【作者】 廖完敏；

【导师】 汪道文；

【作者基本信息】 华中科技大学 ， 内科学， 2011， 博士

【摘要】 肺癌是世界上最常见的癌症死亡原因之一。尽管近年来在诊断技术和治疗方法上均有很大的提高,但是肺癌的预后仍然不佳,原因是三分之二以上的病人在确诊时已经发生了微小转移。因此,探索有效的早期诊断肺癌的方法,争取在外科手术治愈前尽早诊断是当务之急。一种行之有效的方法是,找到肺癌特异性分子标记物,尤其是在最早期阶段就能检测到的与肺癌发生和进展相关的特异性标记物。迄今为止,传统的放射线及痰液细胞学检查对长期吸烟者进行早期筛查并没有降低肺癌死亡率[44,45]。表观遗传学是推动肿瘤发生发展的关键因素之一,而甲基化是最常见的表观遗传改变。近年来的研究表明,基因甲基化是许多类型肿瘤,包括肺癌,发生发展中的早期事件。肺癌中多种基因频繁地发生甲基化,包括肿瘤抑制基因P16、DNA修复基因MGMT、粘附分子E-cadherin及RARp等与肿瘤相关的基因。与遗传学改变不同的是,表观遗传学改变是一种可逆的过程,特异性DNA甲基化转移酶抑制剂5-Aza-dc能使基因去甲基化而激活沉默的基因。CYP2J2基因在肿瘤(包括肺癌)的发生发展中起着非常重要的作用[40-42]。目前,很多研究集中在CYP2J2对病理生理作用上,如CYP2J2在肿瘤、心血管疾病及药物和异物代谢方面的作用[43],而表达调控面的研究尚未见报道。阐明表达调控的机制,有利于肿瘤CYP2J2基因的早期诊断及靶向治疗,因此,对其表达调控的机制进行深入研究非常有必要。遗传学和表观遗传学在调节基因表达方面均起着至关重要的作用,本研究从表观遗传学角度,研究DNA甲基化在CYP2J2基因表达调控中的作用。本研究对CYP2J2基因在肺癌中的甲基化情况进行了探索,我们发现CYP2J2基因在肺癌中处于高甲基化,证实了CYP2J2基因甲基化部分调节其表达,明确了CYP2J2基因甲基化水平与性别有关,更有趣的是CYP2J2基因甲基化与非小细胞肺癌患者预后有关。首先,我们用MSP方法检测了三种肺癌细胞(A549、NCI-H446和NCI-H460)中CYP2J2基因甲基化状态,发现三种肺癌细胞中CYP2J2基因启动子区均发生了甲基化。接着,用去甲基化药物5-Aza-dC干预上述三种肺癌细胞系,用real-time PCR及MSP方法检测干预前后CYP2J2 mRNA表达和启动子区甲基化变化。结果显示：A549和NCI-H460细胞CYP2J2 mRNA表达量较处理前增多,而NCI-H446细胞的表达量无明显改变；而A549和NCI-H460细胞甲基化M带密度较处理前变浅,U带密度较处理前增强,NCI-H446细胞甲基化在处理前后无明显的改变。这些结果表明,CYP2J2基因甲基化部分调节其表达。为了进一步研究在体肿瘤CYP2J2基因甲基化情况,我们用MSP方法检测了56对肺癌组织和癌旁正常组织,及10例非肿瘤患者正常肺组织中CYP2J2基因甲基化。结果显示,56例肺癌中,44例(78.6%)完全甲基化；7例(12.5%)部分甲基化,5例(8.9%)未甲基化；56例配对癌旁正常肺组织中28例(50%)完全甲基化,10例(17.9%)部分甲基化,18例(32.1%)未甲基化；而在10例非肿瘤患者肺组织中无一例甲基化。癌旁正常组织中检测到甲基化,提示1.甲基化发生在肿瘤发生的早期阶段；2.甲基化的发生早于病理形态的改变；3.甲基化可能是肿瘤细胞恶性转化过程中的一部分。在癌组织以及癌旁正常组织中发现CYP2J2基因甲基化,但是对照组中无1例发生甲基化,提示CYP2J2基因甲基化可能是肿瘤的特异性改变。因此,CYP2J2基因甲基化可能作为肿瘤早期诊断的标志。CYP2J2甲基化是否是临床病理因子之间存在相关性分析表明CYP2J2甲基化与肺癌病人年龄、肿瘤病理类型、分期、吸烟及生存率之间的没有明显的相关性,但是男性患者的CYP2J2甲基化频率较女性患者高；另外,CYP2J2基因甲基化与全体肺癌患者(包括小细胞肺癌和非小细胞肺癌)整体生存率之间没有明显的关系,但是对于非小细胞肺癌患者而言,CYP2J2基因高甲基化的两年生存率明显高于CYP2J2基因低甲基化或者未发生甲基化的患者。本研究表明CYP2J2是一种新发现的,在肺癌中高度甲基化的基因。CYP2J2在三种肺癌细胞、56例肺癌组织及相应的癌旁正常组织中均有明显的甲基化,但是在非肿瘤患者的肺组织中几乎没有甲基化；用去甲基化药物5-Aza-dC处理肺癌细胞可以通过部分逆转CYP2J2基因的甲基化状态从而调节CYP2J2基因的表达；通过对CYP2J2甲基化与临床病理因素之间的相关性分析发现女性患者的甲基化频率低于男性患者,而且CYP2J2基因甲基化是非小细胞肺癌患者预后的强力预测因子。综上,我们对CYP2J2基因在肺癌中的甲基化情况进行了比较深入的研究,初步揭示了肺癌中CYP2J2基因频繁的甲基化,并且该基因的甲基化特异性发生于肿瘤相关组织,提示CYP2J2基因甲基化可能与肺癌的发生密切相关,CYP2J2基因甲基化可能成为肺癌早期诊断的标志物,为肺癌的发病机制提供了理论依据,对临床上肺癌的防治具有积极的指导作用。更多还原

【Abstract】 Lung carcinoma is one of the most common causes of cancer deaths in the world. Despite much progress in the treatment and detection methods of lung cancer, the prognosis remains poor. This situation results largely from micrometastasis, which is present in greater than two-thirds of patients at the time of diagnosis. Therefore, it clearly is imperative that efficient diagnostic methods be developed to detect lung carcinoma at the earliest stages, during which curative surgical resection remains feasible. One promising approach to early detection involves the identification of lung carcinoma-specific molecular biomarkers, especially those associated with the initiation and progression of lung carcinoma in its earliest stage. Screening tests on long-term smokers used to date (radiography and sputum cytology) have failed to reduce lung carcinoma mortality.Epigenetic events are a critical force driving initiation and progression of cancer. Methylation is the most common epigenetic change. Recent studies show that silencing of tumor-related genes, resulting from epigenetic alterations, are an early event in many human malignancies, including non-small cell lung cancer (NSCLC). In lung cancer, several sets of genes including the tumor suppressor gene p16, the DNA repair gene O6-methylguanine-DNAmethyltransferase (MGMT), E-cadherin and retinoic acid receptor beta have been shown to be frequentlymethylated and inactivated. Epigenetic alterations in cancer, as opposed to genetic lesions, are potentially reversible. The DNA methylation inhibitor 5-aza-2-deoxycytidine (5-Aza-dC) can reactivate the expression of genes by demethylating.CYP2J2 gene plays an important role in tumor development including lung cancer. Currently, many studies focused on the metabolism of CYP2J2 on the drugs foreign. However, regulation of expression has not been reported. This study investigated methylation of CYP2J2 gene in lung cancer. We found that aberrant promoter hypermethylation of CYP2J2 gene in lung cancer, and promoter methylation of CYP2J2 gene partially regulated mRNA and protein expression. We confirm the relationship of sex and the methylation level of CYP2J2 gene promoter.We went first to evaluate CYP2J2 promoter methylation status by methylation-specific PCR in 3 lung cancer cell lines (A549, NCI-H446 and NCI-H460). They all displayed a clear methylated band. Then we treated lung cancer cell lines with the demethylating agent 5-aza-dC. Treatment with 5-Aza-dC increased the expression of CYP2J2 mRNA in both A549 and NCI-H460 cell lines. There is no significant change before and after treatment in NCI-H446. MSP revealed a reduction of the intensity of the band corresponding to methylated DNA in comparison to untreated cell lines, which was paralleled by an increase of amplification products when primers for unmethylated DNA were used. These results support the conclusion that CpG methylation of the CYP2J2 gene regulated expression in part.To elucidate methylation of CYP2J2 gene in vivo, CYP2J2 methylation was analyzed in normal and matching tumor tissue from 56 patients with lung cancer, and a control group of 10 patients without cancer using a methylation-specific PCR. CYP2J2 promoter hypermethylation was detectable by MSP in 44 of 56 (78.6%) tumor samples,28 of 56 (50%) matching normal lung of lung cancer patients, and none of 10 (0%) normal lung specimens of the control group without lung cancer. Indeed, the observation that methylated CYP2J2 was only present in noncancerous tissues when there was detectable methylation of CYP2J2 in tumor tissue and the lack of any CYP2J2 methylation in normal lung tissue from patients without lung cancer suggest that this methylation event is part of the process of malignant transformation, and, thus, a tumor-specific phenomenon in lung cancer. It is possible that this noncancerous tissue, although it appeared to be histologically normal, is abnormal because of environmental factors such as exposure to cigarette smoke.To further understand the correlation between CYP2J2 methylation and clinicopathological factors, we initially analyzed associations between the methylation of CYP2J2 gene and available clinical information including smoking status sex, age, tumor stage, and histologic subtype of the tumor. No association was found between methylation levels of CYP2J2 gene and most of the risk factors, except for sex. Lung cancers from men exhibited higher methylation levels of CYP2J2 than those from women.As noted above, we identified novel gene frequently methylated in lung cancer. Promoter hypermethylation of CYP2J2 gene was detected in three kinds of lung cancer cell lines, primary lung tumors, and the surrounding normal tissues, whereas hypermethylation of CYP2J2 gene was rarely found in normal sample without cancer patients. Promoter methylation of CYP2J2 gene partially regulated mRNA and protein expression. And, lung cancers from men exhibited higher methylation levels of CYP2J2 than those from women.In summary, we have systemically investigated the methylation of CYP2J2 gene in lung cancer. We propose that CYP2J2 hypermethylation is present in the lung of most patients with lung carcinoma, suggesting that this molecular alteration may be involved in the process of lung carcinogenesis, and that it is a potential target for diagnosis and treatment of lung carcinoma. Moreover, this study provides evidence of sex-specific differences in methylation patterns in lung cancers. And thus, exemplifies the mechanism by which environmental factors may interact with key genes involved in tumorigeness.更多还原