大肠癌组织DCC基因启动子甲基化及表达水平相关性研究

目的:探讨结直肠癌缺失基因 (DCC)在大肠癌组织中的表达特征及其与启动子甲基化的相关性.方法:分别采用免疫组织化学和甲基化特异性PCR (MSP)方法检测71例大肠癌及其相应癌旁组织中DCC蛋白的表达情况及DCC基因启动子甲基化状态.结果:大肠癌组织中DCC蛋白表达水平 (35/71, 49.3%) 显著低于癌旁正常组织 (34/39, 87.2%, P<0.001),且与临床Duke分期相关,P=0.023;大肠癌组织中DCC基因启动子甲基化发生率 (54/71, 76.1%)高于癌旁正常组织 (14/39, 35.9%, P<0.001)和癌旁炎性组织 (10/22, 45.5%, P=0.007),且直肠癌患者DCC基因启动子甲基化发生率 (28/31, 90.3%)高于结肠癌患者 (27/40, 67.5%, P=0.022);大肠癌组织中DCC蛋白失表达组甲基化发生率 (34/36, 94.4%)显著高于DCC蛋白表达组 (20/35, 57.1%, P<0.001).结论:DCC基因启动子甲基化可能是大肠癌中DCC失表达的主要机制之一.     

ADAMTS9基因启动子甲基化在直肠癌中的表达与临床病理特征的相关性

目的检测直肠癌组织中ADAMTS9基因转录和启动子甲基化状态,探讨其临床意义。方法收集2016年1月至2018年12月间于延安大学附属医院普通外科医院行直肠癌根治手术切除的83例直肠癌组织及其相应的癌旁组织标本,通过实时定量PCR和巢式-甲基化特异性PCR检测ADAMTS9基因转录和启动子甲基化状态,分析ADAMTS9基因启动子甲基化与直肠癌临床病理特征的相关性。结果直肠癌组织中ADAMTS9 mRNA表达水平低于配对癌旁组织(χ²=1161,P<0.0001)。83例直肠癌组织中ADAMTS9基因甲基化发生率20.5%,高于癌旁组织7.2%(6/83)(χ²=37.19,P<0.0001)。ADAMTS9基因启动子区异常甲基化增加直肠癌发病风险(OR=10.254,95%CI=3.395~25.631)。直肠癌组织中ADAMTS9启动子区甲基化与肿瘤TNM分期、分化程度相关。结论ADAMTS9基因启动子异常甲基化参与了直肠癌的发生发展过程。     

乳腺癌患者血浆循环DNA中Sox17基因甲基化检测的临床意义

背景与目的：在乳腺癌发生、发展过程中,甲基化异常是导致抑癌基因失活的重要机制,是一种可用于肿瘤诊断及预后判断、有价值的生物标志物。本研究旨在通过检测乳腺癌组织及其相应的血浆循环DNA中Sox17基因的甲基化状况,探讨其在乳腺癌早期诊断和预后判断方面的应用价值。方法：采用甲基化特异性聚合酶链反应(methylation specific-PCR,MSP)法,对86例乳腺癌组织、36例乳腺良性肿瘤的癌旁正常组织及其配对的血浆循环DNA中Sox17基因启动子甲基化进行检测,并结合乳腺癌的主要临床病理特性进行分析。结果：86例乳腺癌组织中Sox17基因启动子的甲基化率为77.9%(67/86),与其相应血浆循环DNA中Sox17基因启动子的甲基化率为61.6%(53/86),36例癌旁正常乳腺组织及血浆中均未检测到Sox17基因异常甲基化。患者血浆循环DNA中Sox17基因启动子的甲基化与肿瘤组织中该基因的甲基化显著相关(r=0.502,P=0.000)。在乳腺癌组织标本中Sox17基因甲基化率与患者肿瘤分期(χ2=6.18,P=0.041)、淋巴结转移(χ2=13.54,P=0.001)显著相关,在血浆标本中,Sox17基因甲基化率与患者肿瘤分期(χ2=27.06,P=0.000)、肿瘤大小(χ2=9.65,P=0.007)及淋巴结转移(χ2=20.80,P=0.000)显著相关,与患者年龄、组织学分级及ER、PR、HER-2/neu等指标差异无统计学意义(P>0.05)。结论：Sox17基因启动子甲基化在乳腺癌的发生、发展中起着重要作用,可能与乳腺癌的预后相关。血浆中Sox17基因甲基化,是一个有潜在应用价值的生物标志物。     

散发性乳腺癌中 DNMT3a、3b 表达及其与 BRCA1基因启动子甲基化和蛋白表达的相关性

目的:探讨DNMT3a、3b蛋白表达与散发性乳腺癌BRCA1基因启动子甲基化状态及蛋白表达的相关性。方法:收集乳腺肿瘤组织及相关临床资料。采用免疫组化法检测200例散发性乳腺癌与25例乳腺纤维腺瘤组织中DNMT3a、3b蛋白的表达情况。甲基化特异性PCR检测108例散发性乳腺癌中BRCA1基因启动子甲基化状态。结果:与乳腺纤维腺瘤相比,乳腺癌组织中DNMT3a表达水平显著增高(P=0.037),DNMT3b表达水平增加不显著(P=0.478)。乳腺癌中BRCA1基因启动子存在甲基化。DNMT3a、3b过表达与乳腺癌病程晚期(TNMⅢ-Ⅳ期)显著相关(P=0.064,P=0.007);DNMT3a与BRCA1基因甲基化正相关(r=0.222,P=0.021),与BRCA1蛋白负相关(r=-0.172,P=0.022)。BRCA1蛋白表达与乳腺癌患者OS和DFS显著相关(P=0.025,P=0.027),DNMT3a表达与散发性乳腺癌OS和DFS存在一定的相关性(P=0.052,P=0.091)。结论:DNMT3a、3b高表达与乳腺癌侵袭转移、病程进展、预后相关。DNMT3a可能参与催化BRCA1基因启动子甲基化导致BRCA1蛋白表达下调的过程。抑制DNMT3a、3b蛋白可能有利于散发性乳腺癌的预防和治疗。     

Atrogin-1基因在贲门腺癌组织中的表达及其异常甲基化

目的：探讨atrogin-1基因在贲门腺癌（gastric cardia adenocarcinoma,GCA）中的异常甲基化及表达,并分析其临床意义。方法: 选用河北医科大学第四医院2004—2008年间的贲门腺癌患者手术标本共139例,分别应用亚硫酸氢盐转换-甲基化特异性PCR（bisulfite conversion-methylation specific polymerase chain reaction,BS-MSP）、RT-PCR和免疫组织化学法检测贲门腺癌组织及相应癌旁（距癌灶边缘3～5 cm）组织中atrogin-1基因的甲基化、mRNA和蛋白表达情况,应用免疫组织化学法检测相应组织中Smad4蛋白的表达。结果: 贲门腺癌组织中atrogin-1基因启动子区的甲基化率\[44.6%（62/139）\] 显著高于癌旁组织\[3.6%(5/139)\]（χ2=63.891,P=0.001）,且atrogin-1基因的甲基化与TNM分期及肿瘤的组织学分化程度密切相关（χ2=6.144, P<0.05）。贲门腺癌组织中atrogin-1基因的mRNA和蛋白表达水平显著低于癌旁组织\[(0.482 5±0.175 4) vs (0.896 9±0.290 1),t=10.62, P=0.01;34.5% vs 82.0%, χ2=4.441,P=0.001\],且与其启动子区的甲基化状态之间有明显的相关性(r=-0.256,P=0.001)。贲门腺癌组织中Smad4蛋白表达的阳性率显著低于癌旁组织（46.0% vs 95.7%; χ2=2.945,P=0.001）,且与atrogin-1蛋白表达之间呈明显的正相关(r=0.604,P=0.001）。结论: Atrogin-1基因启动子区高甲基化导致的基因沉默可能是贲门癌组织中此基因表达降低的机制之一。     

食管鳞癌中hMLH1、E-cadherin、p16^INK4a基因启动子甲基化及其意义

背景与目的:目前认为抑癌基因启动子甲基化导致转录抑制是恶性肿瘤发生的重要机制之一,hgLH1、E-cadherin及p16INK4a基因在多种恶性肿瘤中都已被证实存在较高频率的甲基化.本研究通过检测食管鳞癌组织及癌旁组织中hMLH1、E-cadherin,p16INK4a基因启动子甲基化的发生情况,探讨hMLH1、E-cadherin、p16INK4a基因启动子甲基化在食管鳞癌发生发展中的作用.方法:采用酚-氯仿法提取105例食管鳞癌组织及癌旁组织的基因组DNA,应用甲基化特异性PCR对所提DNA进行hMLH1、E-cadherin、p16INK4a基因甲基化检测.采用EnVison免疫组织化学二步法对癌组织中上述3种基因蛋白表达进行检测.结果:癌组织中E-cadherin、hMLH1、p16INK4a基因启动子甲基化的阳性率分别为57.1%(60/105)、20.9%(22/105)和50.5%(53/105),而癌旁食管组织中相应的3个基因的甲基化率分别为10.5%(11/105)、1.9%(2/105)和7.6%(8/105),均显著低于癌组织.E-cadherin(P=0.021)及p16INK4a(P=0.026)基因甲基化与蛋白表达缺失密切相关,而hMLH1基因甲基化与蛋白表达无显著相关性.E-cadherin基因启动子甲基化与淋巴结转移有关(P=0.016),p16INK4a基因启动子甲基化与低分化癌有关性(P=0.024).hMLH1基因甲基化与各项临床病理特征均无关.结论:食管鳞癌中p16INK4a基因启动子甲基化与相应蛋白表达缺失密切相关,且在低分化癌中更多见;E-cadherin基因启动子甲基化与相应蛋白质表达缺失有相关性,并且有淋巴结转移多见的显著特征,这2个基因的甲基化位点与食管鳞癌密切相关.hMLH1基因甲基化可能并不直接参与食管鳞癌的发生、发展.     

miR-204启动子甲基化作为结直肠癌表观遗传生物标志物的临床研究

目的 探讨miR-204启动子甲基化在结直肠癌(CRC)中的临床意义。方法 收集2018年1月至2020年3月于我院就诊的69例CRC患者血浆样本、肿瘤组织标本和癌旁正常结肠黏膜组织标本；另外招募68例健康志愿者作为正常对照组，采集血浆样本。使用肿瘤基因图谱计划(TCGA)数据库与基因芯片数据(Methyl450K)分析肿瘤和正常组织中识别的CpG位点的甲基化水平。采用定量甲基化特异性PCR法(qMSP)和实时荧光定量PCR法(qPCR)检测样本中miR-204启动子区甲基化状态和基因表达量。结果 TCGA数据表明miR-204表达与宿主基因TRPM3的表达协调下调，并确定了miR-204启动子周围的两个CpG岛。与癌旁组织比较，CRC组织中miR-204启动子甲基化参考百分比(PMR)升高[1.78(0.35,16.61)vs. 1.51(0.45,2.98),P<0.001],且与miR-204表达量呈负相关(r=-0.324,P=0.007),与DNA甲基转移酶1 mRNA呈正相关(r=0.502,P<0.001)。此外，与正常对照组比较，CRC患者血浆cfDNA中m...     

胃癌组织中EphA1的表达与肿瘤转移及患者生存时间的关系

背景与目的:受体酪氨酸激酶Eph基因家族在生长发育和肿瘤发生中发挥重要作用.本课题研究EphA1在胃癌中的表达,并分析其与临床病理指标之间的联系,旨在探索EphA1基因在胃癌发生中的作用.方法:利用实时定量逆转录聚合酶链式反应(real time RT-PCR)检测胃癌组织和正常黏膜中EphA1转录子的表达.通过甲基化特异性PCR检查启动子区相关的CpG岛甲基化状态.并运用免疫组织化学染色测定细胞的EphA1蛋白表达水平.结果:EphA1转录子在34%(19/56)的样本中表达下调,25%(14/56)的样本中表达上调,41%(23/56)的样本中表达差异无统计学意义(P＞0.05).EphA1转录子表达水平与肿瘤大小(P=0.05)、分期(P=0.001)以及淋巴结转移(P=0.011相关.在检查的15例EphA1下调的标本中,12例检出甲基化EphA1 DNA;在5例上调的标本中,仅1例检出甲基化DNA;18例表达无差异的标本中,3例检出甲基化DNA(P=0.001).145例组织标本中EphA1蛋白表达与转录子表达的临床意义相符.随访89例患者分析生存时间发现,EphA1下调者预后比上调者长(P=0.005).结论:EphA1可能在胃癌细胞的侵袭和转移中发挥作用.     

TIMP-3甲基化调控与大肠癌转移的关系研究

目的探讨TIMP-3基因启动子5’CpG岛甲基化状况与大肠癌转移的关系。方法随机收集45例大肠癌病例。用甲基化特异性PCR检测TIMP-3基因启动子5’CpG岛甲基化状况;用免疫组化方法检测TIMP-3蛋白的表达水平;分析TIMP-3基因启动子甲基化与大肠癌转移之间的关系。结果大肠癌肝转移组、淋巴结转移组TIMP-3基因甲基化率分别高于非肝转移组（P〈0．01）、非淋巴结转移组（P〈0．01）;Duck＇s C＋D组甲基化率高于Duck’s A＋B组。大肠癌TIMP-3基因甲基化与TIMP-3蛋白的表达缺失有关（P〈0．01）。结论TIMP-3甲基化是大肠癌中TIMP-3蛋白表达缺失的主要原因,在大肠癌转移中发挥重要的作用,提示预后不良。     

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。     

胃癌、结直肠癌患者血浆神经肽Y水平

目的研究胃癌、结直肠癌患者血浆神经肽Ｙ（ＮＰＹ）水平,探讨血浆ＮＰＹ浓度与癌的关系。方法选择肝肾功能正常的胃癌（１８例）、结直肠癌（２０例）患者,取术前空腹静脉血,采用放射免疫分析测定血浆ＮＰＹ浓度。结果３８例癌患者血浆ＮＰＹ浓度术前为１２５．３±３１．５ｐｇ／ｍｌ,低于健康对照１４５．１±４４．１ｐｇ／ｍｌ（Ｐ＜０．００１）。体重下降＞３ｋｇ,ＮＰＹ降低（Ｐ＜０．０１）;肿瘤＞５ｃｍ,ＮＰＹ也降低（Ｐ＜０．０５）。胃癌分期与血浆ＮＰＹ水平呈负相关（ｒ＝－０．７３,Ｐ＜０．０１）。肠癌分期与血浆ＮＰＹ水平无相关性。血浆ＮＰＹ水平与性别、年龄、血压、浸润深度、分化等级、淋巴结转移状态无关（Ｐ＞０．０５）,与血红蛋白浓度和血浆白蛋白含量无相关性。结论胃癌、结直肠癌患者血浆ＮＰＹ低于对照组,胃癌患者血浆ＮＰＹ降低与肿瘤进展可能有关     

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.     

Plasma matrix metalloproteinase-9 level is better than serum matrix metalloproteinase-9 level to predict gastric cancer evolution.

PURPOSE: Matrix metalloproteinase-9 (MMP-9) in blood is a promising new tumor marker. The aims of the present study are to compare the usefulness of plasma and serum MMP-9 levels for predicting gastric cancer development, invasion, and survival. EXPERIMENTAL DESIGN: In this nested case-control study, 114 gastric cancer patients and 87 healthy controls were enrolled. MMP-9 levels and activities were quantitatively measured by ELISA assay and zymography. The results were compared with the occurrence, clinicopathologic features, and outcomes of gastric cancer patients. The follow-up time for all patients was at least 5 years. RESULTS: Serum MMP-9 levels were significantly higher than plasma MMP-9 levels. Both plasma and serum MMP-9 levels correlated significantly with active MMP-9 identified by zymography (P = 0.002 and P = 0.048, respectively). Plasma MMP-9 level was significantly elevated in gastric cancer patients when compared with control subjects (P < 0.001). Serum MMP-9 levels did not differ between the groups. Receiver-operator characteristics analysis showed the values of sensitivity (82.5%) and specificity (65.5%) at the maximum accuracy for plasma MMP-9 at >or=60 ng/mL (P < 0.001). Elevated plasma MMP-9 correlated significantly with lymph node metastasis [odds ratio (OR), 3.43; P = 0.019], lymphatic invasion (OR, 7.58; P = 0.009), and venous invasion (OR, 4.14; P = 0.033). Patients with elevated plasma MMP-9 levels had poorer survival rates than those with normal plasma MMP-9 levels (P = 0.038). Serum MMP-9 level did not correlate well with gastric cancer-invasive phenotypes or survival. CONCLUSION: Our results suggest plasma MMP-9 level is a better marker than serum MMP-9 level for predicting gastric cancer development and progression.     

三阴性乳腺癌中DNMT1表达与ERα基因启动子甲基化的关系

目的:探讨三阴性乳腺癌中DNMT1表达与ERα基因启动子甲基化的关系,以及二者在三阴性乳腺癌中的临床意义。方法:采用免疫组化法检测了126例三阴性乳腺癌组织中DNMT1蛋白的表达情况,同时采用甲基化特异性PCR法检测了ERα基因启动子区甲基化水平。结果:126例三阴性乳腺癌中,DNMT1阳性表达率为38.1%,ERα基因启动子区甲基化率为54.8%,DNMT1表达水平与ERα基因甲基化呈显著正相关（r=0.220,P=0.013）。与TNM Ⅰ-Ⅱ期组织相比,在TNM Ⅲ-Ⅳ期组织中ERα基因甲基化显著增加（P=0.040）;与无淋巴结转移的组织相比,发生淋巴结转移的组织中DNMT1表达水平显著增加（P=0.043）。ERα基因启动子区甲基化与三阴性乳腺癌患者OS及DFS缩短显著相关（P=0.020,P=0.036）;DNMT1阳性表达可显著缩短患者OS（P=0.034）,同时可使患者DFS出现缩短趋势（P=0.054）。结论:三阴性乳腺癌中DNMT1蛋白参与了ERα基因甲基化,且该事件与三阴性乳腺癌的侵袭转移、病程进展及不良预后相关。     

结直肠癌患者血浆Septin9 DNA甲基化联合FOBT检测的临床应用价值

目的:探讨血浆Septin9 DNA甲基化及粪便隐血试验（FOBT）在结直肠癌（CRC）诊断中的应用价值。方法:回顾性收集2017年6月至2022年1月我院收治并经病理检查确诊的101例结直肠良性腺瘤患者、209例结直肠癌患者分别作为良性腺瘤组和结直肠癌组，选取同期在我院进行体检的98例健康人群作为正常对照组，比较三组一般资料及血浆Septin9 DNA甲基化及FOBT阳性情况，比较不同病理特征、不同临床分期结直肠癌患者血浆Septin9 DNA甲基化、FOBT阳性情况，采用受试者工作曲线（ROC）评估血浆Septin9 DNA甲基化、FOBT单项及联合检测对结直肠癌的诊断价值。结果:三组血浆Septin9 DNA甲基化、FOBT阳性率比较，结直肠癌组高于良性腺瘤组及正常对照组，良性腺瘤组高于正常对照组（P<0.05）；肿瘤低分化、淋巴结转移、脉管/神经侵犯的结直肠癌患者血浆Septin9 DNA甲基化和FOBT阳性率高于肿瘤高分化、中分化及未发生淋巴结转移、未发生脉管/神经侵犯的结直肠癌患者（P<0.05）；随着结直肠癌TNM临床分期升高，血浆Septin9 DNA甲基化及FOBT阳性率升高，差异具有统计学意义（P<0.05）；血浆Septin9 DNA甲基化和FOBT联合诊断结直肠癌的敏感度、曲线下面积（AUC）（87.08%、0.885）均高于两者单独诊断（59.33%、0.744和52.63%、0.643，P<0.05）；血浆Septin9 DNA甲基化诊断结直肠癌的特异度、阳性预测值和阴性预测值分别为89.45%、85.52%和67.68%；FOBT分别为76.38%、70.06%和60.56%，两者联合诊断分别为89.95%、90.10%和86.89%。联合诊断优于单项诊断。结论:随着结直肠病变恶性程度的增加，血浆Septin9 DNA甲基化和FOBT阳性率升高，血浆Septin9 DNA甲基化、FOBT联合检测对结直肠癌具有较高的诊断价值，可作为结直肠癌的实验室诊断指标。     

Tumor-derived aberrant methylation in plasma of invasive ductal breast cancer patients: clinical implications

Progressive p16 methylation has been associated with metastasis and invasive phenotypes in many cancers. Loss of E-cadherin (CDH1) function contributes to breast cancer progression by promoting cell proliferation, invasion and metastasis. Using methylation-specific PCR, aberrant hypermethylation of p16 and CDH1 in tumor and plasma was analyzed and correlated with levels of serum protein markers, carcinoembryonic antigen (CEA) and carcinoma antigen 15-3 (CA15.3), in 36 patients with invasive ductal breast cancer. Aberrant p16 methylation was found in 11% (4/36) of primary tumors and 8% (3/36) of plasma samples. Aberrant CDH1 methylation was detected in 25% (9/36) of primary tumors and 20% (7/36) of plasma samples. p16 and/or CDH1 hypermethylation was found in 31% (11/36) of primary breast carcinomas and 82% (9/11) of breast cancer patients with tumoral methylation showing identical epigenetic changes in plasma. The 25 patients without tumoral methylation did not show epigenetic changes in the plasma. Tumoral p16 methylation was significantly associated with advanced tumor stage (p=0.028; Fisher's exact test), tumor size (p=0.017) and nodal metastasis (p=0.002). However, p16 methylation in plasma was only associated with nodal metastasis (p=0.012). Altogether, aberrant p16 methylation in plasma and elevated serum CEA level were associated with advanced tumor stage (p=0.033), tumor size (p=0.022) and extensive nodal metastasis (p=0.003). With clinical implications, p16 hypermethylation in plasma and/or raised serum CEA levels may prove useful as diagnostic and prognostic markers for breast cancer.     

GATA5甲基化在结直肠癌血浆和粪便中的检测及临床诊断价值

背景与目的：结直肠癌(colorectal cancer,CRC)是全球发病率第三,死亡率第四的恶性肿瘤。遗传学及表观遗传学的改变引发抑癌基因甲基化表达沉默是CRC发生的重要原因,本研究旨在探讨血浆、粪便GATA5启动子甲基化检测在CRC临床诊断中的应用价值。方法：收集34例健康体检者和43例CRC患者的血浆和配对的粪便标本。采用甲基化特异性PCR法(methylation specific PCR method,MSP)检测其血浆、粪便中GATA5甲基化水平,并分析其与临床病理特征相关性。结果：MSP结果显示CRC患者血浆、粪便中GATA5启动子甲基化率(60.74%、76.60%)高于健康者发生率(26.47%、32.35%),差异均有统计学意义(P值分别为0.006 7,0.000 2)。CRC患者血浆甲基化发生率与临床分期(P=0.000 5)、淋巴结转移(P=0.020)密切相关,而粪便GATA5甲基化水平与临床病理特征无统计学意义。结论：检测粪便GATA5甲基化水平并辅以血浆GATA5甲基化水平可作为一种简单、非侵入、敏感及特异的方法应用于CRC患者的临床诊断。     

Hypermethylation of the TPEF/HPP1 gene in primary and metastatic colorectal cancers

The role of promoter methylation in the process of cancer cell metastasis has, however, not yet been studied. Recently, methylation of the TPEF (transmembrane protein containing epidermal growth factor and follistatin domain) gene was reported in human colon, gastric, and bladder cancer cells. Using the Methylight assay, TPEF/HPP1 gene methylation was assessed in primary colorectal cancers (n = 47), matched normal colon mucosa, as well as in the liver metastasis of 24 patients with colorectal cancer, and compared to the methylation status of the TIMP-3, APC, DAPK, caveolin-2, and p16 genes. TPEF was frequently methylated in primary colorectal cancers (36 of 47) compared to the normal colon mucosa (1 of 21) (P < .0001), and TPEF mRNA expression in colon cancer cell lines was restored after treatment with 5-aza-2'-deoxycytidine. The p16 and APC genes were also frequently methylated in primary colorectal cancers (P < .02) compared to the normal colon mucosa. Interestingly, promoter methylation was significantly more frequent in proximal, nonrectal cancers (P < .05). Furthermore, a high degree of methylation of the TPEF gene was also observed in liver metastasis (19 of 24). In summary, we observed frequent TPEF methylation in primary colorectal cancers and liver metastases, indicating that epigenetic alterations are not only present in the early phases of carcinogenesis, but are also common in metastatic lesions. The high frequency of TPEF methylation in this series of colorectal cancers underscores the importance of epigenetic changes as targets for the development of molecular tests for cancer diagnosis.     

Predictive value of vascular endothelial growth factor (VEGF) in metastasis and prognosis of human colorectal cancer.

Vascular endothelial growth factor (VEGF) may affect the phenotype of cancer cells, such as growth velocity and metastatic potential, due to its probable multifunctional property including a mitogenic activity for vascular endothelial cells. The present study was designed to investigate the association of VEGF mRNA expression with progression and metastasis of human colorectal cancer. The level of VEGF mRNA expression was quantified by Northern blot hybridization in tumorous and non-tumorous tissues obtained from 60 primary colorectal cancer patients. The ratio of the former to the latter was defined as the VEGF T/N ratio, and the prognostic significance of this ratio, following surgery, in addition to the relationship to progression and metastatic potential, was evaluated. The value of the VEGF T/N ratio was significantly correlated with the depth of tumour infiltration (P=0.046), the incidence of liver metastasis (P < 0.0001) and lymph node metastasis (P=0.036). Patient prognosis was estimated by the Kaplan-Meier method and the log-rank test. When the VEGF T/N ratio was higher than 4.8 for which the chi2 value of the log-rank test was maximal, the tumour was defined as showing overexpression of VEGF mRNA. Patients with overexpression of VEGF mRNA demonstrated poorer survival than patients without overexpression of VEGF mRNA (P < 0.001). The overall estimated hazard ratio for death in patients with overexpression of VEGF mRNA was 1.94 according to a multivariate analysis (P=0.005). Thus, VEGF is associated with the progression, invasion and metastasis of colorectal cancer, and overexpression of VEGF mRNA in the primary tumour is assumed to be closely correlated with poor prognosis in colorectal cancer patients. Moreover, the VEGF T/N ratio may be used as an independent prognostic marker in colorectal cancer patients.