【作者】 王芳；

【导师】 邬堂春；

【作者基本信息】 华中科技大学 ， 劳动卫生与环境卫生学， 2009， 博士

【摘要】 焦化作业过程中产生的焦炉逸散物(coke oven emission，COE)中含有大量致癌性多环芳烃(polycyclic aromatic hydrocarbons，PAHs)，是引起焦炉工肺癌高发的主要原因。焦炉工肺癌是我国的法定职业性肿瘤之一，目前已进入。一个稳定的高发期，也是我国职业肿瘤防治工作的重点。烟草已公认为肺癌的危险因素，与COE相似，其中也含有大量致癌性的PAHs。但不同的个体，在相似的外暴露环境中DNA损伤程度不同，且只有少部分接触致癌性PAHs者会最终发生肺癌，而且发病的潜伏期存在很大差异，这提示个体易感性的作用不容忽视。核苷酸切除修复(nucleotideexcision repair，NER)是体内识别DNA损伤形式最多、最灵活也最复杂的修复途径，在PAHs引起的DNA损伤修复中发挥重要作用。因此研究NER基因多态性与DNA损伤及肺癌易感性的关联，不仅有利于我们加深对PAHs致癌机制的认识，而且有利于筛检易感人群。对NER基因多态性和DNA损伤及肿癌易感性的关系已有部分研究，但可能由于缺少对整个基因或整个通路“综合变异性”的考虑，忽视了其它有功能的位点，从而影响关联研究中结果的产生。本研究首先以接触高浓度焦炉逸散物的焦炉作业者为研究对象，探讨了NER基因多态性与PAHs引起DNA损伤的关系，然后对启动区SNPs的功能进行了初步探讨，结合上述的结果，又进行了有意义SNPs与肺癌易感性的关联研究，从焦化作业所造成的DNA损伤和肺癌发生这两个层面出发，期望为职业人群的个体防护和肺癌遗传易感性的研究提供科学依据。本研究共分以下三部分：第一部分核苷酸切除修复基因多态性与焦炉工DNA损伤的关联研究PAHs是焦炉逸散物最重要的致癌组份之一，可引起机体DNA损伤，而后者是肿瘤发生的早期生物学事件，NER是体内修复损伤DNA的主要途径，在PAHs所致损伤的修复中发挥着重要作用，因此，研究NER基因多态性对焦炉工DNA损伤的影响对了解职业人群的遗传易感性具有重要意义。本部分采用TaqMan-MGB探针分型技术，根据HapMap数据库信息挑选了NER修复通路中8个基因共32个多态性位点，对475名焦炉作业工人进行了分型。共有4个基因的6个位点多态性与彗星尾矩(Olive tail moment，OTM)有关，1个位于中暴露组，5个在高暴露组。在中暴露组，XPA基因rs1800975位点GG和GA基因型携带者OTM分别为0．30(0．12-0．82)和0．39(0．17-1．86)，均显著性低于AA基因型携带者0．53(0．16-2．62)(P＜0．05)。在高暴露组，与DDB2 rs3781619AA基因型携带者相比，GG基因型携带者OTM明显增高，OTM分别为0．39(0．13-1．67)和0．53(0．17-2．57)，P=0．036。携带有XPC rs2228001位点GG基因型的个体OTM为0．42(0．17-2．54)，明显高于TT基因型携带者0．33(0．13-1．38)，且随G等位基因数目的增多，OTM逐渐增高，P＜0．05。XPC基因rs3731055位点GA和AA基因型携带者OTM分别为0．35(0．13-1．24)和0．25(0．14-1．98)，低于GG基因型携带者0．45(0．17-1．73)，差别有显著性，趋势检验也有意义(P＜0．05)。XPD rs50871GG纯合子携带者DNA损伤水平为0．23(0．13-1．00)，显著低于野生型纯合子TT携带者0．40(0．15-1．22)，P＜0．05；携带有rs50872 TC杂合子的个体OTM为0．33(0．13-1．02)，显著性低于CC纯合子0．46(0．15-1．32)，P＜0．05。在三个暴露组中，我们未发现ERCC1，XPB和XPG基因的多态性对工人OTM有显著性影响。单体型对分析结果显示，低暴露组中，DDB2基因TACGA／TACGA单倍型对OTM值最高，为0．75(0．22-2．21)，与人群中分布最广泛的CTGAG／CTGAG单倍型对相比(0．29(0．08-4．13))，差别有统计学意义，P＜0．05。低暴露中，XPC基因TAA／TAA单体型携带者OTM为0．65(0．19-2．21)高于TGA／TAA单体型对携带者(0．24(0．11-2．06))，P＜0．05。中暴露组中，与XPA基因GTA／GAG单体型对携带者相比，GTA／GTG单体型对携带者OTM明显增高，OTM分别为0．82(0．19-4．28)和0．36(0．17-3．16)；高暴露组中ATA／ATA单体型对携带者OTM为0．62(0．20-2．57)，明显高于GTA／GAG单体型对携带者(0．35(0．13-1．24))，P＜0．05。同时，高暴露组中，XPD基因TCCTC／TCTTC单体型对携带者DNA损伤(0．25(0．13-0．61))明显低于TGCTG／TCCTC单体型对携带者(0．53(0．13-1．77))，P＜0．05；XPF基因AC／AC单倍型携带者的OTM明显高于TC／TC单倍型携带者，OTM分别为0．77(0．20-1．42)和0．35(0．13-2．57)，P=0．010。其它基因的单体型对在三个暴露组中均无统计学差别。进一步分析基因型的联合作用对DNA损伤风险的影响，结果表明在高暴露组中，与有1个或没有危险等位基因的个体相比，有2个、3个或多于4个危险等位基因的个体OTM增高的风险分别为1．74(0．74-4．10)、1．50(0．59-3．84)和2．15(0．61-7．66)，趋势检验有意义，P=0．04。在中暴露组，与只有1个或没有相对保护的等位基因的个体相比，有2个、3个或多于4个相对具有保护作用的等位基因的个体OTM降低的风险是分别是0．62(0．24-1．68)(P=0．35)、0．25(0．10-0．67)(P=0．006)和0．31(0．12-0．83)(P=0．020)，降低的趋势为0．01；在高暴露组中，相对于只有1个或没有保护性等位基因的个体，有2个、3个或多于4个保护性等位基因的个体OTM降低的风险是分别是0．45(0．14-1．41)(P=0．17)、0．20(0．06-0．66)(P=0．008)和0．15(0．04-0．57)(P=0．005)，并且降低的趋势有意义，P trend=0．0007。本部分中我们探讨了NER基因多态性与焦炉逸散物引起的外周血淋巴细胞DNA损伤的关系，发现XPA rs1800975位点GG基因型与中暴露组DNA损伤降低相关联；高暴露组XPD rs50871 GG纯合子携带者、rs50872位点TC杂合子携带者及XPC rs3731055位点GA和AA基因型携带者DNA损伤水平降低；高暴露组DDB2rs3781619 GG基因型及XPC rs2228001 GG基因型与DNA高损伤相关联，NER基因间的联合作用与DNA损伤有明显关联。第二部分XPA及XPC基因启动子区SNPs功能研究启动子区多态的存在引起了序列结构的差异，使特定基因有可能结合不同的转录因子，从而引起转录和翻译效率的不同。我们采用双荧光素酶活性分析系统检测了XPA rs1800975和XPC rs3731055位点对荧光素酶活性的影响，用SYBR Green实时荧光定量的方法检测上述位点在荧光素酶mRNA表达中的作用。结果发现在三种细胞株中，XPA基因5’UTR区含有rs1800975-G的质粒荧光素酶的活性均强于含有rs1800975-A的质粒(P＜0．001)。含有XPC基因rs3731055-A的质粒，其荧光素酶活性在16HBE细胞中强于rs3731055-G的质粒，差别有临界显著性(P=0．08)；在A549和HePG2细胞株中，含XPC基因rs3731055-A的质粒，荧光素酶活性均强于含有rs3731055-G的质粒(P=0．013和P＜0．01)。相对于含有XPA rs1800975-A的质粒，三种细胞株中含XPA rs1800975-G的质粒荧光素酶表达均显著性增高，在16HBE细胞中增高了1．66倍(P=0．020)，A549细胞增高了9．08倍(P＜0．01)，HePG2细胞增高了8．73倍(P＜0．01)。与含有XPCrs3731055-G的质粒相比，含XPC rs3731055-A的质粒在16HBE、A549和HePG2细胞中的荧光素酶表达分别增高了3．11倍(P＜0．01)、2．93倍(P=0．025)和4．08倍(P＜0．01)。本部分的结果提示XPA rs1800975-G及XPC rs3731055-A可以增强荧光素酶的活性及表达。第三部分NER基因多态性与肺癌易感性的关联研究肺癌是多因素参与多阶段发展的复杂过程，是由环境因素和个体遗传因素综合作用的结果。在前面研究的基础上，我们深入分析了可影响焦炉工DNA损伤的SNP与肺癌发病危险性的关联。结果表明，经校正年龄、性别、吸烟、饮酒及肿瘤家族史等影响因素后，DDB2 rs3781619 GA杂合子携带者发生肺癌的危险性是TT纯合子携带者的1．19倍(OR=1．19，95％CI=0．99-1．42)，差异有临界显著性，P=0．067，GG纯合子携带者发生肺癌的危险性是TT纯合子携带者的1．31倍(OR=1．31，95％CI=1．04-1．68)，P=0．040。DDB2 rs3781619基因型与吸烟及肿瘤家族史之间有联合作用，同时具有rs3781619 GA+GG基因型、吸烟及有肿瘤家族史者肺癌发病的风险为8．04(4．72-13．72)倍，P＜0．01。四个SNPs(XPA rs1800975、XPD rs50871、XPDrs50872和DDB2 rs3781619)与肺癌预后均无明显关联。综上所述，本研究以NER基因多态性为主线，探讨了它们与焦炉作业工人DNA损伤的关系，对SNP功能的进行了初步研究，探讨了有意义SNPs与肺癌易感性的关联。本研究的创新之处在于：(1)在校正了内暴露的情况下，利用HapMap数据，结合生物信息学，选取能代表NER通路基因的遗传变异，探讨其与DNA损伤的关系；(2)首次以体外细胞实验初步验证了与DNA损伤有关联的启动区SNPs的功能；(3)在不同人群中验证了关联研究的结果。本研究的不足之处及有待深入研究的地方：(1)DNA损伤到肺癌的发生是一个复杂的过程，本研究中的肺癌样本并不能代表焦炉工肺癌，因此需进一步研究基因多态性与焦炉工肺癌易感性的关联；(2)需在人群样本中检测相关蛋白表达，并结合新技术新方法研究多通路蛋白质间的交互作用；(3)将SNPs与其它分子标记如拷贝数变异或微小RNA等结合起来研究它们与职业人群易感性的关联，为职业人群筛检提供依据。更多还原

【Abstract】 The abundant polycyclic aromatic hydrocarbons (PAHs) in cigarettes and coke ovenemission (COE) have been confirmed to be the main cause of lung cancer. Lung cancerof coke oven workers is one of the eight occupational tumors legislated in our country.However, recent researches demonstrated that DNA damage level may be different evenunder similar environments. Moreover, only a small portion of smokers and workerseventually develop cancer, which indicated the importance of individual susceptibility.The nucleotide excision repair (NER) is one of the most volatile repair pathways, whicheliminates a wide spectrum of DNA lesions including bulky base adducts induced bynumerous chemical compounds like PAHs. Therefore, to investigate the relationshipbetween NER genetic variations and DNA damage level as well as the associationbetween NER gene variations and lung cancer risk would be of great importance forunderstanding the underlying mechanism of PAHs carcinogenesis, screening ofsusceptible population and improving risk assessment among the exposed subjects. Somestudies have investigated the relationship between NER SNPs and DNA damage leveland lung cancer risk, but the results are still inconsistent. It may be attributed to theirsmall studying population or the fact that all reported SNPs in the candidate genes in a whole pathway were not taken into account.In the present study, we firstly used the HapMap data to select Tagging SNPs in theNER pathway and detected the association between these SNPs and DNA damage levelin the coke oven workers; secondly we investigated the potential functions of SNPslocated in the promoter region. Finally we detected the genotypes of the SNPs that canmodify DNA damage level in lung cancer patients.PartⅠThe associations of genetic variations in the NER geneswith PAHs-induced DNA damage in coke oven workersPAHs are the main carcinogenic components of COE and can cause DNA damage,which is considered as the early event in lung cancer development. NER plays animportant role in the repair of PAHs induced DNA damage. Therefore, to investigate therelationship between NER genetic variations and DNA damage level would be importantfor understanding the underlying mechanism of PAHs carcinogenesis and screening ofsusceptible population. In this section, with the HapMap Han Chinese data, wegenotyped 32 SNPs in 8 core genes involved in NER in 475 coke oven workers. SixSNPs in 4 genes were associated with DNA damage level, five in the high exposuregroup and the other one in the intermediate. In the intermediate exposure group, theOlive tail moment (OTM) in carriers of XPA rs1800975 GG and GA genotype (median0.30 and 0.39) was significantly lower than that of the AA genotype carriers (median0.53), P＜0.05. In the high-exposure group, two SNPs were associated with elevatedlevels of DNA damage while three with decreased levels of DNA damage. DNA-damagelevels elevated in subjects with the XPC rs2228001 GG genotype (median 0.42)compared with those with the TT genotype (median 0.33, P＜0.05); meanwhile, carriersof the DDB2 rs3781619 GG genotype had higher DNA-damage levels than those of the AA genotype (median 0.39 and 0.53, respectively, P=0.036). The OTM decreased incarriers of the XPC rs3731055 AA genotype (median 0.25) compared with those of theGG genotype (median 0.45) (P＜0.05). The OTM in carriers of XPD rs50871 GGgenotype was lower than those of the TT genotype (median 0.23 and 0.40, respectively,P trend=0.048), and a similar trend was found in the rs50872 TC and TT genotypecompared with the CC genotype (P trend=0.012). No significance was found in theassociation between polymorphisms in ERCC1, XPB and XPG genes and DNA damagelevel.The diplotype analysis revealed that, in the low exposure group, the DDB2TACGA/TACGA diplotype carriers had the highest OTM (median 0.75) compared withthat of the most widely distributed CTGAG/CTGAG diplotype (median 0.29), P＜0.05. TheOTM elevated in carriers of XPC TAA/TAA diplotype (median 0.65) compared withthose of the TGA/TAA diplotype (median 0.24), P＜0.05. In the intermediate exposuregroup, the OTM in carriers of XPA GTA/GTG diplotype were significantly higher thanthat in carriers with the GTA/GAG diplotype (median 0.82 vs. 0.36), and carriers of theATA/ATA diplotype also had higher OTM (median 0.62) than those of the GTA/GAGdiplotype (median 0.35) in the high exposure group, P＜0.05. In the high exposure group,the DNA damage in subjects with XPD TCCTC/TCTTC diplotype was significantlylower than that in subjects with TGCTG/TCCTC diplotype (median 0.25 vs. 0.53).Meanwhile, carriers of the XPF AC/AC diplotype had higher DNA damage level (median0.77) than carriers of TC/TC diplotype (median 0.35), P=0.010. No other diplotypes inNER genes were found to have significant association with DNA damage level.Further analysis of combinations of genetic variants was assessed by logisticregression models. For the at-risk genotypes, the ORs for individuals with four and more risk alleles tended to be high, though not statistically significant, in all three groupsdivided by 1-hydroxypyrene levels compared with individuals having one or no riskgenotypes. For the relatively protective genotypes, the ORs in individuals with three andmore variant alleles decreased significantly compared with those with one or no variantalleles in the intermediate (OR=0.25 (0.10-0.67) and 0.31 (0.12-0.83) for 3 and≥4 variantalleles, respectively) and high (OR=0.20 (0.06-0.66) and 0.15 (0.04-0.57) for 3 and≥4variant alleles, respectively) exposure groups, compared with the individuals with 0-1alleles in the same group; the trend for increased number of protective variant allele withdecreased OR was statistically significant (P=0.01 for the intermediate and P=0.0007 forthe high exposure groups).These results demonstrated that workers with variant alleles of XPC rs2228001 andDDB2 rs3781619 had higher DNA damage level, while the OTM decreased in subjectswith XPA rs1800975 GA and GG, XPC rs3731055 AA, XPD rs50871 GG and rs50872 TCgenotypes.PartⅡFunctional investigation of SNPs located in the promoter ofXPA and XPCDifferences in the sequence structure due to the variations in the promoter regionmay give rise to the variety of binding affinity of specific transcriptional factor, and thencause the disparity in efficiency of transcription and translation. To explore the possiblefunctional impact of the SNPs on the XPA and XPC gene that had shown their effects onDNA damage level in the multivariate analysis of covariance, plasmids were constructedwith luciferase as reporter gene and transfected in cultured cells. Relative luciferaseactivity (RLA) and luciferase expression were detected. The RLA containing XPArs1800975-G promoter was remarkably higher than that of the rs1800975-A containing promoter in the three types of cell lines (all P＜0.001). Similarly, greater RLA wasobtained in the construct with XPC rs3731055-A allele compared to that with XPCrs3731055-G allele in 16HBE (P=0.08), A549 (P=0.013) and HepG2 (P＜0.01) cell lines.The luciferase expression was significantly up-regulated in the XPA rs1800975-Gcontaining promoter in all three cell lines, with the rs1800975-G allele resulting in 1.66,9.08 and 8.73 times higher in luciferase expression compared to the rs1800975A allele in16HBE (P=0.020), A549 and HepG2 cell lines (P＜0.01). Compared with constructcontaining the XPC rs3731055-G allele, the XPC rs3731055-A allele containingfragment had 3.11, 2.93 and 4.08 fold higher luciferase expression in 16HBE (P=0.08),A549 (P=0.025) and HepG2 cell lines (P＜0.01). These data suggested that the XPArs1800975-G and XPC rs3731055-A allele may enhance promoter activity and mRNAexpression level.PartⅢAssociations between NER SNPs and lung cancersusceptibilityLung cancer is a complex multifactor process, while one of the early events ofwhich is DNA damage. In order to determine whether the SNPs that can modify DNAdamage level were associated with susceptibility of lung cancer, we conducted acase-control study of 1152 patients and 1152 matched cancer free controls in south China.After adjustments of age, gender, smoking status, drink and family history of cancer, wefound that the DDB2 rs3781619 GA and GG genotypes were associated with increasedrisk of lung cancer (OR=1.19, 95%CI=0.99-1.42, P=0.067 and OR=1.31,95%CI=1.04-1.68, P=0.040, respectively). Joint effect was found in the rs3781619genotypes with smoking and family history of lung cancer. Subjects carryingrs3781619GA+GG genotypes, smoking and having family history of lung cancer had significantly higher risk of lung cancer (OR=8.04, 95%CI=4.72-13.72, P＜0.01).However, XPA rs1800975, XPD rs50871 and rs50872 were not significantly associatedwith lung cancer risk. Additionally, there were no significant associations between thefour SNPs (XPA rs1800975, XPD rs50871, XPD rs50872 and DDB2 rs3781619) andprognosis of lung cancer.In summary, we selected Tagging SNPs in the NER genes with the HapMap dataand detected the associations of these SNPs with DNA damage level in the coke ovenworkers and risk of lung cancer, and investigated the potential functions of SNPs in thepromoter region. Our results indicated that NER gene variations may moderate DNAdamage level and lung cancer risk, and SNPs in the promoter may have an impact on thepromoter activity and expression.However, lung cancer is a complex process involving a variety of factors. Thusassociations of variations in other pathways with lung cancer susceptibility in coke ovenworkers need to be detected. Secondly, protein expression and protein-proteininteractions in the related population need to be investigated in numerous pathways.Finally, further researches of the association of combining effect of SNPs and othermolecular markers such as copy number variation and microRNAs with lung cancersusceptibility are warranted in the future.更多还原