散发性大肠癌CpG岛甲基子表型的基因表达

目的探讨CpG岛甲基子表型阳性的散发性大肠癌的基因表达特征。方法对71例散发性大肠癌采用甲基化特异性PCR法行p14ARF、人类mut-s同系物1(hMLH1)、p16INK4a、6-氧-甲基鸟嘌呤-DNA甲基转移酶(MGMT)和肿瘤甲基化位点1(MINT1)共5个基因启动子甲基化的检测,确定CpG岛甲基子表型;进行K-ras、APC、P53、Bax和转化生长因子βⅡ受体(TGFβRⅡ)共5个基因的免疫组化检测。分析散发性大肠癌CpG岛甲基子表型和基因表达之间的关系。结果71例散发性大肠癌中,CpG岛甲基子表型阳性率为21．1％(15／71);K-ras、APC、P53、Bax和TGFβRⅡ蛋白阳性表达率分别为43．7％(31／71)、42．3％(30／71)、47．9％(34／71)、71.4％(50／70)和59．2％(42／71)。CpG岛甲基子表型和APC、P53、Bax、TGFβRⅡ蛋白表达均无显著相关性,但与K-ras蛋白表达显著相关,CpG岛甲基子表型阳性者K-ras蛋白阳性表达率显著高于CpG岛甲基子表型阴性者(66.7％比37．5％, P=0．043)。结论CpG岛甲基子表型阳性的散发性大肠癌中K-ras蛋白呈高表达,提示多基因同时甲基化与K-ras蛋白的激活表达密切相关,两者的关系表明表遗传学机制可以间接引起遗传学改变。     

结直肠腺癌凝血栓蛋白1基因甲基化异常

探讨凝血栓蛋白（THBS）1基因表达，启动子胞嘧啶磷酸鸟嘌呤岛（CpG岛）甲基化与结直肠腺癌及其临床与病理特片的关联，并分析THBS1基因甲基化与其蛋白表达的相关性。     

不同阶段结直肠肿瘤分泌型卷曲相关蛋白基因甲基化及表达

目的探讨分泌型卷曲相关蛋白（sFRP）家族基因启动子CpG岛甲基化在结直肠肿瘤发生、发展和诊断中的作用。方法分别用甲基特异性PCR和逆转录PCR检测72例结直肠腺癌、33例腺瘤、18个变性隐窝灶（ACF）中sFRP基因甲基化及mRNA表达。结果正常大肠黏膜不存在sFRP基因甲基化。sFRP1、2、4、5甲基化率在腺癌中分别为93。1％、83。3％、36．1％和52．8％；在腺瘤中分别为87．9％、81．8％、24．2％和57．6％；ACF中分别为94．4％、77．8％、27．8％和55．6％。腺癌、腺瘤和ACF间sFRP基因甲基化率差异无统计学意义（P〉0．05），肿瘤组织甲基化率均高于正常黏膜及癌旁正常组织（P〈0．05）。正常黏膜表达sFRPl～5基因mRNA。与正常黏膜相比，腺癌中sFRP1、2、4、5分别在90．3％、70．8％、26．4％和61．1％的样本中表达下调（P〈0．05）；腺瘤中sFRP1、2、5分别在75．8％、45。5％和39。4％的样本中表达下调（P〈0．01）。腺癌中sFRP2、4、5表达下调较腺瘤更常见（P〈0．05）。sFRP3基因启动子不含CpG岛，仅极少数肿瘤标本（7／105）表达下调。肿瘤组织中sFRP基因表达下调与基因启动子高甲基化相关（P〈0．05）。结论sFRP基因家族在ACF中已出现高频率甲基化，是结直肠肿瘤发生常见的早期事件，可能导致sFRP基因表达下调。sFRP1、2、5甲基化可能成为早期发现结直肠肿瘤的生物学标记。     

应用全基因组甲基化芯片筛选结直肠腺瘤基因启动子区甲基化标志物

目的应用全基因组甲基化芯片筛选结直肠腺瘤基因启动子区甲基化标志物。方法选择2013年1月至2014年12月在广西壮族自治区人民医院及广西医科大学第一附属医院住院的进展期结直肠腺瘤患者9例(腺瘤组),另选择经肠镜排除炎症性肠疾病、癌前病变及癌的患者20例作为对照组。腺瘤组于内镜下切除结直肠腺瘤标本(直径≥2 cm),对照组取结直肠黏膜标本(结肠黏膜13例,直肠黏膜7例)。提取组织DNA,应用全基因组甲基化芯片进行甲基化检测,通过生物信息学分析筛选差异甲基化基因启动子区甲基化标志物。结果以Δβ≥|0.4|,在基因启动子区共发现1 870个差异甲基化标志物,1 524个为高甲基化标志物,346个为低甲基化标志物,其中包含929个启动子差异甲基化基因。GO分析结果显示,启动子差异甲基化基因主要在化学性突触传递、神经系统发育、细胞外基质组织、细胞外基质结构成分、RNA聚合酶Ⅱ调控区序列特异性DNA结合、序列特异性DNA结合、质膜、蛋白质类细胞外基质、细胞膜等注释条目中出现富集。KEGG_Pathyway分析显示,启动子差异甲基化基因主要参与了神经活性的配体-受体相互作用、尼古...     

非小细胞肺癌11个肿瘤相关基因启动子CpG岛甲基化的研究

目的观测11个基因在非小细胞肺癌中的甲基化谱,探讨肺癌基因诊断的新途径.方法应用甲基化特异性的PCR（methyl-specific PCR,MSP）.方法检测甲基化率,回收产物测序.结果11个基因中有6个基因在癌组织中的甲基化率均＞40%并且高于癌旁组织,6个基因的甲基化率分别是APC 61%,RASSF1a 55%,p73 45%,p16INK4a 43%,CDH13 43%及RAR-β 41%,＞65岁组RASSF1a、p16INK4a和RAR-β甲基化率高于≤65岁组;吸烟史组APC、RASSF1a、p16INK4a和CDH13的甲基化率高于无吸烟史组;腺癌组APC、CDH13和RAR-β甲基化率高于鳞癌组,而鳞癌组p16INK4a甲基化率高于腺癌组;RASSF1a和p16INK4a甲基率随TNM 临床分期而逐渐增加.结论6个基因甲基化率的发生具有肿瘤特异性,并且相关基因的甲基化与NSCLC患者的病理生理密切相关,我们的研究为早期诊断NSCLC提供帮助.     

胰腺癌组织中p16基因启动子区5''CpG岛甲基化及其表达研究

在胰腺癌多步骤、多阶段发生过程中有多种肿瘤相关基因参与。p16基因是其中一个重要的抑癌基因，参与细胞周期的调控，控制细胞的生长和分化。大量研究表明，多种肿瘤细胞中p16基因通过突变、缺失、高甲基化等方式失活，造成p16蛋白功能丧失，从而导致肿瘤细胞恶性生长。免疫组化研究发现，胰腺癌组织中p16蛋白失表达的比例很高，而关于胰腺原发瘤中p16基因甲基化改变及其临床病理意义的研究报道少见，本研究旨在探讨胰腺癌组织中p16基因启动子区5’CpG岛甲基化情况及其与P16蛋白表达的关系和临床意义，研究其在胰腺癌发生中的可能机制。     

DNA甲基化及其在结直肠癌中的研究进展

结直肠癌是我国常见的恶性肿瘤,病死率高,近年发病率呈上升趋势。在肿瘤发生发展过程中,遗传物质的异常改变发挥了重要作用。但表观遗传学的出现,改变了人类对遗传信息的认知,表观遗传学逐渐成为肿瘤研究领域的热点。表观遗传学改变涉及染色质的任何组分,常见的表观遗传学调控包括DNA甲基化、组蛋白修饰、染色质重塑以及非编码RNA调控。DNA甲基化是结直肠癌中最常见的表观遗传学改变。与正常细胞相比,肿瘤细胞的DNA甲基化模式被严重破坏。     

EYA4基因在结直肠癌中的甲基化状态及其意义

背景：EYA4（eyes absent4）是一种非巯基蛋白酪氨酸磷酸酶,参与器官发育、细胞凋亡调控、先天性免疫、DNA损伤修复、血管生成等过程。目的：研究EYA4基因在结直肠癌中的甲基化状态及其意义。方法：选取2006年6月～2007年1月上海交通大学医学院附属仁济医院31例结直肠癌患者,采集其癌组织和相应癌旁非癌组织。以5-氮杂-2’-脱氧胞苷（5-Aza—dC）处理人结肠癌细胞株HT29、HCT116、SW480、SW1116。应用MSP技术检测结直肠癌组织、癌旁非癌组织、HT29、HCT116、SW480、SW1116细胞中EYA4基因启动子的甲基化状态。分析结直肠癌患者临床病理特征与EYA4基因甲基化关系。采用RT—PCR和蛋白质印迹法分别检测结肠癌细胞株中EYA4基因和蛋白的表达水平。结果：结直肠癌组织EYA4基因启动子甲基化率显著高于癌旁组织（77．4％对6．5％,P〈0．01）。EYA4基因和蛋白在启动子甲基化的HT29、HCT116、SW480细胞中不表达,在启动子非甲基化的SW1116细胞中显著表达。以5-Aza—dC处理后EYA4基因和蛋白在LIT29和SW480细胞中表达上调。结论：EYA4基因是结直肠癌发生的甲基化相关基因,有望成为结直肠癌的诊断标记物。     

生物钟基因hClock蛋白在结直肠肿瘤中的表达研究

目的探讨人类生物钟基因hClock蛋白在结直肠肿瘤中的表达及其意义。方法应用免疫组织化学法检测不同Dukes分期结直肠肿瘤及相应癌旁组织中hClock基因蛋白产物（Clock蛋白）的表达,并比较其差异。结果Clock蛋白红结直肠肿瘤中呈现中或强阳性表达,与肿瘤分化程度及Dukes分期无显著相关性（P〉0.05）。癌旁组织呈现弱阳性表达。结论Clock蛋白与结直肠肿瘤的发生有相关性,与侵袭、转移的关系有待更深入地研究。     

XAF1基因与p53基因在结直肠肿瘤中的表达研究

目的观察两个相邻位置的抑癌基因XIAP相关因子1(XAF1)与p53基因在结直肠肿瘤中的表达。方法选取结肠癌细胞株Colo205、Colo320、LoVo、SW1116为体外研究对象,另取41例结直肠癌、28例结直肠腺瘤/息肉以及31例正常人结肠黏膜为体内研究对象。应用逆转录聚合酶链反应检测XAF1mRNA与p53mRNA在体内外的表达,应用免疫组化法检测P53蛋白在结直肠组织中的表达。结果4种细胞株中XAF1表达均较为低下,p53mRNA表达均较高。结直肠肿瘤组织中的XAF1mRNA表达显著低于正常组织(P<0.01),结直肠癌中的表达又显著低于良性肿瘤(P<0.05)。结直肠肿瘤组织中的p53mRNA表达显著高于正常组织(P<0.01),但在良、恶性肿瘤中的表达差异无统计学意义(P<0.05)。P53蛋白在正常组织中未见表达,良、恶性肿瘤组的组织P53蛋白表达之间差异无统计学意义(P>0.05)。结论XAF1基因在结直肠肿瘤中表达降低,且结直肠癌XAF1mRNA表达显著低于良性肿瘤,可作为判断良、恶性肿瘤的鉴别指标。     

Hypermethylation of CpG island in O6-methylguanine-DNA methyltransferase gene was associated with K-ras G to A mutation in colorectal tumor

AIM:To investigate the functions of promoter hypermethylation of O6-methylguanine-DNA methyltransferase (MGMT) gene in colorectal tumorigenesis and progression. METHODS: The promoter hypermethylation of MGMT gene was detected in 27 sporadic colorectal adenomas, 62 sporadic colorectal carcinomas and 20 normal colorectal mucosa tissues by methylation-specific PCR. At the same time, the expression of MGMT protein was carried out in the same samples using immunohistochemistry. Mutant-allele-specific amplification was used to detect K-rasG to A point mutation in codon 12. RESULTS: None of the normal colorectal mucosa tissues showed methylated bands. Promoter hypermethylation was detected in 40.7% (11 of 27) of adenomas and 43.5% (27 of 62) of carcinomas. MGMT proteins were expressed in nucleus and cytoplasm of normal colorectal mucosa tissues. Loss of MGMT expression was found in 22.2% (6 of 27) of adenomas and 45.2% (28 of 62) of carcinomas. The difference between them was significant (P= 0.041). In the 6 adenomas and 28 carcinomas losing MGMT expression, 5 and 24 cases presented methylation, respectively (P = 0.027, P<0.001). Thirteen of the 19 colorectal tumors with K-rasG to A point mutation in codon 12 had methylated MGMT(P= 0.011). The frequencies of K-rasG to A point mutation were 35.3% (12 of 34) and 12.7% (7 of 55) in tumors losing MGMT expression and with normal expression, respectively. CONCLUSION: Promoter hypermethylation and loss of expression of MGMT gene were common events in colorectal tumorigenesis, and loss of expression of MGMT occurs more frequently in carcinomas than in adenomas in sporadic patients. Hypermethylation of the CpG island of MGMT gene was associated with loss of MGMT expression and K-rasG to A point mutation in colorectal tumor. The frequency of K-rasG to A point mutation was increased in tumors losing MGMT expression. It suggests that epigenetic inactivation of MGMT plays an important role in colorectal neoplasia.     

Frequent aberrant promoter hypermethylation of O6-methylguanine-DNA methyltransferase and death-associated protein kinase genes in immunodeficiency-related lymphomas

Aberrant promoter hypermethylation is a mechanism of tumour suppressor gene inactivation. We explored aberrant promoter hypermethylation of multiple genes in 88 human immunodeficiency virus (HIV)-non Hodgkin lymphomas (NHL), 25 post-transplant lymphoproliferative disorders (PTLD) and five common variable immunodeficiency (CVI)-related NHL. Twenty-six of 79 (32.9%) HIV-NHL, eight of 14 (57.1%) PTLD and two of five (40.0%) CVI-NHL showed aberrant hypermethylation of O6-methylguanine-DNA methyltransferase (MGMT). Aberrant hypermethylation of death-associated protein-kinase (DAP-K) occurred in 70 of 84 (83.3%) HIV-NHL, 19 of 25 (72.0%) PTLD and three of five (60.0%) CVI-NHL. These data implicate MGMT and DAP-K hypermethylation in lymphomagenesis of immunodeficient hosts. In particular, promoter hypermethylation of DAP-K represents the most frequent molecular alteration yet identified in immunodeficiency-related lymphomas.     

The hypermethylation and protein expression of p16 INK4A and DNA repair gene O6-methylguanine-DNA methyltransferase in various uterine cervical lesions

Purpose This study is aimed at investigating the significance of gene promoter methylation status and protein expression of p16 ^INK4A and O⁶-methylguanine-DNA methyltransferase (MGMT) in the various uterine cervical lesions.Materials and methods Methylation status by using methylation-specific polymerase chain reaction (MS-PCR) and protein expression by using immunohistochemistry for p16 ^INK4A and MGMT genes were performed in cervical squamous intraepithelial neoplasms (CIN), invasive squamous cell carcinomas (SCC), adenocarcinomas and non-neoplastic cervices.Results None of 20 non-neoplastic cervices showed p16 ^INK4A and MGMT gene hypermethylation, whereas at least one of these genes was hypermethylated with 50.0% (5/10) of CIN I, 65.0% (13/20) of CIN II–III, 70.2% (33/47) of SCC and 85.0% (17/20) of adenocarcinoma. p16 ^INK4A protein was totally negative in non-neoplastic cervices, but positive with 90.0% of CIN I, 100% of CIN II–III and adenocarcinoma, and 78.7% of SCC. MGMT protein was expressed in 10% of non-neoplastic cervices, but significantly increased in SCC (42.5%) and adenocarcinoma (70.0%). The protein expression of p16 ^INK4A and MGMT was not related to their gene promoter methylation status.Conclusions The hypermethylation of p16 ^INK4A and MGMT genes in the uterine cervix may indicate the presence of malignant cells, and p16 ^INK4A immunostaining is useful in grading CIN and diagnosing invasive SCC and adenocarcinoma.     

Decrease in cytosine methylation at CpG island shores and increase in DNA fragmentation during zebrafish aging

Age-related changes in DNA methylation have been demonstrated in mammals, but it remains unclear as to the generality of this phenomenon in vertebrates, which is a criterion for the fundamental cause of senescence. Here we showed that the zebrafish genome gradually and clearly lost methylcytosine in somatic cells, but not in male germ cells during aging, and that age-dependent hypomethylation preferentially occurred at a particular domain called the CpG island shore, which is associated with vertebrates’ genes and has been shown to be hypomethylated in humans with age. We also found that two CpG island shores hypomethylated in zebrafish oocytes were de novo methylated in fertilized eggs, which suggests that the zebrafish epigenome is reset upon fertilization, enabling new generations to restart with a heavily methylated genome. Furthermore, we observed an increase in cleavage of the zebrafish genome to an oligonucleosome length in somatic cells from the age of 12 months, which is suggestive of an elevated rate of apoptosis in the senescent stage.     

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。     

亚砷酸钠对HaCaT细胞MGMT基因启动子区甲基化CpG结合蛋白-2、DNA甲基转移酶1、组蛋白去乙酰化酶1结合的影响

目的 观察亚砷酸钠(NaAsO2)对人肤角质形成细胞株(HaCaT细胞)MGMT基因启动子区甲基化CpG结合蛋白-2(MeCP2)、DNA甲基转移酶1(DNMT1)及组蛋白去乙酰化酶1(HDAC1)结合情况的影响,为深化阐释砷毒作用机制提供依据.方法 分别以0.00(空白对照)、3.13、6.25、12.50、25.00 μmol/L NaAsO2重复间隔处理HaCaT细胞72 h(NaAsO2处理24h,隔天再次相同处理,重复3次),以人表皮鳞癌细胞株(A431)作为阳性对照,定量染色质免疫共沉淀技术(Q-ChIP)检测MGMT基因转录调控区ChIP1、ChIP2区域及MGMT基因编码区ChIP3区域MeCP2、DNMT1、HDAC1结合情况.结果 各组HaCaT细胞MGMT基因转录调控区ChIP1、ChIP2区域MeCP2、DNMT1、HDAC1蛋白结合水平比较,差异有统计学意义(F值分别为7.387、84.634、78.442和19.263、69.649、26.546,P均＜0.05);其中各NaAsO2处理组ChIP1、ChIP2区域MeCP2、DNMT1、HDAC1蛋白结合水平[3.13 μmol/L NaAsO2处理组:(136.00±16.97)％、(145.00±2.83)％、(88.50±19.09)％和(106.50±37.48)％、(112.34±8.73)％、(59.71±8.49)％;6.25 μmol/L NaAsO2处理组:(130.00±42.43)％、(154.50±4.95)％、(101.00±1.27)％和(88.50±3.54)％、(134.32±2.82)％、(102.75±19.91)％;12.50 μmol/LNaAsO2处理组:(141.50±23.33)％、(161.50±7.78)％、(125.00±11.31)％和(119.50±24.75)％、(171.59±3.54)％、(167.61±10.61)％;25.00 μmol/NaAsO处理组:(134.50±43.13)％、(472.50±50.20)％、(383.50±30.41)％和(180.09±12.73)％、(348.50±27.58)％、(158.45±12.02)％]均高于空白对照组[(51.50±9.19)％、(82.00±12.73)％、(25.03±2.91)％和(37.02±4.24)％、(91.56±26.16)％、(19.09±2.90)％,P均＜0.05].各组HaCaT细胞MGMT基因编码区ChIP3区域MeCP2蛋白结合水平比较,差异无统计学意义(F=1.670,P＞0.05),而DNMT1、HDAC1蛋白结合水平比较,差异有统计学意义(F值分别为4.404、9.863,P均＜0.05),其中25.00 μmol/L NaAsO2处理组DNMT1、HDAC1蛋白结合水平[(615.85±29.63)％、(306.09±59.40)％]与空白对照组[(99.70±12.02)％、(92.45±48.79)％]比较,差异有统计学意义(P均＜0.05).结论 MeCP2可结合于砷所致高甲基化MGMT基因转录调控区,通过招募DNMT1及HDAC1使组蛋白去乙酰化,同时DNMT1可结合于MGMT基因编码区,以非甲基化DNA结合蛋白(MBD)依赖的方式招募HDAC1,通过染色质重塑方式导致MGMT基因沉默,可能是砷毒性表现的早期分子事件.