食管癌前病变组织p16及FHIT基因甲基化探讨

目的:探讨食管癌前病变组织p16及FHIT基因启动子区CpG岛的甲基化状况.方法:采用甲基化特异性聚合酶链反应(MSP)方法,对食管癌前病变不同阶段及鳞状细胞原位癌的病变组织进行了甲基化检测,并与同一个体相应的病变旁组织及慢性食管炎和浸润性鳞癌组织的甲基化情况进行了时比分析研究.结果:轻度、中重度不典型增生、鳞状细胞原位癌和浸润癌共95例病变组织中p16基因的甲基化频率分别为22.73%、59.09%、78.57%、64.86%:FHIT基因的甲基化频率分别为22.73%、45.45%、64.29%、67.57%.同一个体相应的95例病变旁对照组织中16例未检测成功,余79例中5例(6.33%)p16基因甲基化,3例(3.80%)FHIT基因甲基化,与病变组织相比,均有统计学差异.10例慢性食管炎组织中1例p16基因甲基化,未发现FHIT基因甲基化.结论:p16及FHIT基因甲基化在癌前病变阶段就已经存在,可能是食管癌发生的早期事件之一.     

食管鳞状细胞癌及癌前病变组织中p16基因甲基化的研究

目的探讨食管鳞状细胞癌及癌前病变组织p16基因CpG岛的甲基化情况,及其在食管癌发生和早期诊断中的价值。方法采用MSP方法,对食管癌前病变不同阶段、鳞状细胞原位癌和浸润癌的病变组织进行了甲基化检测,并与其相应的正常组织和慢性食管炎组织的甲基化情况进行对比分析。结果轻、中、重度不典型增生、鳞状细胞原位癌和浸润癌的甲基化频率分别为22.73%(5/22)、46.15%(6/13)、77.78%(7/9)、78.57%(11/14)和64.86%(24/37)。67例正常对照组织中3例(4.48%)p16基因甲基化,与病变组织相比,差异有统计学意义,P=0.000。10例慢性食管炎组织中1例(10%)p16基因甲基化。结论p16基因甲基化可能是食管癌发生的最早期事件之一。     

食管鳞状细胞癌及癌前病变组织中p16基因甲基化的研究

目的：探讨食管鳞状细胞癌及癌前病变纽织p16基因CpG岛的甲基化情况．及其在食管癌发生和早期诊断中的价值。方法：采用MSP方法，对食管癌前病变不同阶段、鳞状细胞原位癌和浸润癌的病变组织进行了甲基化检测．并与其相应的正常组织和慢性食菅炎组织的甲基化情况进行对比分析。结果：轻、中、重度不典型增生、鳞状细胞原位癌和浸润癌的甲基化频率分别为22．73％(5/27)、46．15％(6/13)、77．78％(7/9)、78．57％(11/14)和64．86％(24/37)。67例正常对照组织中3例(4．48％)p16基因甲基化，与病变组织相比，差异有统计学意义，P=0．000。10例慢性食管炎组织中1例(10％)p16基因甲基化。结论：p16基因甲基化可能是食管癌发生的最早期事件之一。     

Methylation of the FHIT gene in esophageal carcinoma in Kazakh and Han and relationship with prognosis

目的:探讨和比较新疆哈萨克族和汉族食管癌患者FHIT基因甲基化状态及其与食管癌发生及预后之间的关系.方法:采用甲基化特异性PCR法(methylation-specific polymerase chain reaction,MSP)测定哈、汉两民族共71例食管癌及癌旁组织标本FHIT基因启动子区甲基化状况,建立Cox回归模型分析临床病理指标及FHIT基因甲基化水平等因素与预后的关系.结果:36例哈族食管癌患者癌组织和癌旁组织的FHI7基因启动子甲基化率分别为58.3％和19.4％,35例汉族食管癌患者癌组织和癌旁组织的FHIT因启动子甲基化率分别为51.4％和14.3％,两民族癌组织FHIT基因的甲基化率均明显高于癌旁组织(P＜0.01);哈、汉民族食管癌患者癌组织之间、癌旁组织之间FHIT因启动子甲基化率无显著性差异(P＞0.05).预后因素分析表明肿瘤浸润程度、TNM分期、淋巴结转移区域数是影响哈、汉民族食管癌预后的独立危险因素,女性性别是保护因素.结论:FHIT基因启动子区甲基化状态与新疆哈族、汉族食管癌的发生有密切的关系,但可能与食管癌预后无关.     

新疆哈萨克族和汉族食管癌患者FHIT基因甲基化及其与预后的关系

目的：探讨和比较新疆哈萨克族和汉族食管癌患者FHIT基因甲基化状态及其与食管癌发生及预后之间的关系。方法：采用甲基化特异性PCR法（methvlation-specific polymerase chain reaction,MSF）测定哈、汉两民族共71例食管癌及癌旁组织标本FHIT基因启动子区甲基化状况,建立Cox回归模型分析临床病理指标及FHIT基因甲基化水平等因素与预后的关系。结果：36例哈族食管癌患者癌组织和癌旁组织的FHIT基因启动子甲基化率分别为58.3%和19.4%,35例汉族食管癌患者癌组织和癌旁组织的FHIT基因启动子甲基化率分别为51.4%和14.3%,两民族癌组织FHIT基因的甲基化率均明显高于癌旁组织（P〈0.01）;哈、汉民族食管癌患者癌组织之间、癌旁组织之间FHIT基因启动子甲基化率无显著性差异（P〉0.05）。预后因素分析表明肿瘤浸润程度、TNM分期、淋巴结转移区域数是影响哈、汉民族食管癌预后的独立危险因素,女性性别是保护因素。结论：FHIT基因启动子区甲基化状态与新疆哈族、汉族食管癌的发生有密切的关系,但可能与食管癌预后无关。     

食管鳞癌中p16基因启动子区甲基化及其表达

 目的探讨食管鳞癌（ESCC）p16基因甲基化的状况及其表达与食管鳞癌临床病理特征之间的关系。方法采用甲基化特异性PCR方法（MSP）分别检测75例食管癌组织、癌旁组织和切缘组织p16基因启动子区域CpG岛甲基化状态。采用Envision免疫组化法检测食管癌组织及癌旁组织的p16蛋白的表达。结果75例标本中，食管癌组织、癌旁组织和切缘细织p16基因甲基化率分别为41．3％（31／75）、13．3％（10／75）和6．67％（5／75）。癌组织和癌旁组织P16蛋白的阳性表达率分别为29．3％（22／75）和56．7％0（17／30）。31例癌组织p16基因甲基化阳性标本中有2例（6．4％）检测到P16蛋白的表达，而44例癌组织p16基因甲基化阴性标本中有20例（45．5％）检测到P16蛋白的表达。食管癌组织p16基因甲基化率显著高于癌旁组织和切缘组织（P〈0.01），P16蛋白表达与p16基因甲基化呈负相关。p16基因启动子区甲基化与食管癌的组织学分级、肿瘤部位无明显相关，与临床分期、淋巴转移密切相关。结论p16基因甲基化在食管癌发生发展中起着重要作用，食管鳞癌的分期和淋巴结转移与p16基因甲基化之间有密切关系。     

FHIT和p16基因甲基化与肺癌的发生

背景与目的:探讨FHIT和p16基因甲基化与肺癌发生之间的关系. 材料与方法:采用甲基化特异性PCR检测59例原发性肺癌组织,相对应的32例正常组织及11例支气管鳞状化生组织中FHIT基因、p16基因启动子区CpG岛甲基化状况.结果:肺癌组织和正常肺组织中的FHIT基因甲基化率分别为37.3%(22/59)、0.0%(0/32),两组间的差异有统计学意义(P<0.01);支气管鳞状化生组织FHIT基因甲基化阳性率为18.1%(2/11).肺癌组织和正常肺组织中的p16基因甲基化率分别为50.8%(30/59)、0.0%(0/32),两组间的差异具有统计学意义(P<0.01);支气管鳞状化生组织p16基因甲基化阳性率为18.1%(2/11).肺癌患者吸烟组中FHIT、p16基因甲基化联合检测的阳性率为90.6%(29/32),与非吸烟组(33.3%.9/27)相比较,其差异具有统计学意义(P<0.05).结论: FHIT基因和p16基因启动子区甲基化可能与肺癌的发生有关.     

高发区食管癌患者p16基因甲基化及其表达的比较研究

目的:比较p16基因甲基化在食管癌高发区河南林州(北方组)和广东揭阳(南方组)之间的异同,探讨p16基因甲基化在不同气候环境条件下的两地食管鳞癌(ESCC)发生中的作用。方法:采用甲基化特异性PCR方法(MSP)分别检测两地食管癌组织、癌旁组织和切缘组织p16基因启动子区域CpG岛甲基化状态。采用EnVision免疫组化法检测两地食管癌组织及癌旁组织的p16蛋白的表达。结果:南方组75例标本中,食管癌组织、癌旁组织和切缘组织p16基因甲基化率分别为41.3%(31/75)、13.3%(10/75)和6.67%(5/75);癌组织和癌旁组织p16蛋白的阳性表达率分别为29.3%(22/75)和56.7%(17/30);31例p16基因甲基化阳性标本中有2例(6.4%)检测到p16蛋白的表达,而44例p16基因甲基化阴性标本中有20例(45.5%)检测到p16蛋白的表达。北方组65例标本中,食管癌组织、癌旁组织、切缘组织p16基因甲基化率分别为52.3%(34/65)、16.9%(11/65)和7.69%(5/65);食管癌组织和癌旁组织p16蛋白的阳性表达率分别为32.3%(21/65)和66.7%(20/30);34例p16基因甲基化阳性标本中有4例(11.8%)检测到p16蛋白的表达,而31例p16基因甲基化阴性标本中有17例(54.8%)检测到p16基因的表达。两地组内癌组织p16甲基化率均显著高于癌旁组织和切缘组织,p16蛋白表达与p16基因甲基化呈负相关(P<0.01)。两地同类组织比较p16甲基化率和p16蛋白表达率均无显著性差异(P>0.05)。结论:p16基因异常甲基化后功能失活可能是南、北两地食管癌癌变过程的重要事件,在我国南北环境气候条件不同的两地高发区食管癌的发生中均起着重要的作用。本研究为环境因素和p16基因功能之间的生物学关联在食管癌的发生中提供了一定的证据。     

FHIT和p16基因甲基化与胃癌的关系研究

目的探讨FHIT和p16基因甲基化在胃癌发生发展中的作用。方法应用甲基化特异性PCR（MSP）,检测胃癌组织和正常胃黏膜组织中FHIT基因和p16基因的启动子CpG岛的甲基化状态。结果胃癌组织中FHIT和p16基因甲基化阳性率分别为51.4%和56.8%,两者的甲基化均与胃癌患者年龄、性别、Lauren分型、Borrmann分型、淋巴结转移以及TNM分期无关（P〉0.05）,且两者的甲基化不存在明显正相关关系（P〉0.05,γ=0.17）。结论 FHIT基因和p16基因的甲基化修饰在胃癌发生发展中均起重要作用,两者可能是胃癌发生的早期事件。     

DNA methylation and loss of protein expression in esophageal squamous cell carcinogenesis of high-risk area

DNA methylation is an important mechanism for gene silence. The purpose of this study was to investigate aberrant promoter methylation of the p16 and FHIT genes in tissues and plasma and loss of protein expression in esophageal precancerous conditions (EPC) and esophageal squamous cell carcinoma (ESCC) of high-risk area. Methylation-specific PCR(MSP) was employed to examine the DNA methylation in the plasma and tissues of 95 patients of EPC, ESCC and 10 chronic esophagitis (CE). Loss of protein expression of p16 and FHIT was detected immunohistochemically. In total 95 lesion tissues of EPC and ESCC, p16 methylation was found in 53 (55.79%) cases, and 41 of 53 (77.36%) cases were demonstrated deletion of the p16 protein immunohistochemically. FHIT methylation was found in 49 (51.58%) cases, and 40 of 49 (81.63%) were demonstrated deletion of the FHIT protein. Only 1 (10%) case of 10 CE p16 methylation was found in the tissues. In the plasma of total 105 samples, 2 of 23 high grade intraepithelial neoplasia (HGIN) and 12 of 37 ESCC were detected p16 methylation, and 2 of 23 HGIN and 14 of 37 ESCC were detected FHIT methylation. These results indicate that p16 and FHIT methylation may be one of the earliest events and an important mechanism for gene silencing in esophageal squamous cell carcinogenesis. This study may be helpful for screening the candidate molecular markers for early diagnosis of ESCC in high-risk area.     

Aberrant methylation of the FHIT gene in chronic smokers with early stage squamous cell carcinoma of the lung

Fragile histidine triad (FHIT) gene plays an important role in the pathogenesis of lung cancer. However, the clinicopathological significance of CpG island hypermethylation of FHIT gene in non-small cell lung cancer (NSCLC) remains to be elucidated. We studied FHIT methylation in 254 NSCLCs in order to further understand the clinicopathological and prognostic significance of FHIT methylation in NSCLC. Methylation status of the FHIT gene was examined using Methylation-Specific PCR. All statistical analyses were two-sided, with a 5% type I error rate. Hypermethylation of the FHIT gene occurred more frequently in squamous cell carcinoma than adenocarcinoma. For 93 adenocarcinomas there was no statistically significant association between FHIT methylation and age, gender, smoking history, pathologic stage and p16 methylation. However, FHIT methylation in 125 squamous cell carcinomas was associated with exposure to tobacco smoke and p16 methylation, but not with age, gender and pathologic stage. Hypermethylation of FHIT in squamous cell carcinomas occurred more frequently in current smokers (45%) than in never-smokers (13%). FHIT methylation was significantly associated with p16 methylation in current- and ex-smokers (P = 0.02 and P = 0.01, respectively) with squamous cell carcinoma and in patients with pathologic stage I squamous cell carcinoma (P = 0.001). Patients with p16 methylation were 3.74 times [95% confidence interval (CI) = 1.62 - 7.95; P = 0.001] more likely to have FHIT methylation in squamous cell carcinoma. FHIT methylation in squamous cell carcinoma occurred at a 4.62 times (95% CI = 1.26 - 34.97; P = 0.02) higher prevalence in current smokers than in never-smokers. No prognostic effect of FHIT methylation was observed in stage I and stage II NSCLCs. In conclusion, hypermethylation of the FHIT gene did not have a prognostic significance in early stage NSCLCs. The FHIT methylation is associated with the p16 methylation and smoking in squamous cell carcinoma, suggesting that FHIT may cooperate with p16 for the development of squamous cell carcinoma of lung in individuals exposed to tobacco smoke.     

Frequent methylation-associated silencing of a candidate tumor-suppressor, CRABP1, in esophageal squamous-cell carcinoma

Epigenetic alterations and the resulting inactivation of tumor suppressor genes often contribute to the development of various cancers. To identify novel candidates that may be silenced by aberrant methylation in esophageal squamous-cell carcinoma (ESCC), we analysed ESCC cell lines by a recently developed method known as bacterial artificial chromosome array-based methylated CpG island amplification (BAMCA), and selected candidates through BAMCA-assisted strategy. In the course of this program, we identified frequent CpG methylation-dependent silencing of the gene encoding cellular retinoic acid binding protein 1 (CRABP1) in our panel of ESCC cell lines. Expression of CRABP1 mRNA was restored in gene-silenced ESCC cells after treatment with 5-aza 2'-deoxycytidine. The DNA methylation status of the CRABP1 CpG island with clear promoter activity correlated inversely with expression of this gene. CpG methylation of CRABP1 was frequently observed in primary ESCC tissues as well. Restoration of CRABP1 expression in ESCC cells lacking the protein reduced cell growth by inducing arrest at G(0)-G(1), whereas knockdown of the gene in cells expressing CRABP1 promoted cell growth. Among 113 primary ESCC tumors, the absence of immunoreactive CRABP1 was significantly associated with de-differentiation of cancer cells and with distant lymph-node metastases in the patients. These results indicate that CRABP1 appears to have a tumor-suppressor function in esophageal epithelium, and its epigenetic silencing may play a pivotal role during esophageal carcinogenesis. Its expression status in biopsies or resected tumors might serve as an index for identifying ESCC patients for whom combined therapeutic modalities would be recommended.     

Aberrant DNA methylation of P16, MGMT, and hMLH1 genes in combination with MTHFR C677T genetic polymorphism in esophageal squamous cell carcinoma.

To explore the role of aberrant hypermethylation of cancer-related genes, such as P16, MGMT, and hMLH1, in the esophageal squamous cell carcinoma (ESCC) as well as its relation to dietary folate intake and MTHFR C677T polymorphism, we conducted a molecular epidemiologic study in China. One hundred and twenty-five histologically confirmed ESCC patients having undergone surgery in the Yangzhong People's Hospital between January 2005 and March 2006 were recruited. The aberrant CpG island hypermethylation of P16, MGMT, and hMLH1 genes could be found in cancer tissues with frequency of about 88.0%, 27.2%, and 3.2%, respectively, and in remote normal-appearing esophageal tissues with frequency of about 36.8%, 11.2%, and 0.0%, respectively. No hypermethylation was found in the normal esophageal tissues from healthy controls. Compared with those patients without lymph node metastasis, MGMT gene showed a higher proportion of hypermethylation in cancer tissues, whereas P16 gene showed a higher proportion of hypermethylation in remote normal-appearing esophageal tissues in patients with lymph node metastasis. A significant association was found between MTHFR C677T genetic polymorphism and CpG island methylation status of MGMT gene. After adjustment for potential confounders, individuals carrying CT or TT genotype have higher frequency of hypermethylation in MGMT gene in cancer tissues, with odds ratio of 3.34 (95% confidence interval, 1.07-10.39) and 3.83 (95% confidence interval, 1.13-12.94), respectively. This study indicated that the aberrant CpG island hypermethylation of cancer-related genes was associated with ESCC and might be a promising biomarker in diagnosis and prognosis.     

子宫内膜癌中p16基因缺失及CpG岛甲基化的研究

目的　研究子宫内膜癌中 p16基因缺失及CpG岛甲基化情况。 方法　用PCR技术检测 p16基因在子宫内膜癌中的纯合性缺失及第一外显子异常甲基化。结果　 36例癌组织中 9例发生基因缺失 ,12例发生甲基化 ,未发现基因缺失与甲基化同时存在的病例。结论　 1.p16基因缺失与子宫内膜癌组织学分级严重程度和临床分期呈正相关 ;2 .5’CpG甲基化可能是 p16基因失活的重要机理 ,参与子宫内膜癌发生、发展。     

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。     

肾癌中p16基因状态的研究

 目的　研究肾癌中 p16基因状态与肾癌的关系。 方法　用PCR技术检测MTS1/ p16基因在肾癌中的外显子缺失及第一外显子异常甲基化。结果　 33例癌组织中 7例发生基因缺失 ,10例发生甲基化 ,未发现基因缺失与甲基化同时存在的病例 ,p16基因缺失与肾癌组织学分级严重程度和临床分期呈正相关 ;甲基化集中在分期早 ,分化好的病例。结论　 1.p16基因缺失是晚期事件 ,与预后有关 ;2 .p16基因甲基化可能是肾癌发生的早期事件