in tumors without 5q LOH and normal APC expression and those without 5q LOH and reduced or missing APC expression. Importantly, none of the tumors with 5q LOH and normal APC staining were aberrantly methylated, whereas 50% of the cancers with LOH at 5q and reduced or absent staining were hypermethylated. CONCLUSIONS: This report suggests that tumors with 5q LOH and reduced APC expression are more frequently hypermethylated at the APC promoter compared to those tumors with 5q LOH and normal APC expression. The association among APC promoter methylation status, 5q LOH, and reduced or lost APC expression suggests that de novo methylation plays an important role as a "second hit" in silencing APC expression in colorectal neoplasia.     

Aberrant methylation of the Adenomatous Polyposis Coli (APC) gene promoter is associated with the inflammatory breast cancer phenotype

Aberrant methylation of the adenomatous polyposis coli (APC) gene promoter occurs in about 40% of breast tumours and has been correlated with reduced APC protein levels. To what extent epigenetic alterations of the APC gene may differ according to specific breast cancer phenotypes, remains to be elucidated. Our aim was to explore the role of APC methylation in the inflammatory breast cancer (IBC) phenotype. The status of APC gene promoter hypermethylation was investigated in DNA from normal breast tissues, IBC and non-IBC by both conventional and real-time quantitative methylation-specific PCR (MSP). APC methylation levels were compared with APC mRNA and protein levels. Hypermethylation of the APC gene promoter was present in 71% of IBC samples (n=21) and 43% of non-IBC samples (n=30) by conventional MSP (P=0.047). The APC gene also showed an increased frequency of high methylation levels in IBC (in 74% of cases, n=19) vs non-IBC (in 46% of cases, n=35) using a qMSP assay (P=0.048). We observed no significant association between APC methylation levels by qMSP and APC mRNA or protein expression levels. In conclusion, for the first time, we report the association of aberrant methylation of the APC gene promoter with the IBC phenotype, which might be of biological and clinical importance.     

乳腺癌APC基因甲基化状况及其蛋白表达

背景与目的:结肠腺瘤性息肉病基因(adenomatous polyposis coli,APC)蛋白表达产物是Wnt信号转导途径重要组成部分,该基因失活使β-catenin蛋白降解障碍,从而使Tcf/Lef激活,引起基因异常转录,最终产生癌变.启动子区甲基化是导致抑癌基因转录失活的重要机制.本研究探讨乳腺癌APC基因启动子1A区甲基化状态与其蛋白表达的关系,并分析APC基因异常甲基化与乳腺癌临床病理特征的关系.方法:应用甲基化特异性PCR和免疫组织化学方法检测76例乳腺癌及相应癌旁乳腺组织中APC基因启动子1A区甲基化状态以及蛋白表达.结果:癌旁乳腺组织中均未发现APC基因启动子1A区甲基化,乳腺癌组织中APC基因启动子1A区甲基化率为36.8%.癌旁乳腺组织中APC蛋白阳性率为100%,乳腺癌中APC蛋白阳性率为52.4%.乳腺癌组织中APG基因甲基化与APC蛋白表达呈负相关(r=-0.351,P＜0.05),与TNM分期呈正相关(r=0.335,P＜0.05).结论:乳腺癌发展过程中APC基因启动子1A区出现异常甲基化,是导致该蛋白表达缺失的主要原因,是导致该基因失活的重要机制之一.     

子宫内膜样腺癌APC基因表达和甲基化的初步研究

背景与目的:子宫内膜癌是女性生殖系统常见的恶性肿瘤之一。在我国,近年来其发病率亦显著上升。APC基因是一种抑癌基因,在很多组织中都有表达,其表达与卵巢癌有一定的关系。本实验探讨APC基因的甲基化和表达与子宫内膜样腺癌的发生发展的关系。方法:运用甲基化特异性PCR、RT-PCR、免疫组化方法检测了30例正常的增生期子宫内膜组织、30例不典型增生的子宫内膜组织、60例子宫内膜样腺癌组织中APC基因的甲基化情况以及mRNA和蛋白的表达情况。结果:子宫内膜样腺癌组织中APC基因的甲基化率(65.0%)、mRNA表达率(33.3%)及APC蛋白表达率(30.0%)与不典型增生子宫内膜组织(33.3%、63.3%、50.0%)及正常子宫内膜(23.3%、73.3%、66.7%)相比,差异均具有显著性(P<0.05);而不典型增生子宫内膜组织和正常子宫内膜组织中,APC的甲基化率、mRNA表达率及APC蛋白表达率差异无统计学意义(P>0.05)。在这3种组织中,APC基因的甲基化和mRNA的失表达呈正相关关系。结论:APC的甲基化和表达与子宫内膜样腺癌的发生发展相关。     

Monoallelic methylation of the APC promoter is altered in normal gastric mucosa associated with neoplastic lesions

Adenomatous polyposis coli (APC) promoter hypermethylation has been reported frequently in normal gastric mucosa, but it remained to be clarified whether this occurs in every individual. In this study, methylation of the APC promoter was analyzed in histologically normal-appearing gastric mucosa samples by methylation-sensitive single-strand conformation analysis and by a methylation-sensitive dot blot assay. Epithelial cell samples were collected by microdissection from tissue sections. Equal amounts of methylated and unmethylated APC alleles were found in all gastric mucosa samples from patients without any gastric lesions (20 samples). Allele-specific methylation analysis showed that the methylation of the APC promoter was monoallelic; however, which allele was methylated depended on the cell type. Increased or decreased methylation was found in 10 of 36 (28%) normal gastric mucosa samples adjacent to a gastric or esophageal adenocarcinoma. No allelic loss was found at the APC locus. Modification of the methylation status was also found in 3 of 21 (14%) normal-appearing gastric mucosa samples adjacent to intestinal metaplasia. In contrast, all normal mucosa samples in cases with chronic gastritis but without metaplasia or dysplasia showed a monoallelic methylation pattern. Our results indicate the following: (a) In normal gastric mucosa, the APC promoter shows monoallelic methylation, which is not due to imprinting but most likely due to allelic exclusion; (b) the excluded allele differs between foveolar and glandular epithelial cells; (c) the APC methylation pattern is frequently altered in normal-appearing gastric mucosa of gastric or esophageal adenocarcinoma patients; and (d) such alterations also occur in normal gastric mucosa adjacent to intestinal metaplasia.     

Molecular analysis of APC promoter methylation and protein expression in colorectal cancer metastasis

APC (adenomatous polyposis coli) promoter methylation has been linked to the early development of colorectal cancers. However, the role of APC methylation and its effect on protein expression in colon cancer metastasis is largely unknown. In this study, we investigated APC promoter methylation by Methylight analysis and analysed the APC protein levels by immunohistochemistry and western blot analysis in 24 liver metastasis and 39 primary colorectal cancers. Promoter methylation of the APC gene was found to be a frequent event in liver metastasis (10/24) and significantly more frequent compared with primary colorectal cancer (7/39, P = 0.047). APC methylation was not found in 14 matched normal colon tissues. APC protein was detected in the cytoplasm of primary and metastatic cancer cells and non-tumorous colon epithelium. By western blot analysis, APC protein levels were found to be decreased in primary tumour tissues compared with the normal colon mucosa. In contrast, APC protein levels were not decreased in the cancer cells that had metastasized to the liver. APC protein levels were independent of the presence of APC promoter methylation or gene mutations. In summary, APC promoter methylation is a frequent epigenetic alteration in colorectal cancer metastasis. However, we observed no significant association between APC promoter methylation or gene mutation and APC protein expression in colorectal metastasis. Therefore, metastatic cancer cells seem to harbour a heterogenous genetic and epigenetic background, in which cancer cells may exhibit APC promoter methylation that is independent of APC expression.