【作者】 陈佩林；

【导师】 倪晓华；

【作者基本信息】 华东师范大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 肺癌是世界上最常见的肿瘤之一,在所有的恶性肿瘤中,肺癌的死亡率最高。吸烟引起的DNA损伤在肺癌的发生过程中起重要的作用。香烟中的多环芳烃、芳族胺、亚硝胺被认为是引起机体形成DNA加合物的主要致癌物,积累的DNA加合物能增加DNA损伤的可能性,损伤的DNA会阻止DNA的复制和基因的转录,导致基因突变和肿癌的发生。NER(Nucleotide excision repair)是最通常的DNA修复途径,是修复吸烟产生的DNA加合物的主要途径。所以不正常的DNA修复系统能增加基因组的不稳定性以致引起基因的突变乃至肺癌的发生。人ERCC4(Excision repair cross-complementing rodent repair deficiency,complementation group4)IXPF(Xeroderma pigmentosum,complementationgroupF)位于16p13.3-13.13,它的编码产物是NER途径关键酶之一,它与ERCC1蛋白形成复合物,不仅参与识别受损DNA,而且可以特异切割受损DNA片段的5’端。ERCC4基因缺陷能导致基因的不稳定和引起致癌作用。已有的研究显示在中国人群中(n=1010)ERCC4启动子区-644C/T(rs3136038)多态性与肺癌的发生紧密关联。为了阐明这种关联的分子机制,我们研究了ERCC4基因的启动区多态与基因表达调控的关系。生物信息学分析与ERCC4启动区多态位点结合的转录因子,并用EMSA实验加以验证和分析SNP多态是否影响转录因子结合。双荧光素酶报告系统实验显示不同ERCC4启动子单倍型有不同的转录活性,在HELF细胞中,与其它单倍型启动子相比,野生大单倍型启动子-644C/-357A有较高的启动子转录活性。荧光定量PCR实验结果表明烟雾抽取物(CSE)能极大地抑制ERCC4基因的表达。在CSE条件下,细胞转染实验结果说明野生大单倍型-644C/-357A对CSE的损伤有较好的保护作用。EMSA实验结果表明CREB和CRE-BP1可能是结合在ERCC4启动子-644和-357位点的转录因子,大单倍型-644C/-357A能增强与CREB和CRE-BP1的结合能力。-644C/T(rs3136038)基因型与ERCC4表达水平研究结果显示,与野生纯合基因型相比,突变纯合基因型携带者的外周血白细胞有较低的ERCC4表达水平。我们的研究结果表明基因和环境因子共同调节ERCC4基因的表达,我们推测ERCC4转录调控很可能遵循以下模式:化学致癌物→DNA损伤等应激信号→细胞信号转导途径激活→基因表达改变→DNA损伤修复能力提高或降低,不同基因型的ERCC4受外界DNA损伤条件诱导表达能力可能不同。我们的研究有助于阐明肺癌的发生机理。更多还原

【Abstract】 Lung cancer is one of the most common cancers worldwide,which has the highest mortality rate among all the cancers.Tobacco caused DNA damage or lesion plays an important role in lung cancer genesis.Polycyclic aromatic hydrocarbons, aromatic amines,and nitrosamines involved in the tobacco smoke have been implicated as the major mutagenic carcinogens responsible for DNA adduct formation. Accumulated DNA adducts lead to an enhanced probability for DNA damage,which could impede or block translation and replication,followed by potential mutation and cancer ultimately.Nucleotide excision repair(NER),one of the most versatile DNA repair systems,is the primary means by which smoking-induced genomic mangle can be removed.So,impairment of this DNA repair system may arouse susceptibility to some characteristic human disorders,such as lung cancer.Human ERCC4/XPF(Excision repair cross-complementing group 4)locates in 16p13.3-13.13 and it encodes one of the key enzymes in NER pathway.This enzyme forms a complex with protein ERCC1 and the complex can not only recognize damaged DNA but also specifically excise the 5’ end of the damaged DNA fragment. ERCC4 gene deficits may lead to genetic instability and carcinogenesis.A recent study showed that in a Chinese population(n=1010),the-644C/T(rs3136038) polymorphism in the ERCC4 promoter was significantly associated with the risk of lung cancer.To elucidate the underlying molecular mechanism of the association,we investigated the relationship between the polymorphisms in the ERCC4 promoter and ERCC4 mRNA transcription.We predicted the transcription factors that bind the polymorphisms sites by bioinformatics.We further validated this result by EMSAs, and analyzed whether SNPs of-644C/T and -357A/C(rs6498486)on the transcriptional regulation affect binding of transcription factors.The luciferase report system experiment indicated that the transcription activity of haplotypes in the promoter of ERCC4 has significant difference.A promoter fragment containing major alleles(-644C/-357A)showed the highest activity in HELF cells, compared with other haplotypes.Real-time quantitative PCR showed that cigarette smoke extract(CSE) significantly inhibited ERCC4 gene expression.Transfection experiments in the presence of CSE indicated that the major haplotype of-644C/-357A has the best protective effect for CSE treatment.Electrophoretic mobility shift assays(EMSA)revealed that CREB and CRE-BP1 possibly were the transcription factors that bind the region containing positions -644 and -357.The major haplotype of -644C/-357A of in ERCC4 promoter could significantly enhanced binding ability with CREB and CRE-BP1.Relation between the -644C/T(rs3136038)genotypes and ERCC4 expression level showed that the minor allele homozygote TT carriers had lower expression level of ERCC4 gene than the major allele homozygote CC.Our results suggested that genetic and environmental factors may interact to regulate ERCC4 expression.We postulated that the transcription regulation of ERCC4 gene might abide by the following pattern:chemical carcinogens→stress signals including DNA damage→activation of cellular signal wansduction pathway→change of gene expression→enhanced or decreased DNA repair ability.The ERCC4 with different genotypes might have different expression levels to external conditions of DNA damage.Our research may contribute to elucidating the etiology of lung cancer.更多还原