河北省食管癌高发区贲门腺癌Syk基因甲基化状态研究

目的:研究胃贲门腺癌(gastric cardiac adenocarcinoma,GCA)中脾酪氨酸激酶(spleen tyrosine kinase,Syk) 基因启动子甲基化及其蛋白表达情况.方法:采用改进的甲基化特异性PCR(methylation specific PCR,MSP)和免疫组织化学SP法检测GCA组织及相应癌旁正常组织中Syk基因的DNA甲基化和蛋白表达情况. 结果:47例GCA组织中Syk基因启动子的甲基化发生率为31.9%(15/47),显著高于癌旁正常组织(0/47)(P<0.001);Ⅲ、Ⅳ期贲门癌患者中Syk基因甲基化发生率(35.7%)高于Ⅰ、Ⅱ期 (26.3%),低分化腺癌组的甲基化发生率(37.0%)高于高、中分化组 (25.0%),但差异均无统计学意义.47例GCA组织中Syk蛋白的表达率为17.0%(8/47),与相应癌旁正常组织的Syk蛋白表达率(74.4%,35/47)相比差异有统计学意义(P<0.001);Ⅲ、Ⅳ期患者的Sky蛋白表达率(10.7%)低于Ⅰ、Ⅱ期患者的蛋白表达率(26.3%),低分化腺癌组的蛋白表达率(11.1%)低于高、中分化腺癌组的蛋白表达率(25.0%),但差异均无统计学意义.结论:Syk基因启动子区甲基化导致的基因沉默可能是GCA发生的机制之一,可作为反映GCA生物学行为的检测指标.     

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。     

肝细胞癌ASC基因启动子区甲基化及其mRNA表达研究

目的:探讨肝细胞肝癌(HCC)ASC基因启动子区甲基化与mRNA表达及其临床病理特征的关系.方法:运用甲基化特异性聚合酶链反应(MSP)和实时荧光定量PCR技术,检测58例肝细胞癌及其相应癌旁组织、15例肝硬化肝组织、5例慢性病毒性肝炎肝组织和5例正常肝组织中ASC基因启动子区甲基化状态及其mRNA的表达水平.结果:58例肝细胞癌组织中有40例(69.0%)发生ASC基因启动子区甲基化,其相应癌旁组织中有27例(46.6%)发生该基因启动子区甲基化,而在肝硬化、肝炎和正常肝组织中均未检测到该基因启动子区甲基化.ASC基因在肝细胞癌组织中甲基化频率高于其相应癌旁组织(P=0.015).ASC基因启动子区甲基化与肿瘤直径(P=0.001)、生长方式(P=0.003)以及国际抗癌联盟第6版TNM(TNM6)分期(P=0.001)有关.以ASC基因在正常肝组织中的表达量为参照,58例肝细胞癌组织中有33例出现ASC基因mRNA低表达或表达缺失,其相应癌旁组织中有14例出现低表达或表达缺失,而在肝硬化及肝炎肝组织中ASC基因mRNA均呈正常表达.与癌旁组织相比,肝细胞癌组织中ASC基因mRNA表达明显下调(P<0.05).在发生ASC基因启动子区甲基化的40例肝细胞癌组织中有26例出现mRNA低表达.结论:ASC基因启动子区甲基化是肝细胞癌中的频发事件,可能在肝细胞癌的进展中起重要作用.     

非小细胞肺癌中FHIT基因异常甲基化对其蛋白、mRNA表达的影响

背景与目的 脆性组氨酸三联体(fragile histidine triad,FHIT)是一种新的抑癌基因,其启动子甲基化在肿瘤的发生和发展过程中发挥重要作用.本研究的目的 是通过检测非小细胞肺癌(non-small cell lung cancer,NSCLC)中抑癌基因FHIT启动子CpG岛异常甲基化和蛋白、转录表达情况及其相互关系,探讨FHIT基因在NSCLC发生发展中的作用.方法 应用甲基化特异性PCR(Methylation-specific PCR,MSP)、免疫印迹(Western Blot)及RT-PCR测定52例NSCLC组织及其癌旁正常组织(>5 cm)中FHIT的甲基化、蛋白表达和转录水平.结果 NSCLC癌组织中FHIT甲基化率为38.46%(20/52),在癌旁正常组织中FHIT甲基化率为7.6996(4/52),两者差异存在统计学意义(P<0.05);癌组织中FHIT蛋白表达率为28.8%(15/52),癌旁正常组织FHIT蛋白表达率为88.5%(46/52),两者差异存在统计学意义(P=0.000);FHIT mRNA在癌组织中表达率为51.9%(27/52),在癌旁正常组织中全部表达,且表达量明显高于癌组织(P=0.000).结论 在NSCLC中,FHIT基因启动子甲基化频率明显升高,其蛋白表达率明显下降,mRNA表达率下降,提示FHIT启动子甲基化在肺癌的发生和发展中起着一定作用,而且FHIT基因的甲基化与蛋白及mRNA的表达存在一定的关系.     

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.     

食管癌TβRⅠ和TβRⅡ基因甲基化及mRNA表达的相关性研究

目的:探讨食管鳞状细胞癌(ES-CC)组织中转化生长因子β受体Ⅰ、Ⅱ(TβRⅠ、Ⅱ)基因启动子甲基化及其mRNA表达的关系.方法:改进的甲基化特异性聚合酶链式反应(MSP)和逆转录-聚合酶链反应(RT-PCR)技术检测65例ESCC及59例癌旁组织中Tt3R Ⅰ和TI]RⅡ基因启动子的甲基化状态和41例ESCC及15例癌旁组织中TβR Ⅰ和TβRⅡ基因mRNA的表达水平,分析2个基因启动子甲基化与临床参数和mRNA表达的关系.结果:TβR Ⅰ在ES-CC和癌旁组组的甲基化率分别为52.3%(34/65)和40.7%(24/59),差异无统计学意义,χ2=1.680.P=0.1 95.TβRⅡ在ESCC和癌旁组组的甲基化率分别为70.8%(46/65)和47.5%(28/59),ESCC组的甲基化率显著高于癌旁组,γ2=6.984.P=0.008.TβR Ⅰ和TβRⅡ基因启动子甲基化与临床资料无关,P>0.05.ESCC组TβRⅠ和TβRⅡmRNA表达水平均明显低于癌旁组织(t1=-3.393,P1=0.001;t2=-2.358,P2=0.022).ESCC组中发生甲基化和未发生甲基化的mRNA表达水平差异无统计学意义,t1=-1.287,P1=0.206;t2=-0.922,P2=0.362.结论:TβR Ⅰ基因甲基化可能与ESCC的发生、发展无关,而TβR Ⅱ基因启动子甲基化可能是引起基因转录失活,导致ESCC形成的原因之一.     

肝细胞肝癌上皮型钙黏素基因表达和启动子甲基化研究

目的 探讨上皮型钙黏素(E-cad)基因表达和启动子CpG岛甲基化与肝细胞肝癌(HCC)的关系.方法 用甲基化特异性PCR技术检测36例HCC肿瘤组织及其癌旁非肿瘤组织中,E-cad基因启动子CpG岛甲基化状况,E-cad mRNA表达用RT-PCR技术检测.结果 癌旁组织中E-cad mRNA表达水平显著高于肿瘤组织(P<0.001),Ⅲ～Ⅳ期肿瘤组织中的表达水平显著低于Ⅰ～Ⅱ期肿瘤(P=0.025),较大肿瘤(>5 cm)组织中的表达水平显著低于较小肿瘤(P=0.035),包膜完整的肿瘤组织中E-cad mRNA表达水平显著高于包膜不完整的肿瘤(P=0.001);E-cad基因在HCC肿瘤组织中的甲基化率显著高于癌旁组织(P=0.035),在包膜完整的肿瘤中的甲基化率显著低于包膜不完整的肿瘤(P=0.015);E-cad甲基化阳性的肿瘤组织中,其mRNA表达水平显著低于E-cad非甲基化阳性的肿瘤(P<0.000).结论 E-cad基因表达下降可能与HCC进展相关,其启动子甲基化是该基因表达下降的重要原因之一.     

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。     

贲门腺癌中TSP1启动子区高甲基化与TGF-β1表达的相关性分析

目的:探讨贲门腺癌中凝血酶敏感蛋白1(thrombospondin1,TSP1)基因的甲基化状态及其与转化生长因子β1(transforming growth factor-β1,TGF-β1)蛋白表达之间的相关性.方法:分别应用甲基化特异性PCR(methylation-specific PCR,MSP)、RT-PCR和免疫组织化学法检测贲门腺癌组织及相应癌旁组织的TSP1基因甲基化、mRNA和蛋白表达情况,应用免疫组织化学法检测对应组织中TGF-β1蛋白表达情况.结果: 96例贲门腺癌组织中有34例发生了TSP1基因甲基化,甲基化发生率为35.4%,显著高于癌旁正常组织(P<0.001).Ⅲ期和Ⅳ期贲门腺癌患者中TSP1基因发生甲基化的比率显著高于Ⅰ期和Ⅱ期患者(P<0.05).贲门腺癌组织中TSP1 mRNA及蛋白表达显著低于癌旁正常组织(P<0.05),且与其甲基化状态之间有明显相关性.TGF-β1蛋白在贲门癌腺组织中的表达明显升高,96例贲门癌腺组织中有50例(52.1%)表达阳性,与相应癌旁正常组织相比差异有统计学意义(P<0.001);且随肿瘤分期的升高和肿瘤分化程度的降低,TGF-β1的阳性表达率明显升高(P<0.05).TSP1基因在贲门腺癌中的高甲基化与TGF-β1蛋白表达之间有明显的相关性(P<0.05).结论:TSP1基因启动子区高甲基化和TGF-β1蛋白过表达可能参与了贲门腺癌的发生、发展过程,TSP1基因启动子区发生甲基化导致的基因沉默可能是贲门腺癌发生的机制之一.     

胃癌组织中PCDH8基因甲基化及Dnmt1表达的关系

目的 探讨PCDH8（protocadherin 8）基因在人胃癌组织中的甲基化状态及其与甲基转移酶1（Dnmt1）表达之间的关系,分析其与胃癌发生、发展的关系。方法 取60例手术切除的胃癌组织及其相应癌旁正常胃黏膜组织（距癌组织5 cm以上）,以甲基化特异性聚合酶链式反应（methylationspecific PCR,MSP）检测60例胃癌组织及其癌旁正常胃黏膜组织中PCDH8基因启动子区甲基化的状态;以免疫组化SP法分别检测PCDH8基因蛋白及Dnmt1蛋白的表达水平。结果 胃癌组织中PCDH8甲基化率为55.0%（33/60）,癌旁正常胃黏膜组织中PCDH8的甲基化率为3.3%（2/60）,两者比较差异有统计学意义（χ2=38.76,P〈0.001）,PCDH8甲基化的发生率与患者年龄、性别及TNM分期等差异均无统计学意义（P均〉0.05）,而与有无淋巴结转移的差异有统计学意义（χ2=17.47,P〈0.001）。PCDH8蛋白在癌旁正常胃黏膜组织中的表达（90.0%）明显高于胃癌组织（11.7%）（χ2=73.65,P〈0.001）,Dnmt1蛋白在胃癌组织中的阳性表达率（73.3%）明显高于癌旁正常胃黏膜组织的表达率（5.0%）（χ2=58.79,P〈0.001）。胃癌组织中PCDH8蛋白与Dnmt1蛋白的表达呈负相关（r=-0.544,P〈0.001）。结论PCDH8基因甲基化及Dnmt1的高表达可能参与了胃癌的发生和发展。     

DCC promoter hypermethylation in esophageal squamous cell carcinoma

Deleted in Colorectal Cancer (DCC) is a putative tumor suppressor gene, whose loss has been implicated in colorectal tumorigenesis. Decreased or loss of DCC expression has been demonstrated in a number of human cancers, including esophageal cancer. In this study, we analyzed esophageal squamous cell carcinoma (ESCC) cell lines and primary ESCCs as well as normal esophageal tissues for DCC methylation by bisulfite sequencing, methylation-specific PCR (MSP) and/or quantitative methylation-specific PCR (qMSP). When a qMSP cut-off value for positivity was set to 1.0, DCC methylation was detected in 10 of 12 ESCC cell lines tested, 74% of primary ESCCs (n = 70), 0% of corresponding normal esophageal tissues (n = 20) and 0% of normal esophagus from healthy individuals (n = 19). DCC expression was undetectable in the majority of ESCC cell lines, and treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine reactivated gene expression. DCC overexpression suppressed colony formation in ESCC cell lines, suggesting that DCC may function as a tumor suppressor gene in the esophagus. However, DCC methylation was not associated with any clinical or pathologic parameters measured. We have demonstrated that DCC methylation is a frequent and cancer-specific event in primary ESCCs, suggesting that DCC and associated pathways may represent a new diagnostical therapeutic target.     

The expression of DCC protein in female breast cancer

Background. The deleted in colorectal cancer (DCC) gene has been shown to be frequently deleted or its expression reduced or absent in glioblastomas, colorectal, gastro-intestinal, pancreatic and prostatic tumors. In the present study, we investigated the expression of DCC in surgical specimen from 75 patients with primary breast cancer. Methods. The expression of the DCC, estrogen receptors (ER), and progesterone receptors (PR) was studied in 75 surgical specimens of primary breast cancer using an immunohistochemical method. To evaluate the outcomes of the breast cancer patients, we followed up the patients during minimum of 10 years. Results. Reduced or loss of expression of DCC was identified in 45 out of 75 samples. There were significant differences between cases without metastasis or local recurrences versus these with metastasis or local recurrences (p = 0.006), and between patients alive with no evidence of malignancy versus those with recurrence or dead of disease (p = 0.005). There were no significant differences between the DCC status and age, sex, tumor location, stage, grade, or proportion of patients who received adjuvant therapy. Conclusions. These findings suggest that a decrease in DCC expression may influence the prognosis of breast carcinoma patients.     

Decreased expression of DCC mRNA in human colorectal cancers

The mRNA expression levels of DCC gene, which is cloned from the deleted region of chromosome 18q in colorectal cancers and thought to be a tumor-suppressor gene, was evaluated in tissue specimens surgically resected from patients with colorectal cancer by RT-PCR. This method was chosen as the expression level of DCC mRNA is below the detectable level for Northern-blot analysis. Semi-quantitative measurements of DCC mRNA were performed based on a standard curve defined by serial dilution of DCC cDNA. As a result, the expression level of DCC mRNA was found to be lower in 17 out of 30 colorectal cancers than in adjacent non-cancerous tissues. Inclusion of smooth muscle in tissue specimens was observed to have little disturbing effect on comparisons between cancerous and non-cancerous regions. In addition, all 4 specimens of colorectal cancer with liver metastasis showed the decreased expression level of DCC mRNA, suggesting that functional loss of DCC in cancerous tissues may play an important role in metastatic events.     

大肠癌中DCC基因201密码子点突变的研究

目的：探寻结直肠癌缺陷基因（ＤＣＣ基因）２０１密码子在大肠癌中的突变规律。方法：采用等位基因特异性ＰＣＲＡＳ－ＰＧＲ结合ＳａｌⅠ酶切方法检测３５例大肠癌组织及配对的癌旁粘膜ＤＣＣ基因２０１密友子突变情况。结果：ＤＣＣ基因２０１密码子纯合突变率大肠癌（４０％）显高于癌旁粘膜（２．８％）,（Ｐ〈０．０５）。且与肿瘤侵袭深度、Ｄｕｋｅｓ分期相关,至少有１７例（４９％）大肠癌与相应癌旁粘膜相比增获一个密     

DCC (Deleted in Colorectal Cancer) Inactivation in Hematological Malignancies

DCC (Deleted in Colorectal Cancer) is a putative tumor suppressor gene located on chromosome band 18q21. Allelic deletions of one DCC locus have been found in more than 70% of colorectal carcinomas. Loss of DCC expression has been detected in 80% of all colorectal cancers and in many other types of tumor. DCC is expressed in normal bone marrow and peripheral lymphocytes, nevertheless DCC expression was absent or greatly reduced in 30% of acute leukemias and in 25% of Chronic Myelogenous Leukemias (CML). DCC encodes a transmembrane glycoprotein closely related to the adhesion molecules of the Neural Cell Adhesion Molecule (N-CAM) family. Glycoproteins of this family function like cell surface receptors and are involved in the regulation of many functions including cell recognition and cell differentiation. Highly specialized adhesion molecules participate in the regulation of hemopoiesis by mediating the interactions of hemopoietic cells with the components of the bone marrow microenvironment. Therefore, loss of DCC, as well as loss or alteration of other adhesion receptors, could contribute to leukemogenesis by impairing the interactions of the hemopoietic cells with the bone marrow microenvironment.     

Allele loss from 5q21 (APC/MCC) and 18q21 (DCC) and DCC mRNA expression in breast cancer.

Thirty-four primary, untreated sporadic breast cancers were examined for loss of heterozygosity (LOH) at tumour suppressor loci involved in colorectal cancer: APC/MCC at 5q21 and DCC at 18q21. LOH was identified in 28% informative patients at 5q21 and 31% at 18q21. LOH at 5q21 and 18q21 was compared with allele loss at 17p13 and concurrent LOH at two or more of the loci was noted in 24% of tumours. Expression of a 12 kb DCC mRNA was demonstrated in 14/34 (42%) of the cancers and in all five tumours with LOH at the DCC locus there was an additional 11 kb DCC mRNA. Abnormalities of three loci involved in colorectal cancer (5q21, 17p13 and 18q21) therefore also occur in sporadic breast cancer. The accumulation of such genetic abnormalities may confer a growth advantage important in the development of breast cancer.     

Role of the dependence receptor DCC in colorectal cancer pathogenesis.

More than a decade ago, the DCC (deleted in colorectal cancer) gene was proposed as a putative tumor suppressor gene. Data supporting this proposal included observations that one DCC allele was deleted in roughly 70% of colorectal cancers, some cancers had somatic mutations of the DCC gene, and DCC expression was often reduced or absent in colorectal cancer tissues and cell lines. Despite subsequent studies which have supported DCC's potential role as a tumor suppressor gene, the rarity of point mutations identified in DCC coding sequences, the lack of a tumor predisposition phenotype in mice heterozygous for DCC inactivating mutations, and the presence of other known and candidate tumor suppressor genes on chromosome 18q have raised questions about DCC's candidacy. Following its initial characterization, the DCC protein was identified as a transmembrane receptor for netrins, key factors in axon guidance in the developing nervous system. At first glance, the established role of DCC and netrin-1 during organization of the spinal cord could be viewed as a further challenge to the position that DCC inactivation might play a significant role in tumorigenesis. However, recent observations on DCC's functions in intracellular signaling have renewed interest in the potential contribution of DCC inactivation to cancer. In particular, data indicate that, when engaged by netrin ligands, DCC may activate downstream signaling pathways. Moreover, in settings where netrin is absent or at low levels, DCC can promote apoptosis. Here, we review DCC's candidacy as a tumor suppressor gene, with an emphasis on how recent molecular analyses of DCC have offered support for the notion that DCC may function as a tumor suppressor gene.