CHEK2与REV3L基因多态的肺癌易感性关联与机制研究

【作者】 张舒羽；

【导师】 卢大儒；

【作者基本信息】 复旦大学 ， 遗传学， 2010， 博士

【摘要】 肺癌的发生是一个多因素、多阶段、长期相互作用的结果。肺癌的发生与环境暴露引起DNA损伤后的修复能力密切相关,而细胞周期检控点是维持细胞基因组稳定性的一个重要机制,其中DNA损伤检控点能检测细胞在生命活动过程中出现的DNA损伤并引发细胞周期阻滞,为修复损伤提供足够的时间。CHEK2基因在细胞周期调控中发挥了重要作用；当DNA出现损伤时,ATM与ATR激活CHEK2蛋白,后者将这一信号传递给能磷酸化E2F-1、BRCA1和P53等多个蛋白,启动细胞周期检验点调控。目前已发现CHEK2基因突变与乳腺癌、前列腺癌、结肠癌等多种肿瘤的发生具有密切的关系。我们推测CHEK2基因启动子上的遗传变异可能会影响CHEK2基因最终的表达量,影响个体肺癌易感性的差异,为了检验这一假设,我们采用了病例-对照的研究方法,选择了该基因近端启动子上-122C>G与-48G>A两个单核苷酸多态(SNP)位点,利用Illumina分型方法对500例中国汉族肺癌患者和517例年龄、性别与之相匹配的正常对照进行了基因分型,研究了不同等位基因型和基因型与肺癌风险的相关性；然后在另外575例肺癌患者和589例对照人群中验证该多态和肺癌易感性的相关性。我们还进一步通过荧光素酶、凝胶迁移率电泳、染色质免疫共沉淀等方法深入的探索了阳性位点的功能。在单位点分析中,我们发现CHEK2的启动子多态-48G>A(rs2236141)位点的等位基因型分布在病例和对照中存在显著的统计学差异,P值为0.0018。非条件logistic回归分析表明,rs2236141位点的突变杂合基因型可以显著降低患肺癌的风险(与GG基因型相比,GA基因型的校正OR=0.65,95% CI=0.40-0.85)。而rs2236142多态与肺癌风险没有显著相关性。在验证性关联分析中,我们发现rs2236141多态的基因型分布在病例和对照中也存在显著的统计学差异(P=0.014)。两次关联分析结果合并后,rs2236141多态的基因型分布在病例和对照中存在显著的统计学差异(P=0.001)；rs2236141位点的突变杂合基因型可以显著降低患肺癌的风险1.4倍(P=4×10-4)。功能研究表明,在正常人外周血和肺组织中,A等位基因型的个体CHEK2 mRNA水平高于G等位基因型个体。由于G→A转换可能减少一个甲基化位点(CG→CA),因而该多态可能影响表观遗传学调控。荧光素酶试验表明,G/A等位型启动子表达没有显著差异；但在甲基化条件下,等位型A等位型启动子对于显著高于A等位型。凝胶迁移率电泳实验和表面等离子共振实验表明甲基化的G等位型启动子能增强转录因子的结合。采用体内染色质共沉淀实验表明,结合在该多态附近的转录因子为Sp1,且该转录因子起负调控作用。本研究表明CHEK2基因启动子rs2236141多态与中国人群中肺癌的发病风险具有显著相关性；该多态位点为功能位点,突变型等位基因与负调控的Sp1蛋白结合力较弱,因而表达更高,具有保护作用。跨损伤DNA合成(Translesion DNA Synthesis,TLS),属于一种复制后修复过程,主要包括DNA聚合酶κ、η、ι、ζ等。跨损伤DNA合成途径是生物体的一种应急机制,能够在DNA损伤未被修复的状态下进行复制延伸。REV3L (hREV3)是DNA聚合酶的ζ的催化亚基,是跨损伤DNA合成途径的主要参与者。近年来研究表明REV3L基因能维持基因组稳定性,但也不可避免的会引起DNA突变。因而REV3L基因的多态有可能影响REV3L基因最终的表达量,从而影响肺癌的易感性。为了检验这一假设,我们采用了病例-对照的研究方法,选择了该基因上的15个多态位点,对南京地区500例肺癌患者和517例正常对照进行了基因分型,研究不同等位基因型和基因型与肺癌风险的相关性；然后在来自上海地区的575例肺癌患者和589例对照人群中验证该多态和肺癌易感性的相关性。我们还进一步通过荧光素酶、表面等离子共振、细胞免疫荧光实验、克隆形成实验等方法深入的探索了阳性位点的功能。在单位点分析中,我们发现REV3L基因上rs465646、rs459809和rs1002481三个多态位点的基因型分布在病例和对照中存在显著的统计学差异。其中3’UTR 460 T>C(rs465646)多态位于3’UTR的一段保守区域中,其余两个SNP位于功能尚不清楚的内含子中。Rs465646多态的C等位型携带者显著降低肺癌发病风险(P=0.015)。我们在验证性关联分析中,我们发现rs465646多态的基因型分布在病例和对照中也存在显著的统计学差异(P=0.049)。生物信息学预测表明,REV3L基因3’UTR上有多个microRNA(miRNA)结合位点；T>C多态会影响多个miRNA的结合。我们用表面等离子共振的方法,发现该多态位点附近与miR-25和miR-32均有结合。荧光素酶实验表明,共转染miR-25和miR-32均能显著降低含有REV3L基因3’UTR的报告基因的表达,表明上述miRNA能负调控REV3L基因的表达；此外,等离子表面共振实验表明,T等位型RNA与上述两种miRNA的结合能力更强。报告基因实验结果显示C等位型表达显著高于T等位型。烟草提取物能诱导REV3L基因表达,同时伴随着miR-32表达的显著降低,而miR-25表达没有显著变化；这表明miR-32可能在REV3L的转录后调控中起主要作用。REV3L基因的过表达能降低细胞的克隆形成,减少微核率,这表明REV3L基因具有稳定基因组、抑制肿瘤增殖的作用。我们转染两种等位型RNA来竞争内源miRNA,结果表明转染T等位型RNA后细胞克隆形成更少、微核率显著降低,这表明携带T等位型个体肿瘤基因组不稳定性增高、肿瘤发展更快。我们还在46对肺癌-癌旁组织中检测了REV3L基因的表达,结果表明REV3L基因mRNA在肺癌中表达显著低于癌旁。本研究表明REV3L基因3’UTR rs465646多态为功能位点通过改变miRNA的结合,影响了基因的表达水平,从而使得携带两种基因型的个体肺癌发病风险不同。更多还原

【Abstract】 Lung cancer is the leading cause of cancer-related deaths worldwide and tobacco smoking is one of the most important causes of lung cancer. Since the accumulation of mutations and chromosomal aberrations are the hallmarks of cancer cells, it has been hypothesized that cancer susceptibility derives from hypomorphic variants of DNA repair and checkpoint control genes. The loss of genome-maintenance mechanisms such as DNA repair and cell cycle checkpoint control may result in chromosomal abnormalities.Checkpoint kinase 2 (CHEK2), a tumor-suppressor gene, plays an essential role in the DNA damage checkpoint response cascade. The list of CHEK2 mutations in cancer cells is continuously expanding and the CHEK2 gene has been postulated as a susceptibility gene for a number of other common cancers, including prostate, colorectal cancers and hematopoietic neoplasms. In recent years, CHEK2 mutations have been found in lung cance. Since CHEK2 expression is ubiquitous in mammalian cells, individual differences in lung cancer susceptibility may be determined to be the consequence of impaired CHEK2 expression. We therefore hypothesized that polymorphisms in the CHEK2 gene promoter may modify its transcription and create a susceptibility to lung cancer.We first investigated two polymorphisms in the proximal promoter of the CHEK2 gene and evaluated its association with the risk of lung cancer in a case-control study using 500 incident lung cancer cases and 517 cancer-free controls. We found that CHEK2 rs2236141-48 G>A was significantly associated with lung cancer risk (P= 0.0018). Similar results were obtained in a follow-up replication study in 575 lung cancer patients and 589 controls (P= 0.042). Quantitative PCR showed that individuals with the G allele had lower levels of CHEK2 transcripts in peripheral blood mononuclear cells and normal lung tissues. The-48 G→A variant eliminated a methylation site and thereby relieve the transcriptional repression of CHEK2. Therefore, this polymorphism affected downstream transcription through genetic and epigenetic modifications. Luciferase reporter assays demonstrated that the major G allele significantly attenuated reporter gene expression when methylated. Electro-mobility shift assays (EMSA) and surface plasmon resonance (SPR) revealed that the methylated G allele increased transcription factor accessibility. We used in vivo chromatin immunoprecipitation (ChIP) to confirm that the relevant transcription factor was Spl. Using lung tissue heterozygous for the G/A SNP, we found that Spl acted as a repressor and had a stronger binding affinity for the G allele. These results support our hypothesis that the CHEK2 rs2236141 variant modifies lung cancer susceptibility in the Chinese population by affecting CHEK2 expression. Lung cancer remains the leading cause of cancer-related deaths worldwide. The environment, especially tobacco smoke has proved to be the most important trigger of lung cancer. However, only a fraction of smokers develop lung cancer, suggesting synergy of genetic susceptibility with environment.Cells constantly encounter various carcinogens or cytoxic agents. These compounds directly or indirectly provoke DNA damage creating mutations, DNA cross-links or DNA strand breaks, which lead to DNA instability. Eukaryotic cells from yeast to human possess DNA post-replication repair (PPR) or DNA damage tolerance system. Translesion DNA synthesis (TLS) and homologous DNA recombination (HR) are two major postreplicational repair pathways. The ubiquitous translesion DNA synthesis consists of a series of specialized polymerases, including polymeraseκ,ζ,ηandι.REV3Lp, the catalytic subunit of DNA polymerase zeta is the major participant in translesion DNA synthesis (TLS). Recent evidence suggests that REV3L plays an important role in the maintenance of genome stability despite its mutagenic characteristics. Such function makes it a genetic susceptibility gene for cancers. Here, we investigated 15 common polymorphisms in the REV3L gene in a case-control study of 500 incident lung cancer patients and 517 cancer-free controls in a Chinese population. Single locus analysis revealed that three SNPs (rs465646, rs459809 and rs1002481) were significantly associated with the risk for lung cancer. One of the strongest associations comes from the 3’UTR 460 T>C polymorphism (rs465646). The C-allele was significantly associated with reduced lung cancer risk (P= 0.015). Similar results were obtained in a follow-up replication study in another 575 lung cancer cases and 589 controls (P= 0.049). This 3’UTR 460 T>C variation was predicted to modulate the binding of several micorRNAs. We found that the T-allele demonstrated stronger binding affinity for miR-25 and miR-32, which was accompanied by significantly weaker reporter expression level. Cigarette smoke extracts could upregulate and downregulate the expression of REV3L and miR-32, respectively. Overexpression of REV3L attenuated foci and micronucleus formation, indicating the tumor-suppressing role of REV3L. Our data also shows a significant reduction of REV3L gene expression in 46 lung carcinomas when compared to normal adjacent tissues. These results support our hypothesis that the REV3L rs465646 variant modifies lung cancer susceptibility in the Chinese population by affecting microRNA-mediated regulation.Extending our previous association study, we have successfully identified the risk contributing polymorphism in the REV3L gene and a possible underlying mechanism in carcinogenesis.更多还原