肺癌患者RASSF1A启动子甲基化状态及其基因表达水平

目的探讨肺癌RAS相关区域家族1A(RASSF1A)启动子CpG岛甲基化状态和基因表达水平与肺癌发生的关系。方法用甲基化特异性PCR检测RASSF1A启动子CpG岛甲基化状态,实时定量PCR检测RASSF1A mRNA的表达水平。结果在肺癌组织中RASSF1A启动子甲基化频率为53.33%(24/45),在正常肺组织中发生甲基化的频率为13.04%(3/23)(P<0.05)。正常肺组织中RASSF1A mRNA全部表达,肺癌组织中表达缺失率为28.89%(13/45),且表达量低于正常肺组织(P<0.05);不同年龄、性别、肿瘤大小、恶性程度、肿瘤分类中其表达量无显著差异;RASSF1A甲基化与其mRNA表达水平下降密切相关。结论肺癌中RASSF1A基因启动子甲基化频率明显升高,其表达普遍下调或缺失,提示RASSF1A启动子甲基化在肺癌发生、发展中起一定作用。     

宫颈癌中RASSF1A基因微卫星的不稳定性

Ras相关区域家族1A(ras association domain family 1A,RASSF1A)基因是新近克隆出来的一种肿瘤抑制基因[1],其在调节细胞周期和细胞凋亡方面具有重要作用。RASSF1A基因失表达见于多种人类肿瘤中。本研究选择RASSF1A基因内的两个微卫星多态标记,对110例宫颈组织进行微卫星不稳定     

肺及胃肠道神经内分泌肿瘤相关基因研究进展

目前对肺及胃肠道神经内分泌肿瘤（NETs）相关基因研究较多,LOH、比较基因组杂交(CGH)、微卫星不稳定(MSI)、甲基化特异性PCR(MSP)等方法都是目前研究肿瘤遗传学采用的热门技术,这些方法已证实了MEN1基因的杂合性缺失（LOH）、DNA拷贝数丢失、启动子甲基化。消化道NETs 11q、18q的DNA拷贝数的丢失;小细胞肺癌（SCLC）的微卫星不稳定（MSI）和SCLC在RARβ, CDH1, RASSF1A基因启动子区的高频甲基化也已存在较肯定的实验结果。本文对肺及胃肠道神经内分泌肿瘤相关基因的研究作一概述,包括研究方法、基因改变的分子机制和基因组不稳定性。     

p16、RASSF1A在肝细胞性肝癌患者肿瘤组织及血浆中异常甲基化的检测及临床意义

目的探讨原发性肝细胞性肝癌(HCC)患者肿瘤组织及血浆中p16及RASSF1A启动子区的甲基化状态及其在HCC早期无创诊断中的意义。方法采用甲基化特异性PCR技术检测60例HCC患者肿瘤组织、癌旁组织、血浆及60例正常肝脏患者肝组织及血浆中p16、RASSF1A基因启动子区域的甲基化状态,分析其与肝癌患者临床病理参数之间的关系。结果 60例HCC患者血浆、肿瘤组织及癌旁中p16基因甲基化率分别为68.3%(41/60)、63.3%(38/60)和41.7%(25/60);RASSF1A基因异常甲基化检出率分别为73.3%(44/60)、70.0%(42/60)和36.7%(22/60);60例正常肝脏患者肝组织及血浆p16、RASSF1A未检测到基因启动子区域的甲基化,差异有统计学意义(P=0.000)。HCC患者外周血浆和癌组织中p16、RASSF1A基因的甲基化率与患者年龄、性别、AFP、有无肝炎病毒感染(HBV)、有无肝硬化、Child分级、肿瘤个数、包膜完整与否、有无癌栓、是否复发、病理分级、肿瘤分期无统计学相关性。结论 HCC患者肿瘤组织及血浆DNA中可检测到RASSF1A基因和p16基因的甲基化,外周血p16基因和RASSF1A基因的甲基化检测对肝癌筛查有重要意义,可能成为HCC新的肿瘤分子标记物。     

人RASSF1A基因的真核表达载体的构建及其表达

目的 通过构建人RASSF1A基因的真核表达载体,为研究其在肿瘤细胞凋亡、细胞周期阻滞、细胞增殖抑制等方面奠定基础.方法 采用RT-PCR从人外周血的单个核细胞RNA中扩增出人RASSF1A基因的全长cDNA,利用DNA重组技术将其插入到真核表达栽体pcDNA3.1(+)中获得重组质粒.利用脂质体将重组质粒转染入人鼻咽癌细胞株CNE-2中,采用RT-PCR.westem-blot检测RASSF1A的瞬时表达.结果 酶切图谱分析及基因测序证明人RASSF1A基因已被完整、正确地插入到pcDNA3.1(+)质粒载体中,RT-PCR及Western-blot结果显示转染细胞的RASSF1A表达水平上调.结论 成功构建了RASSF1A基因的真核表达栽体pcDNA3.1(+)/RASSF1A.     

卵巢癌患者血液中RASSF1A基因甲基化的检测及其意义

目的 探讨循环肿瘤DNA中RASSF1A基因的甲基化及其与卵巢癌的关系。方法 应用甲基化特异性PCR（MSP）方法,对51名正常健康人、51例卵巢癌患者和51例卵巢良性肿瘤患者循环DNARASSF1A基因的甲基化进行了检测。结果 51名正常健康人和51例卵巢良性肿瘤患者循环DNARASSF1A基因甲基化的发生率均为0;51例卵巢癌患者循环肿瘤DNARASSF1A甲基化的发生率为43．1％（22／51,P〈0．05）。RASSF1A甲基化与卵巢癌组织学类型无明显相关性（P〉0.05）。临床Ⅰ期和Ⅱ期循环肿瘤DNARASSF1A基因甲基化的发生率明显低于临床Ⅲ期和Ⅳ期（P〈0．05）;高和中分化组RASSF1A基因甲基化的发生率低于低分化组（P〈0．05）。结论 RASSF1A基因甲基化在卵巢癌的发生和发展过程中起重要作用。卵巢癌患者的循环肿瘤DNA中可以检测到RASSF1A基因的甲基化,与卵巢癌的临床分期和组织学分级有关。循环肿瘤DNA中RASSF1A甲基化的检测有助于卵巢癌的诊断和预后判定。     

RASSF1A及其在肿瘤发生中的作用

RASSF1A（Ras-assotiation domain family 1 A）基因是一个新型抑癌基因。目前的研究已经在许多肿瘤中发现了这个基因的失活。虽然这个基因的失活可因基因缺失或突变引起，但最常见的原因还是该基因的启动子区甲基化紊乱。这种表形遗传学改变被证实是肿瘤形成的一个早期事件，是肿瘤形成的主要因素之一。RASSF1A有多个结构域，被认为是一种潜在的Ras癌蛋白效应分子，能与活化的Ras结合，调节凋亡及细胞周期信号通路，在细胞的凋亡、增殖、分化及维持细胞的稳定中发挥多种生物学效应，与肿瘤的发生发展密切相关。     

Ras相关结构域家族蛋白1A基因与肿瘤

Ras作为一个在人类肿瘤研究中重要的癌基因,已成为目前研究得最为深入的基因之一。研究发现,Ras基因除了具有促进细胞牛长、增殖的作用外还具有一个非常重要的功能——抑制细胞生长、促进细胞凋亡和衰老。2000年,一个与鼠Ras效应蛋白Nore1和Maxp 1高度同源的蛋白编码基因——Ras相关结构域家族蛋白1A(RAS associalion domain family proteinlA,RASSF1A)基因的发现引起了研究者们的广泛关注,对其结卡勾、功能、分布和表达情况的研究发现,     

孕妇外周血中RASSF1A基因甲基化状态研究

目的探讨RASSF1A基因作为孕妇外周血中胎儿特异性标记物的可行性。方法随机选取43例的外周血样本,其中妊娠晚期孕妇标本40例、健康未妊娠女性标本3例,以及早期妊娠行流产的绒毛组织样本5例。利用甲基化敏感限制性内切酶PCR(MSRE-PCR)及荧光定量PCR的方法,检测血细胞、绒毛和血浆DNA的RASSF1A基因甲基化状态。结果健康未妊娠妇女血浆及孕妇血细胞中均未检出甲基化RASSF1A基因;绒毛组织中甲基化RASSF1A基因检出率为100%;孕妇血浆中甲基化RASSF1A基因检出率为92.5%。妊娠晚期孕妇血浆RASSF1A基因含量Ct平均值为30.43±2.37。结论在母体和胎儿DNA中,RASSF1A基因甲基化状态有明显差异,用实时荧光定量PCR可对孕妇血浆RASSF1A基因进行定量,提示了RASSF1A基因是无创产前诊断的一个潜在标记物。     

髓母细胞瘤与幕上原始神经外胚叶肿瘤中RASSF1A基因的甲基化差异

目的通过研究髓母细胞瘤与幕上原始神经外胚叶肿瘤（SPNET）中RASSF1A基因的甲基化改变,探讨颅内原始神经外胚叶肿瘤（PNET）的不同亚型中该基因的表遗传学差异及其意义。方法收集25例原发髓母细胞瘤,9例原发SPNET,3株髓母细胞瘤细胞系和2株SPNET细胞系。采用甲基化特异性聚合酶链反应（MSP）检测RASSF1A基因启动子区的甲基化状态。应用去甲基化试剂5-aza-2’deoxycytidine处理存在基因表达缺失的细胞系,探讨基因表达与甲基化之间的关系。结果100％（25／25）的原发髓母细胞瘤、6／9的原发SPNET及全部PNET细胞系中均检测到RASSFIA基因的甲基化。相反,该基因甲基化在全部正常组织（包括2例小脑,5例大脑）中均未检测到。并且,RASSF1A在SPNET中的甲基化率明显低于髓母细胞瘤（Fisher精确检验,P=0．014）。在经去甲基化试剂处理的PNET细胞中,该基因表达得以恢复,证明甲基化与该基因沉默相关。结论RASSF1A甲基化是肿瘤特异性的,RASSF1A甲基化与PNET的发生有一定关联,不同亚型的PNET之间RASSF1A基因的不同甲基化状态提示髓母细胞瘤和SPNET是表遗传学上存在差异的两类肿瘤。     

Impact of RASSF1A gene methylation on clinico-pathological features of tumor and non-tumor tissue of breast cancer

BackgroundBreast cancer is the most common malignancy in women caused by genetic and epigenetic changes. Promoter DNA methylation in tumor suppressor gene plays a major role in breast cancer. The study determined the association of promoter DNA methylation of RASSF1A gene with clinicopathological features in tumor and non-tumor tissue.Materials and methodsA cross sectional study was conducted in the Department of Pathology, Government Institute of Medical Sciences, Greater Noida and Molecular Pathology Laboratory, Department of Pathology, Jawaharlal Nehru Medical College, Datta Meghe Institute of Medical Sciences. Two sections, one from tumor and the other from non-tumor tissue, were obtained and processed for DNA extraction and bisulphite conversion. Methylation specific PCR was done and results of RASSF1A promoter methylation were statistically correlated with clinicopathological features.ResultsOf the 27 breast cancer tissue, 22 showed invasive ductal carcinoma, one showed invasive lobular carcinoma, another showed ductal carcinoma in situ and three cases showed malignant phyllodes tumor of breast. DNA promoter methylation was found in all the cases. 93% of tumor tissue samples and 67% of the non-tumor tissue samples were found to be aberrantly methylated. Tumor size and histological grade were found to be significantly (p-val <0.05) associated with the RASSF1A gene promoter methylation.ConclusionA significant association of higher tumor size and tumor histological grade with promoter methylation of RASSF1A gene exists suggestive of its being an important determinant of prognostic staging. This critical event in tumorigenesis may be of clinical utility in assessing breast cancer progression.Micro abstractThe study focuses on the RASSF1A gene promoter methylation and its impact on the clinicopathological features in Indian breast cancer patients highlighting the differences from other genetically different population. We found that RASFF1A gene methylation has significant impact on tumor size and tumor grade. The work carries high significance because it addresses the DNA methylation of tumor suppressor gene in relevance of breast cancer. It may also be the first such report on Indian patients with breast cancer.     

血浆中多基因联合甲基化检测在肺癌诊断中的应用

 目的:探究血浆中CDH13、RASSF13A、DLEC13、SEPT13、RUNX13等抑癌基因启动子甲基化及其联合检测在肺癌诊断中的价值。从中选出诊断效能高的组合。方法: 采用巢式甲基化特异性PCR（nest methylation specific PCR,nMSP）法,检测106例健康人血浆样本、106例肺癌组织和癌旁组织以及其对应的106例术前血浆样本、中基因启动子区的甲基化状态。对血浆基因组DNA修饰后进行多重置换扩增 （multiple displacement amplification, MDA）,以解决血浆DNA模板不足的问题。结果: 肺癌组织样本中的CDH13、RASSF13A、DLEC13、SEPT13、RUNX13基因启动子甲基化率分别为51.9%、44.3%、54.7%、36.8% 、24.5%。对应的血浆样本中的CDH13、RASSF1A、DLEC1、SEPT9、RUNX3基因启动子甲基化率分别为46.2%、41.5%、50.9%、31.1%、19.8%。Kappa一致性检验结果表明肺癌组织与血浆的甲基化检出率一致。CDH13、DLEC1、RASSF1A、SEPT9组合对肺癌的诊断效能明显高于其它组,准确度ACC为82.08%,Youden指数为0.6415（灵敏度为79.49%,特异度为81.13%）。 结论: 血浆多基因联合甲基化检测有望应用于肺癌早期诊断。     

RASSF1基因转录本A和C的表达与卵巢癌关系的研究

目的探讨RASSF1基因转录本A和C在多个卵巢癌细胞系和卵巢癌组织中所起的作用。方法应用逆转录聚合酶链反应(RT PCR)和激光捕获显微切割技术检测3个卵巢癌细胞系和80例人原发卵巢上皮性恶性肿瘤组织中RASSF1A和RASSF1CmRNA的表达。结果RASSF1A mRNA在卵巢癌SK OV3细胞中表达缺失;RASSF1A和RASSF1C在人卵巢癌组织中的表达率分别为40.0%(32/80)和91.3%(73/80)。RASSF1AmRNA的表达在浆液性癌、黏液性癌和内膜样癌组织中分别是41.2%(20/48),38.1%(8/21)和36.4%(4/11),P>0.05;临床Ⅰ期和Ⅱ期分别为71.4%和75.0%(10/14,9/12),明显高于临床Ⅲ期和Ⅳ期(26.7%、12/45和14.1%,1/9),P<0.05;高和中分化组分别为58.6%和50.0%(17/29,10/20),明显高于低分化组(16.1%,5/31),P<0.05。结论卵巢癌细胞和人原发卵巢癌组织中存在RASSF1AmRNA表达的缺失,RASSF1A的表达与卵巢癌的临床分期和组织学分级有关,可能作为一种新的抑癌基因在卵巢癌的发生和发展过程中起重要作用,RASSF1AmRNA的缺失提示预后不良。     

The role of RASSF1A methylation in cancer

Tumour suppressor gene inactivation is critical to the pathogenesis of cancers; such loss of function may be mediated by irreversible processes such as gene deletion or mutation. Alternatively tumour suppressor genes may be inactivated via epigenetic processes a reversible mechanism that promises to be more amenable to treatment by therapeutic agents. The CpG dinucleotide is under-represented in the genome, but it is found in clusters within the promoters of some genes, and methylation of these CpG islands play a critical role in the control of gene expression. Inhibitors of the DNA methyltransferases DNMT1 and DNMT3b have been used in a clinical setting, these nucleotide analogues lack specificity but the side effects of low dose treatments were minimal and in 2004 Vidaza (5-azacitidine) was licensed for use in myelodysplastic syndrome. Methylation inhibitors are also entering trials in conjunction with another class of epigenetic modifiers, the histone deacetylase inhibitors and this epigenetic double bullet offers hope of improved treatment regimes. Recently there has been a plethora of reports demonstrating epigenetic inactivation of genes that play important roles in development of cancer, including Ras-association domain family of genes. Epigenetic inactivation of RASSF1A (Ras-association domain family 1, isoform A) is one of the most common molecular changes in cancer. Hypermethylation of the RASSF1A promoter CpG island silences expression of the gene in many cancers including lung, breast, prostate, glioma, neuroblastoma and kidney cancer. Several recent studies have illustrated the diagnostic and prognostic potential of RASSF1A methylation. This presents RASSF1A methylation as an attractive biomarker for early cancer detection which, for most cancers, results in improved clinical outcome. DNA methylation analysis is applicable to a range of body fluids including serum, urine, bronchioalveolar lavage and sputum. The ease with which these body fluids can be acquired negates the need for invasive procedures to obtain biopsy material. This review will discuss the feasibility of using RASSF1A methylation as a diagnostic and prognostic marker in cancer management.     

Quantitative assessment of promoter hypermethylation during breast cancer development

The aberrant methylation of cytosine residues in the promoter region of growth regulatory genes is now widely recognized as an additional mechanism for gene inactivation in cancer cells. In this study we analyzed the methylation status of four growth regulatory genes (p16, RASSF1A, cyclinD2, 14-3-3zeta) during breast cancer progression. For this purpose invasive and noninvasive tumor cell populations as well as hyperplastic cell proliferations were isolated from a series of archival breast tissue specimens (n = 57) using laser-assisted microdissection. A new real-time polymerase chain reaction-based assay was used for the sensitive and quantitative determination of the cell-specific methylation status. We found that aberrant promoter methylation was already prevalent in pure intraductal carcinoma with different frequencies and different methylation levels for the four genes analyzed. For RASSF1A and 14-3-3zeta promoter methylation was also demonstrated in epithelial hyperplasia and intraductal papillomas. By contrast, aberrant methylation of cyclinD2 and p16 was restricted to cancerous epithelium. Increased methylation of the cyclinD2 gene was significantly associated with a higher van Nuys grade. Furthermore, when intraductal and invasive tumor cells were compared, significant quantitative changes in the methylation level were detected primarily within the cyclinD2 gene. These results demonstrate that promoter methylation is an early and frequent event in breast cancer development, but displays great quantitative and gene-specific differences, and changes in a gene-specific manner during tumor progression.     

Hypermethylation of RASSF1A in human and rhesus placentas

The pseudomalignant nature of the placenta prompted us to search for tumor suppressor gene hypermethylation, a phenomenon widely reported in cancer, in the human placenta. Nine tumor suppressor genes were studied. Hypermethylation of the Ras association domain family 1 A (RASSF1A) gene was found in human placentas from all three trimesters of pregnancy but was absent in other fetal tissues. Hypermethylation of Rassf1 was similarly observed in placentas from the rhesus monkey but not the mouse. An inverse relationship between RASSF1A promoter methylation and gene expression was demonstrated by bisulfite sequencing of microdissected placental cells and immunohistochemical staining of placental tissue sections using an anti-RASSF1A antibody. Treatment of choriocarcinoma cell lines, JAR and JEG3, by 5-aza-2'-deoxycytidine and trichostatin A led to reduction in RASSF1A methylation but increased expression. These observations extend the analogy between the primate placenta and malignant tumors to the epigenetic level.