APE1/Ref-1基因启动子区多态与肺癌和胶质瘤的遗传易感性分析及功能研究

【作者】 吕娟；

【导师】 金力； 卢大儒；

【作者基本信息】 复旦大学 ， 遗传学， 2010， 博士

【摘要】 肺癌是人类最常见的恶性肿瘤之一,在全球的总体发病率和死亡总人数均位居各类癌症之首,其5年存活率不到15%,对人类健康和生命威胁极大。吸烟是导致肺癌的最主要环境因素,而同时肺癌的发生具有明显的遗传易感性,提示基因及其与环境因素的相互作用在肺癌的发生发展过程中具有重要作用。脱嘌呤脱嘧啶核酸内切酶(APE1/Ref-1)是一种具有DNA损伤修复和氧化还原调控活性的多功能蛋白,参与了细胞增殖与生长、细胞周期调控、凋亡、血管生成等许多重要的生物学过程。越来越多的证据表明APE1/Ref-1在肿瘤的发生发展过程中起重要作用。目前,国际上关于APE1/Ref-1基因多态与肺癌遗传易感性的研究主要集中在基因编码区的一个非同义变异Asp148Glu,但在多项研究中得到了不一致的结论,且缺乏相关的分子生物学功能研究。为了揭示中国人群APE1/Ref-1基因多态与肺癌风险的关系,寻找发现潜在的功能性致病位点,我们采用病例-对照的研究方法,选取了APE1/Ref-1基因的3个SNP位点,首先在中国汉族人群500例肺癌患者和517例年龄、性别相匹配的正常对照中进行了基因分型和关联分析。结果发现位于APE1／Ref-1基因启动子区的rs1760944(-141T／G)多态的等位基因频率分布在病例和对照组中存在显著差异(P=0.02)。经过置换检验校正后,这种显著性依然存在(P=0.0489)。非条件逻辑回归分析发现,GG基因型可显著降低肺癌的患病风险(校正OR=0.62,95%CI=0.43-0.91；P=0.043)。我们的研究没有发现rsl 130409位点(Asp148Glu)与肺癌的相关性,但是在与rs1760944进行联合效应分析时发现,rs1130409的突变等位基因与rs1760944 G等位基因的联合作用对降低当前吸烟者的肺癌患病风险具有等位基因剂量依赖效应。为了进一步考察rs1760944多态与肺癌风险的相关性,我们将第一阶段研究得到的阳性结果在第二阶段包括572例肺癌患者和547例健康对照的独立样本中进行了重复验证,并得到了一致的结果。样本合并后,病例和对照组中rs1760944多态的等位基因和基因型频率分布差异均比在两个单独样本中的差异更显著,P值分别达到0.0001和0.002。逻辑回归分析的结果与单独研究一致,-141GG纯合个体同样显示了较低的肺癌风险(校正OR=0.63,95%CI=0.48-0.81)。趋势检验发现,这种保护效应随着G等位基因数目的增加而增强(P for trend=0.00035)。携带有至少一个G拷贝的个体与非携带者相比,可以降低大约23%的肺癌患病风险(校正OR=0.77,95%CI=0.64-0.93；P=0.006)。此外,我们发现rs1760944多态与吸烟之间存在显著的交互作用(Pinteraction=0.031),GG基因型在当前吸烟者中表现为更明显的保护作用(校正OR=0.49,95%CI=0.32-0.74)。胶质瘤是中枢神经系统中最常见的恶性肿瘤之一,具有发病率、复发率、死亡率高和治愈率低的特点。鉴于胶质瘤的恶性表型以无法控制的细胞迅速增殖、细胞凋亡速度减慢、局部侵袭和血管生成为主要特征,而这些过程可能与APE1/Ref-1的功能活性有关,加上各种类型肿瘤在发病机制上具有一定的相似性,我们推测APE1/Ref-1基因rs1760944多态与肺癌风险的相关性可能也存在于胶质瘤中,且目前国际上尚无这一多态与胶质瘤易感性的相关研究。因此,我们对该多态与胶质瘤的遗传易感性关系,在包含241例胶质母细胞瘤、284例星形细胞瘤(胶质母细胞瘤除外)、241例其他类型胶质瘤病例以及824例正常对照的中国人群中进行了分析。结果发现,rs1760944多态的等位基因及基因型频率分布在胶质母细胞瘤病例组和总体对照组中存在显著差异,P值分别为0.03和0.041。逻辑回归分析显示,携带-141GG基因型的个体相对于TT基因型携带者来说,患胶质母细胞瘤的风险降低了46%(校正OR=0.54,95%CI=0.34-0.87),且这种保护效应随着G等位基因数目的增加而增强(P for trend=0.014),与在肺癌中表现的保护作用一致。但是,我们的研究没有发现该多态与星形细胞瘤及其他类型胶质瘤存在关联。以上研究结果表明,APE1/Ref-1基因多态与肺癌及胶质母细胞瘤的遗传易感性有关,且其在肺癌中的效应可能在一定程度上受到与吸烟等环境因素相互作用的影响。通过第一部分病例-对照研究,我们发现APE1/Ref-1基因启动子区rsl760944(-141T／G)多态与中国人群肺癌及胶质母细胞瘤的易感性显著相关。鉴于-141T／G多态位于APE1/Ref-1基因的核心启动子区,距离转录起始位点上游仅141bp,我们推测该多态是一个潜在的功能性位点,其对癌症风险的效应可能是由于引起了APE1/Ref-1基因转录调控与表达的改变。因此,我们通过一系列分子生物学功能研究探讨了-141T／G多态对APE1/Ref-1表达水平的影响及潜在分子机制。生物信息学预测显示,-141T／G位点所在的启动子区域有多种转录因子的结合,其遗传变异可能影响转录因子Oct-1与该位点及其侧翼序列的结合能力,表现为T到G的变异破坏了两个Oct-1的结合位点。实时定量PCR检测发现,携带-141GG基因型个体的外周血单核细胞(PBMCs)和正常肺组织中APE1/Ref-1的mRNA水平均低于TT基因型携带者。荧光素酶报告基因检测显示,含有-141G等位基因启动子的重组载体pGL3-G在多种细胞中的荧光素酶表达低于pGL3-T,提示-141G型启动子具有较弱的转录活性。凝胶迁移实验(EMSA)发现含有-141G等位基因的生物素标记寡核苷酸探针序列与细胞核蛋白抽提物的亲和力较弱,表现为非标记的T型探针可更大程度地竞争抑制标记探针-核蛋白复合物的形成。通过表面等离子共振实验(SPR),实时监测这两种等位型寡核苷酸序列与核蛋白的结合反应,也得到了相同的结果。提示-141G变异可能影响了某种转录因子与启动子区的结合能力。凝胶超迁移(Supershift)实验显示转录因子Oct-1的特异性抗体可在体外与探针DNA-核蛋白结合,形成超迁移复合物。染色质免疫共沉淀(ChIP)实验在肺组织和细胞内检测到Oct-1与APE1/Ref-1启动子区-141T／G位点及侧翼序列的结合。此外,在细胞中过表达Oct-1蛋白可以显著增强-141T等位基因启动子的转录活性而对G型启动子活性的增强作用较弱,提示Oct-1可以作为正调控因子反式激活APE1/Ref-1基因的表达,并且这种转录激活作用具有等位基因特异性。我们的研究还发现,在苯并芘(BaP)诱导条件下,可显著增强细胞中APE1/Ref-1及Oct-1的mRNA水平,同时可诱导激活pGL3-T／G载体的荧光素酶报告基因表达,并且pGL3-T启动子受诱导的能力更强,提示APE1/Ref-1基因的表达可受到吸烟等环境因素的影响,且-141T／G不同等位型启动子受影响的程度可能存在差异。本部分研究探寻了-141T／G多态的潜在生物学功能,发现该多态可能是通过影响转录因子Oct-1与启动子区DNA的结合,引起APE1/Ref-1表达水平的改变,从而进一步影响肺癌及胶质母细胞瘤的易感性,提示-141T／G多态可能是一个真正的致病位点。我们的研究同时为阐明APE1/Ref-1影响肿瘤发生发展过程的作用机制提供了一些新的证据,有助于相关肿瘤的易感性及病因学研究。更多还原

【Abstract】 Lung cancer is among the most common and life-threatening malignancies worldwide, with the highest incidence and mortality rates and an overall 5-year survival rate of<15%. Although tobacco smoking has long been established as the predominant risk factor for lung cancer, genetic variation also contribute to inter-individual susceptibility to lung cancer, suggesting the pivotal role of genetic determinants including the gene-environment interaction in lung cancer etiology.Apurinic/apyrimidinic endonuclease 1/Redox effector factor-1 (APE1/Ref-1) is a ubiquitous multifunctional protein that possesses both DNA repair and redox regulatory activities. In view of its unique multiple functions, it is not surprising that APE1/Ref-1 plays a critical role in many biological processes, such as cell proliferation and growth, cell cycle control, apoptosis, and angiogenesis.Although it was originally identified as a DNA repair enzyme, accumulating evidence supports a role of APE1/Ref-1 in tumor development. Previous studies about APE1/Ref-1 polymorphisms and their associations with lung cancer susceptibility mainly focused on the nonsynonymous single nucleotide polymorphism (nsSNP), Asp148Glu, in the coding region; however, most findings have been inconsistent, and the functional relevance of this nsSNP has not been elucidated. To investigate association between APE1/Ref-1 polymorphisms and lung cancer risk in Chinese populations, we first genotyped three variants of APEl/Ref-1 among 500 patients with incident lung cancer and 517 age- and sex-matched cancer-free control participants to screen for any risk-associated SNPs.As a result, we found that the allele distribution of rs1760944, a-141 T-to-G variant in the promoter, is significantly different between case patients and control subjects (P=0.02). The significance remained after applying 100,000-time permutation tests (P=0.0489). Further logistic regression analyses revealed that the homozygous-141GG genotype was associated with decreased risk of developing lung cancer [adjusted odds ratio (OR)=0.62; P=0.043]. No altered risk was observed for the prior reported Asp148Glu polymorphism (rsl 130409). However, the joint effects of rs1130409 variant allele and rs1760944 G allele reduced lung cancer risk in the current smokers in a significant locus dose-response manner.Then we performed another independent case-control study of 572 lung cancer patients and 547 control participants to confirm this finding and similar results were obtained. Combined data from the two studies comprising a total of 1,072 lung cancer patients and 1,064 cancer-free controls generated a more significant association (adjusted OR=0.63,95%CI 0.48-0.81; P=0.002). Trend tests showed that the protective effect followed a trend of increasing magnitude by number of G alleles (P for trend=0.00035). Subjects possessing at least one copy of the G allele had～23% reduction in risk of lung cancer (adjusted OR=0.77,95%CI 0.64-0.93; P=0.006). We also observed a statistically significant interaction between rs1760944 and smoking status (P interaction=0.031), and the protective effect of the GG genotype against lung cancer was more evident in the current smokers.Gliomas are the most common primary tumors in the central nervous system, with high rates of incidence, recurrence and mortality. In view of gliomas are characterized of uncontrolled rapid cell proliferation, reduced apotosis rate, local invasion and angiogenesis, which are all relevant to APE1/Ref-1’s function and since the pathogenesis of different tumor types has certain similarities, we speculate that APE1/Ref-1 rs1760944 may also confer individual susceptibility to glioma. To test this hypothesis, we investigated the association between rs1760944 and glioma risk in a case-control study of 241 glioblastoma cases,284 astrocytoma cases (glioblastoma not included),241 other glioma cases, and 824 healthy controls.We observed statistically significant differences in allele and genotype distributions of rs1760944 between glioblastoma patients and control subjects (P= 0.03 and 0.041, respectively). Consistent with the protective effect of the -141 G allele inlung cancer, individuals with the homozygous -141GG genotype exhibited 42% reduced risk of glioblastoma (adjusted OR=0.54,95%CI 0.34-0.87), compared with the TT homozygotes.The protection followed a trend of increasing magnitude by number of G alleles (P for trend=0.014). However, no significant association of rs1760944 with risk of astrocytoma and other glioma was found in our study.These results indicate that genetic variantions in APE1/Ref-1 may modify susceptibility to lung cancer and glioblastoma. Potential gene-smoking interaction may influence its susceptible role in lung cancer. Since the -141T/G SNP (rs1760944) is located in the promoter of APE1/Ref-1, only 141 bp upstream from the transcription initiation site, it appears to be functional by possibly affecting APE1/Ref-1 transcriptional activity. To test this hypothesis, we performed further functional assays.Notably, bioinformatics analysis showed that the -141T-to-G polymorphism destroyed two binding sites for octamer-binding transcription factor-1 (Oct-1), thus it may impair the binding affinity of Oct-1 to the adjacent region of the SNP in the APE1/Ref-1 promoter. We observed an interindividual variation in APE1/Ref-1 mRNA levels in human peripheral blood mononuclear cells (PBMCs) and normal lung tissues, with a higher prevalence of the protective G allele in subjects displaying lower APE1/Ref-1 expression. Our transfection experiments also found that the plasmid containing the -141 G allele displayed significantly decreased luciferase expression than that with the T allele in both human lung adenocarcinoma (H1299) cells and human embryo lung fibroblasts (HELF).Electrophoretic mobility shift assays (EMSAs) showed that the -141G-allele probe had less affinity for the nuclear proteins from H1299 cells, since the formation of the DNA-protein complex was dramatically inhibited by unlabeled T-allele probes. Differences in the binding responses of nuclear proteins to the two oligonucleotides were also measured in real time using surface plasmon resonance (SPR) analyses and similar results were obtained, indicating that the -141G allele impaired the binding affinity of some transcription factor. Supershift assays revealed that the DNA-protein complex bands formed in EMSAs were supershifted by Oct-1-specific antibodies. Chromatin immunoprecipitation (ChIP) reconfirmed in vivo binding of Oct-1 to the APE1/Ref-1 promoter at position -141. These findings demonstrate that the protein of interest was Oct-1, and that the difference in APE1/Ref-1-promoter activities between the -141 T and G alleles was presumably due to their distinct binding affinity to Oct-1.Transactivation assays, through ectopic overexpression of Oct-1, were highly suggestive for a role of Oct-1 in preferential transactivation from the -141T allele promoter, whereas the -141G allele harbored little ability to be transcriptionally activated by Oct-1, suggesting that Oct-1 may function as a transacting factor to exert allele-specific influence on APE1/Ref-1 expression. We also found that BaP can upregulate the mRNA levels of APE1/Ref-1 and Oct-1 in H1299 and HELF cells. The pGL3-T/G transfected H1299 and HELF cells exhibited increased luciferase reporter expression after treatment with BaP. Moreover, the -141T allele-carrying construct exhibited higher capacity to be induced and still maintained significantly higher expression levels, compared with the G allele construct. This result suggests that the expression of APEl/Ref-1 might be induced by carcinogen exposure, such as smoking, in an allele-specific manner.Our study explored the functional significance of the -141T/G polymorphism. We found that it may alter APE1/Ref-1 expression by affecting the binding affinity of Oct-1 to the promoter, thus playing a causal role in lung cancer and glioblastoma susceptibility.These data also provide new insights into the mechanism by which APE1/Ref-1 exert an important effect on tumorigenesis.更多还原