氯化镉诱发16HBE细胞恶性转化不同阶段P16基因甲基化研究

目的对氯化镉（CdCl2）诱发16HBE细胞系恶性转化不同阶段P16基因启动子区甲基化状况进行研究，探讨镉的表遗传致癌机制。方法从各组CdCl2恶性转化不同阶段及接种裸鼠成瘤的16HBE细胞中提取全基因组DNA，采取甲基化特异性PCR法（Methylation—specific PCR，MSP）检测该基因组P16基因启动子区的甲基化状况，与非转化的16HBE对照细胞进行比较，并用去甲基化因子5-Azac（5-Aza-2＇deoxycytidine）处理有异常甲基化的细胞。结果CdCl2恶性转化及接种裸鼠成瘤的16HBE细胞P16抑癌基因启动子区CpG岛存在异常高甲基化现象，且随着细胞恶性程度的增加，甲基化现象有明显升高的趋势，同时发现有异常高甲基化的细胞经去甲基化处理后甲基化现象消失。结论P16基因启动子区的高甲基化导致抑癌基因表达关闭，无法正常发挥细胞增殖周期对细胞分裂和生长的负调控，造成细胞周期失控而导致无限制地细胞增殖，这可能是氯化镉诱导16HBE细胞恶性转化及接种裸鼠成瘤的一种表遗传致癌作用。研究结果可部分解释镉化合物的细胞转化作用及其可能的表遗传致癌机制，以及进一步对甲基化的表型逆转和药物治疗研究提供了重要依据。     

脑胶质瘤组织NPAS2时钟基因启动子区甲基化与预后的关系

目的探讨脑胶质瘤组织NPAS2时钟基因启动子区甲基化与预后的关系。方法采用甲基化特异性PCR检测102例脑胶质瘤组织及其癌旁正常组织NPAS2时钟基因启动子区甲基化状态,并分析脑胶质瘤组织NPAS2时钟基因启动子区甲基化状态与患者临床特征、5年总生存期的关系。结果脑胶质瘤组织NPAS2时钟基因启动子区甲基化频率明显高于癌旁正常组织（P＜0.05）。WHO分级Ⅲ~Ⅳ级脑胶质瘤组织NPAS2时钟基因启动子区甲基化频率明显高于Ⅰ~Ⅱ级脑胶质瘤组织（P＜0.05）。肿瘤全切除与否（HR=0.585,95%CI：0.353~0.969,P＜0.05）、肿瘤直径（HR=1.970,95%CI：1.196~3.243,P＜0.05）、WHO分级（HR=1.786,95%CI：1.110~2.874,P＜0.05）、脑胶质瘤组织NPAS2时钟基因启动子区甲基化状态（HR=2.115,95%CI：1.223~3.656,P＜0.05）是影响患者5年总生存期的独立危险因素。结论NPAS2时钟基因启动子区甲基化与脑胶质瘤恶性程度有关,其甲基化修饰是脑胶质瘤患者长期预后不良的独立危险因素。     

胃癌中E-cad的表达及启动子甲基化的临床意义

目的探讨E-cad（上皮型钙黏素）在胃癌和癌旁组织中的表达及其启动子甲基化状态与胃癌发生、发展、转移和预后等生物学特性的关系。方法应用免疫组织化学SP法和MSP（甲基化特异PCR法）,检测38例癌旁组织和72例胃癌组织中E-cad的表达情况及其启动子甲基化状态。结果癌旁组织均表达E-cad,而胃癌组织E-cad阴性表达率为63.89%（46/72）,其表达缺失与胃癌的分化程度、临床分期、肿瘤浸润深度密切相关（P〈0.05）。在癌旁组织中无E-cad启动子甲基化,而胃癌组织E-cad启动子甲基化发生率为56.94%（41/72）。随着肿瘤的去分化,临床分期增高,肿瘤浸润深度增加和淋巴转移E-cad启动子甲基化率逐渐增加（P〈0.05）。结论E-cad表达缺失和启动子甲基化与胃癌的发生、发展及预后密切相关,启动子甲基化是E-cad表达缺失的重要原因。     

前列腺癌中EphA1基因表达的初步研究

目的:检测前列腺癌中EphA1基因转录及受体表达水平,探讨其表达异常的机制及其临床意义。方法:利用RT-PCR法检测雄激素依赖性前列腺癌细胞系LNCaP和雄激素非依赖性前列腺癌细胞系PC-3中EphA1基因mRNA表达情况,采用甲基化特异性PCR(MSP)方法进行甲基化状态分析;用免疫组化方法检测19例前列腺癌和22例良性前列腺组织标本中EphA1受体表达情况。结果:LNCaP和PC-3细胞中均未检测到EphA1基因转录;PC-3细胞中EphA1基因启动子区内CpG岛存在高甲基化现象,而LNCaP细胞则不存在;前列腺癌和良性前列腺增生组织中EphA1受体表达率分别为36.8%和45.5%,其差异无统计学意义(P>0.05)。结论:前列腺癌细胞LNCaP和PC-3中无明显EphA1基因表达,甲基化可能是导致该基因下调的机制之一。EphA1基因的甲基化在前列腺癌进展及由雄激素依赖向雄激素非依赖转化过程中可能发挥一定作用。     

宫颈癌p16基因甲基化研究新方法

目的建立SYBR GreenⅠ实时荧光定量PCR检测宫颈癌p16基因甲基化的方法，以期达到早期诊断宫颈癌。方法正常基因组DNA经修饰后进行PCR，构建含有DNA甲基化β-aetin的重组质粒，克隆阳性质粒作为标准品用SYBR GreenⅠ实时荧光定量PCR进行检测，建立检测的标准曲线。利用建立好的方法进行检测临床60例宫颈癌组织中p16基因启动子区域5’CpG岛甲基化状态。结果结果显示正常对照组织及癌旁p16基因无甲基化，60例宫颈癌标本的甲基化率为28．33％（17／60）。结论SYBR GreenⅠ实时荧光定量PCR用于p16基因甲基化检测为宫颈癌的早期诊断提供了一种有效的方法。     

Ras相关区域家族1A基因甲基化与恶性肿瘤关系的研究进展

Ras相关区域家族1A(RASSF1A)基因是新近发现的新型候选抑癌基因,其正常表达能抑制肿瘤的发生。启动子区域CpG岛异常甲基化可以导致其失活,并在肿瘤的发生、发展中起着重要作用。RASSF1A的甲基化状态检测具有重要的临床意义,有望为肿瘤的早期诊断提供新的思路,而逆转RASSF1A的甲基化则可能成为肿瘤治疗的新方向。     

Methylation of TIMP3 in esophageal squamous cell carcinoma

AIM： To measure the frequency of DNA methylation of the tissue inhibitor of metalloproteinase 3 （TIMP3） promoter and relate this to any change of gene expression in esophageal squamous cell carcinoma in patients from a region of high incidence in China. METHODS： Cancer cell lines were treated with or without the demethylating reagent 5-aza-2′-deoxycytidine. Methylation of the TIMP3 promoter was assessed in three regions by melt curve analysis and its expression was assessed by real-time RT-PCR. Tumors and proximal resection margins were obtained from 64 patients with esophageal squamous cell carcinoma from a region of high incidence in China. Methylation was assessed by melt curve analysis and expression by immunohistochemistry.
RESULTS： Methylation in one of the three promoter regions assessed correlated with gene silencing in esophageal cell lines. A degree of methylation of TIMP3 was found in only four esophageal squamous cell carcinomas, and partial loss of TIMP3 protein expression in just one.
CONCLUSION： Methylation and loss of expression of TIMP3 occurs infrequently in esophageal squamous cell carcinoma in a region of high incidence in China.     

Blood-based DNA methylation of DNA repair genes in the non-homologous end-joining (NEHJ) pathway in patient with glioma

To investigate the blood-based DNA methylation of repair genes including LIG4, XRCC4, XRCC5, XRCC6 and XRCC7 that involved in non-homologous end-joining (NEHJ) DNA repair pathway in patients with glioma. Blood samples were obtained from 114 glioma patients, 96 normal controls, and 81 glioma patients after radiotherapy and chemotherapy. Blood-based DNA methylation of the five NHEJ repair genes was assayed by methylation-specific polymerase chain reaction (MSP). The DNA methylation level of XRCC5 and XRCC7 in glioma group are significantly higher than those of normal group (P<0.001). Moreover, radiotherapy treatment significantly increased methylation level of XRCC5 and XRCC7 compared to glioma group. No significant difference for the methylation of the other three genes, LIG4, XRCC4 and XRCC6 were detected among three groups. In conclusion: our findings indicate that DNA methylation modification plays an important role to regulate the gene expression of XRCC5 and XRCC7, from the results that the gene methylation level of the glioma group is higher than that of the normal group. Increased methylation of XRCC5 and XRCC7 in blood samples of glioma patients and patients with radiotherapy and chemotherapy suggests that blood-based methylation level of XRCC5 and XRCC7 could be a potential indicator for evaluating of the effect of radiotherapy and chemotherapy for glioma patient.