全血RUNX3启动子甲基化对胃癌早期诊断的意义

目的: 探讨全血RUNX3启动子甲基化对胃癌早期诊断的意义.方法: 采用甲基化特异性聚合酶链反应(MSP)法, 检测80例胃癌患者的癌组织及其对应的癌旁组织及全血中RUNX3启动子甲基化情况.结果: 胃癌组织标本中RUNX3基因启动子甲基化率为45%, 癌旁组织中甲基化率为0%,全血中甲基化率为40%. 胃癌组织、全血中RUNX3基因启动子甲基化, 差异无统计学意义( P>0.05). 全血标本中RUNX3启动子甲基化表达情况与分化程度、是否有淋巴结转移密切相关, 与年龄、性别、肿瘤部位浸润深度无关.结论: 全血RUNX3启动子区甲基化对胃癌早期诊断有重要指导意义, 与胃癌的发生、发展密切相关.     

胃癌死亡相关蛋白激酶基因甲基化对其信使核糖核酸和蛋白表达的影响

目的 研究胃癌组织死亡相关蛋白激酶(DAPK)基因启动子区甲基化对原发性胃癌组织中DAPK mRNA及蛋白表达的影响.方法 采用逆转录(RT)-PCR法检测62例原发性胃癌及癌旁组织DAPK mRNA表达,甲基化特异性PCR(MSP)法检测DAPK启动子区CpG岛甲基化状态,对其中34例胃癌组织甲基化阳性者的DAPK蛋白表达进行Western印迹法检测.结果 胃癌组织中DAPKmRNA和蛋白表达水平明显低于癌旁组织,分别为0.2863±0.2027比0.5736±0.1968、0.2616±0.0913比0.6529±0.1808,差异均有统计学意义(P值均＜0.01).DAPK在胃癌组织和癌旁组织中的甲基化频率分别为54.8%和17.7%,差异有统计学意义(P＜0.01).在胃癌组织中,甲基化组DAPKmRNA表达明显低于非甲基化组(0.1399±0.0835比0.4640±0.1569,P＜0.01).DAPK基因甲基化与胃癌TNM分期显著相关(P=0.04).结论 原发性胃癌组织DAPK mRNA和蛋白表达缺失或低下与其启动子甲基化程度增高显著相关.     

胃癌p16基因启动子异常甲基化的临床意义

目的 观察胃癌组织中p16基因启动子区异常甲基化状态,并探讨其甲基化改变与临床特征的关系.方法 采用甲基化特异的聚合酶链反应检测胃癌组织及相应癌旁正常组织中p16基因启动子区甲基化状态.结果 胃癌组织p16基因的异常甲基化率显著高于相应的癌旁正常组织(P＜0.01).胃癌组织中该基因的异常甲基化与患者年龄、性别、肿瘤大小、病理分期、组织分化程度、肿瘤浸润深度及淋巴结转移等临床特征无显著相关性.结论 p16基因启动子区的异常甲基化是胃癌发生发展中的频繁事件,在胃癌的发生中具有肿瘤特异性.     

错配修复基因hMLH1甲基化与胃癌的关系

目的:通过研究散发性胃癌中错配修复基因hMLH1 mRNA的表达及其启动子区5'CpG岛甲基化状态,探讨hMLH1基因异常甲基化在胃癌发生过程中的作用.方法:应用甲基化特异性PCR(methylation specific PCR,MSP)检测60例胃癌组织及其癌旁黏膜组织中hMLH1基因启动子区的甲基化状态,逆转录PCR(RT-PCR)检测两种组织中hMLH1 mRNA表达情况.结果:胃癌组织hMLH1 mRNA表达水平明显低于癌旁组织(t=4.082,P<0.01),hMLH1基因启动子区高甲基化18例(30%),其癌旁黏膜组织中未发现有甲基化.hMLH1基因启动子区甲基化与胃癌的临床病理参数之间无明显的相关性.hMLH1 mRNA表达阴性的21例病例中,17例(81%)发生甲基化,而hMLH1 mRNA表达阳性的39例中仅有1例(2.5%)发生甲基化,hMLH1 mRNA表达降低与甲基化之间存在明显的相关性(χ2=8.0182,P=0.0046).结论:胃癌组织中hMLH1基因启动子区甲基化与其mRNA表达缺失密切相关,是导致hMLH1基因错配修复功能缺陷的重要原因之一.     

血管紧张素转移酶mRNA在胃癌中的表达

目的 定量分析血管紧张素转移酶(ACE)mRNA在胃癌组织和癌旁组织的表达及其临床意义.方法 采用实时荧光定量PCR检测34例胃癌组织及对应癌旁组织ACE mRNA表达水平,分析其与分化程度、临床分期和转移状态等临床病理特征的相关性.结果 ACE mRNA在胃癌组织中的表达显著高于癌旁组织(P=0.003).胃癌组织ACE mRNA表达与分化程度、临床分期和转移状态等临床病理特征无相关性(P＞0.05).结论 ACE mRNA在胃癌组织中呈明显高表达,提示其与胃癌的发病密切相关.     

胃癌Runx3基因的甲基化

目的:探讨Runx3基因甲基化与胃癌的关系方法:采用MSP法检测38例配对胃癌组织、癌旁正常组织和转移淋巴结中Runx3基因甲基化的情况.结果:73.7%的胃癌组织中存在Runx3基因异常甲基化,而相应的癌旁正常组织和转移淋巴结中该基因的甲基化率分别为21.1%和65.8%.癌组织和转移淋巴结中Runx3基因甲基化的发生率显著高于癌旁正常组织(P<0.05).胃癌组织中,该基因甲基化与肿瘤大小显著相关(P =0.021),但与肿瘤大体类型、分化程度、浸润深度及生长方式等临床病理特征无关.结论:Runx3基因异常甲基化是胃癌发生、发展过程中的频繁事件,通过检测胃黏膜组织及淋巴结中该基因的甲基化情况,可能会对胃癌的早期诊断及判断淋巴结的微转移提供一定的参考价值.     

Caveolin-2基因甲基化状态在胃癌组织中的检测

目的:探讨Caveolin-2基因启动子区域5'CpG岛甲基化与胃癌发生发展的关系.方法:运用甲基化特异性PCR(MSP)技术检测33例胃癌组织及5例距肿瘤5 cm以上的癌旁正常胃组织中Caveolin-2基因启动子区域5'CpG岛甲基化状态.结果:5例正常胃组织Caveolin-2基因启动子区域5'CpG岛均为甲基化阴性.33例胃癌组织中29例甲基化阳性,甲基化率为87.9%(29/33),其中20 例癌组织(60.6%)表现为完全甲基化,9例癌组织(27.3%)表现为部分甲基化.4例癌组织(12.1%)表现为甲基化阴性.统计学结果显示癌组织中Caveolin-2基因启动子区域5'CpG岛甲基化率显著高于正常组织.结论:胃癌组织中存在较高的Caveolin-2基因启动子区域5'CpG岛甲基化率,甲基化可能与胃癌的发生有关.     

胃癌组织中BNIP3基因甲基化及其与DNMT1表达的关系

目的探讨人胃癌组织DNMT1表达及其与BNIP3基因甲基化状态的关系。方法收集120例手术切除并经病理学证实的胃癌组织及其相应的癌旁正常组织,采用甲基化特异性PCR(MSP)方法检测胃癌组织及其相应癌旁组织BNIP3基因甲基化状态,采用免疫组化SP法检测BNIP3基因及DNMT1基因的蛋白表达水平。结果 120例胃癌组织中有72例发生了BNIP3基因甲基化,甲基化阳性率为60.0%,其相应癌旁正常组织有5例发生了BNIP3甲基化,甲基化阳性率为4.2%,差异有统计学意义(χ~2=85.839,P0.01),胃癌组织中BNIP3基因甲基化状态与患者性别、年龄、肿瘤大小、肿瘤分期无相关性(P0.05),而与分化程度及淋巴结转移情况有相关性(P0.05),胃癌组织中BNIP3蛋白的表达率为17.5%(21/120),显著低于其癌旁正常组织(80.0%,96/120),差异有统计学意义(χ~2=93.809,P0.01),胃癌组织中DNMT1蛋白的表达率为75.0%(90/120),显著高于其癌旁正常组织(5.8%,7/120),差异有统计学意义(χ~2=119.195,P0.01)。胃癌组织中BNIP3蛋白与DNMT1蛋白的表达呈负相关(r=-0.542,P0.01)。结论 DNMT1高表达可能导致BNIP3蛋白表达减少,从而参与了胃癌的发生。     

肝细胞癌中wwox基因启动子甲基化与蛋白表达的关系

目的:探讨wwox基因启动子甲基化及蛋白表达与肝细胞性肝癌的关系.方法:通过甲基化特异性PCR(methylation specific polymerase chain reaction,MSP)方法及免疫组织化学(immunohistochemestry,IHC)法分别检测60例肝细胞性肝癌组织和癌旁组织中wwox基因启动子甲基化状态和蛋白表达水平.结果:癌组织及癌旁组织中wwox基因启动子甲基化阳性率分别为41.67%(25/60)和8.33%(5/60)(P=0.000).WWOX蛋白在癌和癌旁组织中的表达具有显著性差异[35.00%(21/60)vs70.00%(42/60),P=0.001].wwox基因启动子甲基化和蛋白表达与肝外转移、肿瘤直径、肿瘤细胞分化密切相关(P=0.007,0.014,0.011);WWOX蛋白表达与临床分期、肿瘤直径、肿瘤细胞分化密切相关(P=0.018、0.023、0.001).wwox基因启动子甲基化与蛋白表达显著负相关(γ=-0.408,P=0.001).结论:启动子区甲基化是wwox基因失活的重要机制.wwox启动子区异常甲基化可能参与了肝癌的发生发展,在肝癌的进展发挥重要作用.     

Mechanism and pathobiologic implications of CHFR promoter methylation in gastric carcinoma

AIM： TO investigate the aberrant methylation of CHFR promoter in human gastric cancer （GC） and its impact on the expression of CHFR mRNA and protein, as well as its correlation with clinical and histological features of human GC. METHODS： Methylation-specific polymerase chain reaction （MSPCR） was used to detect the methylation status of CHFR promoter in 20 primary GC samples and paired normal gastric mucosa. The CHFR mRNA and protein expressions were investigated both by RT-PCR and by Western blotting. The CHFR protein expression in 39 GC samples was immunohistochemically examined. RESULTS： The DNA methylation of the CHFR gene was found in 9 of the 20 GC samples （45%） and the down-regulation of CHFR mRNA and protein was significantly associated with the methylation status of the CHFR gene （P = 0.006）. In 20 samples of corresponding non-neoplastic mucosa, no DNA methylation of the CHFR gene was detected. The CHFR gene methylation in poorly differentiated GC samples was significantly higher than that in well-differentiated GC samples （P = 0.014）. Moreover, the negative CHFR protein expression rate in paraffin-embedded GC samples was 55.07% （38/69）, the positive rate in poorly differentiated GC samples was 36.73% （18/49）, which was significantly lower than 65.00% （13/20） in well-differentiated GC samples （X^2 = 4.586, P = 0.032）. CONCLUSION： Aberrant methylation of the CHFR gene may be involved in the carcinogenesis and development of GC, and is the predominant cause of down-regulation or loss of CHFR mRNA or protein expression. As aberrant methylation of CHFR promoter is correlated with tumor differentiation, it may help to predict the prognosis of GC and CHFR may become a novel target of gene therapy for GC in the future.     

Runx3基因在胃癌中的表达及其表达下调机制

目的:观察Runx3基因在人胃癌组织及相应正常胃组织中的表达分布特点,并对其表达下调的机制进行初步研究．方法:对25例胃癌标本进行DNA、RNA和蛋白的提取,利用单链构象多态性(SSCP)和微卫星法,检测Runx3基因突变和缺失情况．用 MSP(methylation specific PCR)检测25例胃癌标本及细胞株(MGC803,SGC7901)的甲基化状态．分别以蛋白印迹法(western blot)和逆转录聚合酶链反应(RT-PCR),检测Runx3蛋白质和mRNA水平．结果:在25例胃癌标本中发现一例杂合性缺失,未发现Runx3第三外显子突变．MSP法检测胃癌标本中55％(14/25)发生启动子区域的甲基化,同时在胃癌细胞株MGC803中Runx3 基因发生了部分甲基化．Western blot和RT- PCR发现Runx3在胃癌组织中表达明显低于相应正常胃组织(蛋白:50 178±14 404 vs 95 020±43 136,P<0．01;mRNA:0．66±0．31 vs 1．06±0．34,P<0．01)．结论:Runx3基因在胃癌组织中表达较正常组织明显下调,提示该基因与胃癌的发生和发展有关．Runx3基因表达下调的机制主要是因为启动子区域甲基化．     

Rationales for expression and altered expression of apoptotic protease activating factor-1 gene in gastric cancer

AIM: To elucidate the relationship between apoptotic protease activating factor-1 (Apaf-1) gene and gastric cancer. METHODS: Thirty-five postoperative cancer and adjacent normal tissue samples were collected in the present study. Expression of the Apaf-1 gene in these samples was analyzed by semi-quantitative RT-PCR. Loss of heterozygosity (LOH) was used to determine whether there was loss of Apaf-1 gene in domain of 12q22-23 in the samples. Promoter methylation of Apaf-1 gene in the samples was analyzed by methylation specific (MSP) PCR. RESULTS: The expression of Apaf-1 mRNA in gastric cancer tissue samples was 51%. The LOH frequency of D12S346, D12S1706, D12S327, D12S1657 and D12S393 was 33%, 8%, 58%, 12% and 42%, respectively. Fifty percent LOH was found at two sites and 17% LOH at three sites. Apaf-1 mRNA expression decreased significantly in 13 cases (rs = 0.487, P = 0.003). The rate of Apaf-1 promoter methylation was 49% in gastric cancer tissue samples and 23% in para-cancerous tissue samples. Promoter methylation occurred significantly in 16 of 18 gastric cancer tissue samples with decreased expression of Apaf-1 mRNA rs = 0.886, P = 10-6). CONCLUSION: The expression of Apaf-1 gene is low in gastric cancer tissues. Methylation of Apaf-1 gene promoter and LOH in domain of 12q22-23 are the main reasons for the expression and altered expression of Apaf-1 gene.     

Hypermethylation of Syk gene in promoter region associated with oncogenesis and metastasis of gastric carcinoma

AIM: To investigate the relationship between methylation of Syk (spleen tyrosine kinase) gene in promoter region and oncogenesis, metastasis of gastric carcinoma. The relation between silencing of the Syk gene and methylation of Syk promoter region was also studied. METHODS: By using methylation-specific PCR (MSP) technique, the methylation of Syk promoter region in specimens from 61 gastric cancer patients (tumor tissues and adjacent normal tissues) was detected. Meanwhile, RT-PCR was used to analyse syk expression exclusively. RESULTS: The expression of the Syk gene was detected in all normal gastric tissues. Syk expression in gastric carcinoma was lower in 14 out of 61 gastric cancer samples than in adjacent normal tissues (chi(2)=72.3, P<0.05). No methylation of Syk promoter was found in adjacent normal tissues. hypermethylation of Syk gene in promoter was detected 21 cases in 61 gastric carcinoma patients. The rate of methylation of Syk promoter in gastric carcinoma was higher than that in adjacent normal tissues (chi(2)=25.1, P<0.05). In 31 patients with lymph node metastasis, 17 were found with Syk promoter methylation. A significant difference was noted between two groups (chi(2)=11.4,P<0.05). CONCLUSION: Hypermethylation leads to silencing of the Syk gene in human gastric carcinoma. Methylation of Syk promoter is correlated to oncogenesis and metastasis of gastric carcinoma. Syk is considered to be a potential tumor suppressor and anti-metastasis gene in human gastric cancer.     

胃癌组织中肿瘤相关基因甲基化及其表达与叶酸和代谢酶MTHFR基因多态性的关系

目的:探讨人胃癌组织中癌基c-myc,抑癌基因p16INK4A,p21WAF1,错配修复基NhMLH1和hM2SH2的甲基化状态及其表达与叶酸、MTHFR基因多态性的关系．方法:胃癌38例手术切除标本的癌区、癌旁和外周正常黏膜组织,运用FOL ACS:180自动化学发光系统测定叶酸含量,PCR-RFLP技术检测MTHFR基因677(C→T)和1298(A→C)两个常见多态,并分别以Real—time RT-PCR和甲基化特异性PCR (MSP)技术检测肿瘤相关基因的表达和甲基化状态．结果:c-myc表达升高,p16INK4A,hMLH1和hMSH2表达降低的胃癌黏膜组织其基因启动子区异常甲基化．p21WAF1,hMSH2表达降低, p16INK4A高甲基化者叶酸水平明显降低,c-myc低甲基化和表达升高者中均存在低叶酸水平．MTHFR 677CC基因型的胃癌黏膜组织p16INK4A甲基化升高且表达降低,而其余肿瘤相关基因的甲基化及其表达与MTHFR两个常见多态均无明显相关性．结论:DNA甲基化在胃癌的发生、发展中具有重要作用,叶酸水平和MTHFR基因多态性通过影响部分肿瘤相关基因的甲基化状态而调控其表达．     

巢式MSP法检测胃癌患者血浆p16、MGMT基因甲基化

目的:探讨胃癌患者血p16和MGMT基因启动子区甲基化状态.方法:采用巢式甲基化特异性PCR法(nestedmethylation-specific PCR,nMSP)对6例胃癌患者血浆中p16和MGMT基因启动子区甲基化状态进行研究.同时以16例健康体检作为对照.结果:在受检的69例胃癌患者中,p15、MGMT基因启动子区甲基化率分别为30.4%(21/69)、17.4%(12/69).对照组未见p16、MGMT基因启动子区甲基化.胃癌患者血浆中存在p16和MGMT基因启动子区甲基化,其中p16基因启动子区甲基化与对照组相比差异显著,具有统计学意义.结论:检测血浆循环DNA中p16、MGMT基因启动子区的甲基化状态,可为胃癌的早期分子诊断提供了有用信息.     

Hedgehog通路中Ptchl基因在人胃癌中高甲基化状态分析

目的 研究Hedgehog通路中Ptchl基因表达及其甲基化状态在胃癌发生中的变化.方法 分别抽提10例胃癌组织及其癌旁＞3 cm组织和胃癌细胞株AGS的总RNA和基因组DNA.实时定量逆转录多聚酶链反应(QRT-PCR)检测Ptch基因的mRNA表达.应用软件分析Ptchl基因5'调控序列的CpG岛状态,亚硫酸氢盐测序PCR(BSP)分析甲基化水平.结果 对Ptchl mRNAla转录体转录起始位点(计为0点)上游-3950 bp和下游+2050 bp进行CpG岛分析,发现存在两个CpG岛,第1个为-1139 bp～+860 bp,第2个为+875 bp～+1692 bp.以第1个CpG岛的-870 bp～+229 bp区段内的19个CpG位点的BSP测序结果显示,胃癌细胞株AGS全部发生甲基化,胃癌组织中甲基化程度为16%～100%,平均64%±32%,癌旁组织甲基化程度为0%～42%,平均13%±14%,两组问差异有统计学意义(P＜0.05).Spearman秩相关分析发现,Ptchl基因甲基化同其表达呈负相关(r=-0.558,P=0.011).结论 Ptchl基因高甲基化参与胃癌的发生,可能为胃癌新的肿瘤标志物.     

Metastatic suppressor genes inactivated by aberrant methylation in gastric cancer

AIM： TO screen out the differentially methylated DNA sequences between gastric primary tumor and metastatic lymph nodes, test the methylation difference of gene PTPRG between primary gastric tumor and metastatic lymph nodes, and test the regulatory function of 5-aza2-deoxycytidine which is an agent with suppression on methylation and the level of methylation in gastric cancer cell line. METHODS： Methylated DNA sequences in genome were enriched with methylated CpG islands amplification （MCA） to undergo representational difference analysis （RDA）, with MCA production of metastatic lymph nodes as tester and that of primary tumor as driver. The obtained differentially methylated fragments were cloned and sequenced to acquire the base sequence, which was analyzed with bioinformatics. With methylation-specific PCR （MSP） and RT-PCR, methylation difference of gene PTPRG was detected between primary tumor and metastatic lymph nodes in 36 cases of gastric cancer. Methylation of gene PTPRG and its regulated expression were observed in gastric cancer cell line before and after being treated with methylation-suppressive agent. RESULTS： Nineteen differentially methylated sequences were obtained and located at 5＇ end, exons, introns and 3＇ end, in which KL59 was observed to be located at 9p21 as the first exon of gene p16 and KL22 to be located at promoter region of PRPRG. KL22, as the probes, was hybridized with driver, tester and 3-round RDA products respectively with all positive signalsexcept with the driver. Significant difference was observed in both methylation rate of gene PTPRG and PTPRG mRNA expression rate between primary tumor and metastatic lymph nodes. Demethylation of gene PTPRG, with recovered expression of PTPRG mRNA, was observed after gastric cancer cell line being treated with methylation-suppressive agent. CONCLUSION： Difference exists in DNA methylation between primary tumor and metastatic lymph nodes of gastric cancer, with MCA-RDA as one of the good analytical methods. Significant     

肿瘤-睾丸抗原MAGE-1、MAGE-3、SSX-2及NY-ESO-1在胃癌中的表达

目的　检测肿瘤 睾丸 (cancer testis ,CT)抗原MAGE 1、MAGE 3、NY ESO l及SSX 2基因mRNA在胃癌组织中的表达 ,探索其在胃癌免疫治疗中的应用价值。方法　用逆转录聚合酶链反应 (RT PCR)方法对胃癌患者癌组织、相应癌旁组织和对照组织(慢性胃炎和正常胃组织 )中的MAGE 1、MAGE 3、NY ESO 1及SSX 2基因mRNA进行检测 ,结合临床指标进行分析 ,随机选取RT PCR阳性扩增产物进行序列测定。结果　在在检测的 5 0例胃癌组织中 ,MAGE 1、MAGE 3、NY ESO 1及SSX 2基因mRNA阳性率分别是 48% (2 4/5 0 )、42 % % (2 1/5 0 )、11% (6/5 0 )和 8% (4 /5 0 ) ;多个CT抗原基因mRNA在胃癌中同时表达 ,至少表达一种CT抗原者达76% (3 8/5 0 ) ,表达两种或两种以上者为 40 % (2 0 /5 0 ) ,表达三种或三种以上者为 10 % (5 /5 0 ) ,同时表达四种者为 6% (3 /5 0 )。而相应的癌旁组织和对照组织中均未检测到四种目的基因的表达。DNA测序结果表明RT PCR产物确为这四种基因的目的片段。MAGE 1、MAGE 3、NY ESO 1及SSX 2基因的表达与年龄、性别、肿瘤大小、分化程度、浸润深度和淋巴结转移等无显著相关性 (P >0 .0 5 )。结论　CT抗原 (MAGE 1、MAGE 3、NY ESO 1及SSX 2 )基因在胃癌组织中呈高特异表达 ,这使得以这些基因编码的蛋