热休克蛋白27、70基因遗传变异与DNA损伤、肺癌易感性和预后的关联性研究
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摘要
肺癌是目前全世界最常见的恶性肿瘤之一,其发病率呈逐年增长的趋势,由肺癌导致的死亡是全部恶性肿瘤的首位。肺癌的发生是多因素参与的多阶段过程,是环境因素和遗传因素共同作用的结果。DNA损伤是肺癌发生的重要早期分子生物学事件,焦化作业过程中产生的焦炉逸散物(coke oven emission, COE)中含有大量多环芳烃类化合物(polycyclic aromatic hydrocarbons, PAHs),可引起机体DNA损伤,是导致职业性焦炉工肺癌高发的重要原因。大量职业流行病学研究表明,即使是具有相似的外环境PAHs暴露水平的焦炉工人,DNA损伤程度存在明显差异,此外,只有少部分接触者最终发生肺癌,且发病的潜伏期长短不一,这提示个体的遗传背景决定了个体在接触环境PAHs暴露后的早期遗传毒性和肺癌易感性的差异。
大多数肺癌患者在发病时己处于晚期,5年生存率仅约15%。肺癌患者的TNM分期和治疗方式是影响其预后的最主要临床特征,研究表明,位于同一TNM分期的不同个体接受同一化疗方案或药物的疗效存在明显差异,提示个体的基本特征及遗传背景决定了个体对放化疗的敏感性及患者的预后。
热休克蛋白(heat shock proteins, HSPs)细胞内重要的机体应激保护性蛋白质,在受到热、氧化应激、毒物或其他应激反应之后迅速大量表达。HSPs作为分子伴侣,可参与新生蛋白质的合成、折叠、装配和转运至特定的细胞结构发挥其生物学作用,还可帮助变性或受损蛋白质的复性或降解。Hsp27和Hsp70是研究最为广泛的HSPs家族成员,两者可能通过发挥下列作用参与肿瘤的发生、发展和预后：(1)保护细胞或生物免遭各种有害应激因素的损害,从而赋予细胞从各种应激中恢复的能力、提高细胞的存活率；(2)作为分子伴侣维持细胞内蛋白质的正确结构和生物学活性,防止变性蛋白的不可逆性聚集,维持细胞内环境稳态；(3)可通过与重要的凋亡相关蛋白质相互作用参与抑制内源性或外源性细胞凋亡通路；(4)可通过与DNA修复酶相互作用而参与DNA损伤的修复过程。
肺癌发生和预后的个体差异性可能与关键致病基因的遗传变异有关,我们推测位于Hsp27和Hsp70基因功能区域的单核苷酸多态性(single nucleotide po lymorphism, SNP)位点可能通过调节基因的表达量而影响其功能的发挥,进而影响个体的肺癌发病风险及肺癌患者的预后。本研究结合人群流行病学和体内外生物学研究,从“基因再测序_标签SNPs的选择→人群流行病学研究→基因型-表型研究SNPs功能研究”等多个层面系统地探讨了影响Hsp27和Hsp70表达的功能性SNPs对焦炉作业人群DNA损伤、肺癌易感性和预后的影响,以期为探索职业人群的个体防护措施和寻找肺癌发病及预后的生物学标志物提供科学依据。本研究共分为五部分：
第一部分HSPB1基因多态性与肺癌易感性、预后的关联性研究Hsp27是小分子量热休克蛋白家族中的重要成员,在机体受到体内外应激因素刺激后迅速大量表达。研究表明Hsp27具有抑制细胞内源性和外源性凋亡通路的作用,且可作为许多肿瘤(如胃癌、肝癌、前列腺癌等)的预后标志物。本部分首先通过对60例健康的中国汉族人群的HSPB1基因进行再测序,根据测序结果构建出该基因的单体域(Haploblock),选择出3个tagSNPs,分别为-1271G>C、-1230G>A和-870T>C位点。在武汉1152对肺癌病例对照人群中对上述3个tagSNPs进行基因分析,多元logistic回归分析发现,-1271GC和-1271CC基因型与-1271GG基因型相比能提高肺癌发病风险(校正后OR分别为1.26和1.27,P值分别为0.013和0.073),且随着-1271C等位基因个数的增加,肺癌发病风险逐渐提高(Rrend=0.024)。其他2个tagSNPs位点与肺癌发病风险无明显差异(P>0.05)。我们继而在上海南京的500对肺癌病例对照人群中验证了-1271G>C多态性与肺癌发病风险的关系,多元logistic回归分析发现,在此人群中,-1271GC和-1271CC基因型与-1271GG基因型相比均能显著增加肺癌发病风险(校正后OR分别为1.44和1.65,P值分别为0.011和0.017),且有明显的等位基因剂量效应关系(Ptrend=0.004)。
我们进一步分析上述3个HSPB1基因tagSNPs位点与武汉248例晚期NSCLC患者预后的关系。采用Kaplain-Meier方法分析发现,与-1271GG基因型相比,携带-1271GC和-1271CC基因型患者的中位生存期(median survival time, MST)显著延长(Log-rank P=0.004)。采用多元Cox回归模型校正年龄、性别、吸烟状态、病理学分型、TNM分期、手术治疗、化疗和放疗等因素后发现,与-1271 GG基因型相比,-1271GC和-1271CC基因型能分别降低29%和45%的患者死亡危险度(校正后HR分别为0.71和0.55,P值分别为0.045和0.012)；随着携带-1271C等位基因数目的增加,患者死亡危险度逐渐降低(Ptrend=0.007)。未发现另外2个tagSNP位点与晚期NSCLC患者预后的关系(P>0.05)。我们继而在335例南京晚期NSCLC患者人群中验证了上述结果,采用Kaplain-Meier方法分析发现,与-1271GG基因型相比,携带-1271GC基因型或-1271CC基因型患者的MST显著延长(Log-rankP=0.029)。多元Cox回归分析发现,与携带-1271GG基因型相比,携带-1271GC基因型的患者能降低27%的死亡危险度(校正后HR=0.73,P=0.029)；将-1271GC和-1271CC基因型合并后分析发现,与-1271GG基因型相比,携带-1271GC+CC基因型患者的MST明显增加(Log-rank P=0.009),死亡危险度显著降低(校正后HR=0.75,P=0.032)。
本部分我们构建了中国汉族人群HSPB1基因的单体域并挑选出3个tagSNPs进行后续研究,分别在2个独立的肺癌病例对照研究人群和晚期NSCLC患者预后人群中分析发现：-1271G>C多态性的-1271C等位基因能提高中国汉族人群肺癌的发病风险、降低晚期NSCLC患者的死亡危险度。该研究结果提示HSPB1基因-1271G>C多态可能作为预测肺癌发病和预后的遗传标志物。
第二部分HSPAIL-HSPAIA-HSPAIB基因簇多态性与肺癌易感性、预后的关联性研究
HSPA1L-HSPA1A-HSPA1B基因簇位于人6p21.33的HLA-III区域,均编码产生诱导性的Hsp70蛋白。Hsp70蛋白在静息状态下低表达于胞浆,而在应激状态下大量表达于细胞核中,发挥对细胞内遗传物质和蛋白质的保护作用。研究发现在大多数肿瘤组织中均有Hsp70的高表达,肿瘤病人循环系统中的Hsp70抗原和抗体也可能作为肿瘤诊断和预后的重要生物学标志。
本部分研究结合HapMap计划公布的此区域的中国汉族人群SNPs数据信息和本实验室前期对60例中国汉族人群HSPA1L-HSPA1A-HSPA1B基因簇的再测序结果,共同挑选出该区域的6个tagSNPs位点进行后续研究。在武汉1152对肺癌病例对照人群中进行上述位点的基因分型,多元logistic回归分析发现,以HSPA1B基因+39CC基因型为对照,+39CT和+39CT+TT基因型均能显著增加肺癌发病风险(校正后OR均为1.39,P值分别为0.013和0.010)；随着+39T等位基因个数的增加,肺癌发病风险提高(Ptrend=0.026)。HSPA1B基因-912C>T和+39C>T多态位于同一个单体域中。对该基因的单体型结果发现,携带-912T-+39T单体型个体与携带-912C-+39C单体型个体比较,肺癌发病风险明显增加(校正后OR=1.37,P=0.008)。未发现其他5个tagSNPs位点(HSPA L_+2763C>T、HSPA1L_+2437A>G、HSPA1A_-110A>C、HSPA1A_+190G>C、及HSPA1B_-912C>T位点)与肺癌发病风险的关系(P>0.05)。我们继而在北京1000对肺癌病例对照人群中验证了HSPA1B基因+39C>T多态性与肺癌发病风险的关系,多元logistic回归分析发现,以HSPA1B基因+39CC基因型为对照,+39CT和+39CT+TT基因型均能显著增加肺癌发病风险(校正后OR分别为1.30和1.28,P值分别为0.038和0.048)。
我们进一步研究了上述6个tagSNPs位点与武汉281例晚期NSCLC患者预后的关系,采用Kaplain-Meier方法分析发现,与HSPA1B基因-912CC基因型相比,携带-912CT或-912TT基因型患者的MST显著缩短(Log-rank P=0.005);与HSPA1B基因+39CC基因型相比,携带+39CT基因型患者的MST显著缩短(Log-rankP=0.028)。多元Cox回归分析发现,与-912CC基因型相比,-912CT和-912TT基因型能分别增加62%和92%的患者死亡危险度(校正后HR分别为1.62和1.92,P值分别问0.001和0.017)；与+39CC基因型相比,携带+39CT+TT基因型能临界增加51%的患者死亡危险度(校正后HR=1.51,P=0.066)。对HSPA1B基因单体型和单体型对的分析结果发现,以-912C-+39C单体型为参照,携带-912C-+39T单体型的晚期NSCLC患者的死亡危险度险明显增加(校正后HR=1.51,P=5.9x10-4)；以CC／CC单体型对为参照,携带CC/TC和CC/TT单体型的晚期NSCLC患者的死亡危险度明显增加(校正后HR分别为1.57、1.78,校正后P分别为0.004、0.033)。
本部分我们挑选了HSPA1L-HSPAIA-HSPA1B基因簇的6个tagSNPs,分别在两个独立肺癌病例对照研究人群分析发现,HSPA1B基因+39C>T位点的+39T等位基因能提高肺癌发病风险。HSPA1B基因启动子区-912T等位基因和HSPA1B基因5'UTR区+39T等位基因还能增加晚期NSCLC患者的死亡危险度,此结果还需要在另一独立晚期NSCLC患者中进行验证,并分析此2个多态性位点的生物学功能。
第三部分BCL2、BAG1、APEX1基因多态性与肺癌易感性、预后的关联性研究
Hsp70主要通过与细胞内多种蛋白质结合而发挥细胞保护作用。BAG-1(Bcl-2-associated athanogene 1, Bcl-2关联永生蛋白1)是细胞内重要的凋亡抑制蛋白,其可以作为共伴侣(co-chaperone)结合在Hsp70蛋白的N端,并促进Hsp70蛋白的C端底物结合区与相应蛋白如Bcl-2对结合。Bcl-2也是细胞内重要的凋亡抑制蛋白,BAG-1可结合Bcl-2蛋白并增强其抑制凋亡的作用。上述三种蛋白(Hsp70、Bcl-2和BAG-1)相互作用,共同发挥抗凋亡的作用。此外,Hsp70还可与DNA修复蛋白HAP-1 (Human AP endonuclease 1,人无嘌呤无嘧啶核酸内切酶)相互作用增强其核酸内切酶活性。
本部分根据HapMap数据库公布的中国汉族人群SNPs数据信息选择与Hsp70相互作用的BCL2、BAG1、APEX1基因功能区域的tagSNPs位点进行研究。在武汉1152对肺癌病例对照人群中分析发现,以BCL2基因+21AA基因型为对照,+21AG和+21AG+GG基因型能显著降低肺癌发病风险(校正后OR分别为0.76和0.77,P值分别为0.025和0.027)；随着+21G等位基因个数的增加,肺癌发病风险逐渐降低(Ptrend=0.039)。以BCL2基因c.*1204GG基因型为对照,c.*1204GA和AA基因型均能临界降低肺癌发病风险(校正后OR分别为0.83和0.79,P值分别为0.050和0.087),携带c.*1204GA+AA基因型个体肺癌的发病风险显著降低(校正后OR=0.82,P=0.029)；随着c.*1204A等位基因个数的增加,肺癌发病风险逐渐降低(Ptrend=0.035)。未发现其他4个SNPs位点(如BCL2基因-938C／A位点、BAG1基因c.车1612G／C位点、APEX1基因Ile64Val和Asp148Glu位点)与肺癌发病风险的关系。将HSPA1B基因+39C>T位点与BCL2基因+21A>G、c.*1204G>A位点联合分析发现,随着携带危险性基因型数目的增多,肺癌的发病风险逐渐增加(Ptrend<0.001)。
我们进一步研究了上述三个基因的tagSNPs位点与武汉281例晚期NSCLC患者预后的关系,采用Kaplain-Meier方法分析发现,与BCL2基因c.*1204GG基因型相比,携带c.*1204GA或AA基因型患者的MST显著延长(Log-rank P=0.027)。采用多元Cox回归分析发现,与携带c.*1204GG基因型相比,c.*1204GA和c.*1204AA基因型能分别降低31%和33%的患者死亡危险度(校正后HR分别为0.69和0.67,P值分别为0.013和0.068)；随着携带c.*1204A等位基因数目的增加,患者死亡危险度逐渐降低(Ptrend=0.018)。将HSPA1B基因-912C>T和+39C>T位点与BCL2基因c.*1204G>A位点联合分析发现,随着携带危险性基因型数目的增多,晚期NSCLC患者的MST逐渐减少(Log-rank P<0.001),死亡危险度逐渐增加(Ptrend<0.001)。
本部分结果提示BCL2基因+21A>G多态的+21G等位基因和c.*1204G>A多态的c.*1204A等位基因能减低肺癌的发病风险,后者还能延长晚期NSCLC患者的MST,减低其死亡危险度。HSPA1B与BCL2基因阳性位点在提高肺癌发病风险和晚期NSCLC患者死亡危险度方面具有联合作用。本部分的人群结果还有待于在更大样本的独立人群中进行验证,并利用体内外生物学实验中分析阳性位点的功能。
第四部分HSPB1、HSPA1B、BCL2基因阳性SNPs位点与PAHs所致的DNA损伤的关联性研究
炼焦过程中产生的焦炉逸散物中含有大量致癌性PAHs,可导致机体的DNA损伤,后者是肿瘤发生的早期生物学事件,也是职业性焦炉工肺癌发病的主要原因。由于时间的限制,本课题组尚未收集到足够的焦炉工肺癌人群,但对这一特定职业PAHs暴露人群的遗传背景和外周血DNA损伤程度进行检测,将作为一个良好的反映“基因型-表型”关系的分析模型。
本部分研究共收集了某焦化厂309例工人,其中包括207例直接暴露于焦炉逸散物至少半年以上的焦炉作业工人(暴露组)和102无职业PAHs暴露的工人(对照组),采用血浆BPDE-白蛋白加合物水平作为内暴露水平对所有人群的环境暴露剂量进行校正。对本研究前三部分中发现的阳性SNPs位点(HSPB1基因-1271G>C、HSPA1B基因-912C>T和+39C>T、BCL2基因+21A>G和c.*1204G／A位点)进行基因分析,探讨与机体DNA损伤程度的关系。研究发现,焦炉作业工人外周血淋巴细胞Olive尾矩(Olive Tail Moment, OTM) (1.22±1.10)显著高于对照组(0.60±0.92)(P<0.001)；多元协方差分析校正年龄、工龄、吸烟和BPDE白蛋白加合物水平后发现,在暴露组中,与携带HSPB1基因-1271GG基因型相比,携带-1271GC、-1271CC、-1271GC+CC基因型的职业焦炉作业人群的OTM值明显增加(校正后P值分别为0.033、0.061、0.027)；随着携带-1271C等位基因数目的增加,暴露组个体外周血淋巴细胞OTM值逐渐增加(Ptrend=0.031)。随着携带HSPA1B基因-912T等位基因数目的增加,对照组个体外周血淋巴细胞OTM值逐渐增加(Ptrend=0.038)。未发现HSPA1B基因-912C>T和+39C>T位点及BCL2基因+21A>G和c.*1204G>A位点各基因型与暴露组和对照组外周血淋巴细胞OTM值的关系(P值均>0.05)。
本部分的研究结果提示,在机体受到外源性有害物质(如PAHs暴露)时,携带HSPB1基因-1271C等位基因个体的DNA损伤程度明显增加,DNA修复能力降低。结合第一部分的结果可以推测这可能是-1271C等位基因增加肺癌发病风险的重要原因,还可能是使携带-1271C等位基因的晚期NSCLC肿瘤患者提高了对放化疗的敏感性及其预后的重要原因。
第五部分HSPB1、HSPA1B基因调节区阳性SNPs位点的功能研究
位于基因翻译起始点上游含有启动子区和5’端非翻译区(5'untranslated region,5'UTR),前者的DNA转录元件可以和相应的转录因子结合而启动基因的转录,后者的二级结构具有维持mRNA稳定性的作用。因此,位于这2个区域的SNPs位点可能通过影响上述调节机制而改变基因的表达水平。本部分主要对第一部分研究发现的位于HSPB1基因启动子区-1271G>C位点和第二部分发现的位于HSPA1B基因启动子区-912C>T位点和5'UTR区+39C>T位点对相应蛋白表达水平的影响进行功能研究。
我们采用报告基因试验检测了上述三个位点对报告基因荧光素酶活性的影响,采用Western blotting方法检测了-1271G>C基因型对肺癌患者肺癌组织及癌旁组织中Hsp27蛋白表达的影响。结果发现在16HBE、H446、A549和HepG2四种细胞株中,含-1271C等位基因的HSPB1基因启动子驱动的荧光素酶活性均明显低于含-1271G等位基因的HSPB1基因启动子(P值均<0.05)。Western blotting检测发现Hsp27蛋白在肺癌组织的表达量明显高于在癌旁组织中的表达；在肺癌组织中,携带上述三种不同-1271G>C基因型个体的Hsp27蛋白表达没有显著性差异；在癌旁组织中,携带-1271CC基因型个体的Hsp27蛋白表达临界低于携带-1271GG基因型个体(P=0.057)。
将含4种不同HSPA1B基因-912C>T、+39C>T位点组成的单体型(即-912C-+39C、-912C-+39T、-912T-+39C、-912T-+39T单体型)的报告基因质粒转染16HBE、A549和HepG2三种细胞株后发现,-912C-+39T、-912T-+39C、-912T-+39T单体型驱动的驱动的荧光素酶活性均明显低于含-912C-+39C单体型的报告基因质粒(P均<0.05)；-912T-+39T单体型驱动的荧光素酶活性均明显低于含-912C-+39T、-912T-+39C单体型的报告基因质粒(P均<0.05)；-912T-+39C单体型驱动的荧光素酶活性均明显低于含-912C-+39C单体型的报告基因质粒(P均<0.05)；含-912C-+39T、-912T-+39C单体型的报告基因质粒驱动的荧光素酶活性无显著性差异(P均>0.05)。荧光素酶活性高低依次为：-912C-+39C>-912C-+39T=-912T-+39C>-912T-+39T。将只含有HSPA1B基因+39C>T多态性位点(分别携带+39C和+39T等位基因)的报告基因质粒转染上述三种细胞株后发现,携带+39T等位基因驱动的荧光素酶活性均明显低于+39C等位基因(P均<0.05)。
本部分的结果提示含-1271C等位基因的HSPB1基因启动子驱动的荧光素酶的表达和活性明显降低,且在癌旁组织中,携带-1271CC基因型个体的Hsp27蛋白表达临界低于携带-1271GG基因型个体；含-912T和+39T等位基因的HSPA1B基因启动子驱动的荧光素酶的表达和活性明显降低。
综上所述,本论文以肺癌病例对照人群和晚期NSCLC患者为研究对象,探讨HSPB1基因(表达Hsp27蛋白)和HSPA1L-HSPA1A-HSPA1B基因簇(表达Hsp70蛋白)及Hsp70相互作用基因(BCL2、BAG1、APEX1)的tagSNPs位点与肺癌发病风险和晚期NSCLC患者预后的关系；进一步从基因型-表型上分析了阳性位点与焦炉工DNA损伤的关系,并对HSPB1和HSPA1B基因5’端调节区阳性SNPs进行功能实验,探索了阳性位点对目基因表达的影响。本研究的创新之处在于：(1)首次发现HSPB1及HSPA1B基因多态性对肺癌发病和预后的影响(2)从肺癌的早期生物学效应(DNA损伤)、肺癌发生、肺癌预后三个方面出发,比较完整的阐明功能性SNPs的作用；(3)在多中心的独立样本中验证了流行病学研究的结果；(4)将职业性PAHs暴露的焦炉作业工人的遗传背景和DNA损伤水平联系起来,建立了一个良好的反映基因-环境相互作用的“基因型-表型”研究模型。
本研究的不足之处及有待深入研究的地方：(1)本研究发现HSPA1B和BCL2基因多态性均与肺癌发病风险和预后有关,此结果还需要在其他人群中进行验证,对HSPA1B和BCL2位点的生物学功能和基因-基因相互作用的机制还有待于进一步研究和证实；(2)本研究还需要进一步研究焦炉逸散物致焦炉工DNA损伤的生物学机制和作用靶点,为焦炉工人的个体防护措施提供依据；(3)从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,需进一步采用队列研究证实研究影响焦炉工肺癌易感性的环境和遗传生物学标志,为职业人群筛检方法的建立和肺癌发生发展机制的进一步阐明提供科学依据。
Lung cancer is the most frequently occurring malignancy with increasing incidence and is the leading cause of mortality in cancer-related deaths in China and world-wide. Individual's genetic background and the environmental pollutant both contribute to lung carcinogenesis, which is a multiple factors related multi-step process. The abundant polycyclic-aromatic-hydrocarbons (PAHs) in coke-oven emission (COE) may cause DNA damage for coke-oven workers. DNA damage is a crucial early biological event for lung carcinogenesis and the main cause for lung cancer of coke oven workers. Numerous occupational epidemiological studies have revealed that, coke oven workers with a similar environmental PAHs exposure level had dramatically different DNA damage levels. Furthermore, only a small number of those exposed develop lung cancer with distinct latency periods, suggesting that genetic heterogeneity may play a role in determining the individual's genetic toxicity and lung cancer susceptibility when exposing to carcinogenic PAHs.
Most lung cancer patients are often diagnosed at an advanced stage with only a 15% five-year survival rate. The TNM (tumor node metastasis) stage and therapy strategies are the most important clinical prognostic factors for lung cancer patients. However, lung cancer patients with similar therapy strategies and the same stages had dramatically different responses to anticancer therapies and had distinct survival outcomes, likely due to the genetic heterogeneity of gene/protein expression profiles.
Heat shock proteins (HSPs) are a group of stress inducible cyto-protective proteins that can be stimulated by heat shock, oxidative stress, cytotoxic pollutant and etc, which can initiate the cellular programmed heat shock response and follow with the synthesis of HSPs. HSPs function as molecular chaperones, binding to the nascent polypeptides to assist them proper refolding, assembling and translocation, and facilitating the misfolding peptides to repair or targeting damaged polypeptides for degradation. Hsp27 and Hsp70 were the widely studied and crucial members of the HSPs super-family, and can both participate in cancer development, proliferation and prognosis through the following pathways. First, Hsp27 and Hsp70 can protect and enable the cell to recover and survive from harmful stress stimili. Second, Hsp27 and Hsp70 can keep the correct structure and biological activities of proteins and prohibit the irreversible aggregation of degenerate proteins, which help to maintain the cellular homeostasis. Third, Hsp27 and Hsp70 can interact with many key apoptosis-related proteins and exhibit anti-apoptotic functions in both intrinsic and extrinsic apoptosis pathways. Finally, Hsp27 and Hsp70 may also participate in the DNA repair pathways by interacting with Uracil-DNA glycosylase (UDG), Human AP endonuclease 1 (HAP1), human mutL homolog 1 (hMLH1) and human mutL homolog 2 (hMLH2) proteins.
The heterogeneity of lung cancer susceptibility and prognosis may be due to the genetic variants of the key process genes. As a result, we hypothesized that the single nucleotide polymorphisms (SNPs) which exist in the HSPB1 genes and HSPA1A-HSPA1B-HSPA1L gene cluster may affect the expression levels and/or functional activities of Hsp27 and Hsp70 proteins and contribute to lung cancer susceptibility and survival. In the present study, we carried out the population based epidemiological studies and both the in vivo and in vivo biological researches, including gene resequencing, tagSNPs identification, case-control and case-only survival studies, genotype-phenotype correlation study, and the functional analyses. This study aimed to identify the associations between functional genetic variants of the hsp27 and hsp70 genes and the DNA damage levels of coke-oven workers, lung cancer susceptibility and survival, which would provide scientific research methods and rules for identifying genetic biomarkers for lung cancer risk and prognosis.
PartⅠAssociations of genetic variations in the HSPB1 gene with lung cancer risk and prognosis.
Heat shock protein 27 (Hsp27) is the key member of the small HSPs family, which can be elevated by various environmental and pathophysiologic stumili. Previous studies have confirmed the anti-apoptotic properties of Hsp27 in both intrinsic and extrinsic apoptosis pathways. Hsp27 has been reported to be an independent prognosis biomarker for many cancers, such as gastric, liver, and prostate carcinoma. In this part, we resequenced the full-length HSPB1 gene in 60 randomly selected healthy Han Chinese, constructed the haploblock structure of the HSPB1 gene, and selected 3 tagSNPs (-1271G>C,-1230G>A,-870T>C) by the Haploview4.0 software. We genotyped the three selected tagSNPs in 1152 paired Chinese lung cancer cases and controls from Wuhan city. The multivariate logistic regression analysis, after adjusted for age, sex, smoking status, pack-years and family history of cancer, revealed that, when compare with the-1271GG genotype, subjects with the-1271GC and-1271CC genotypes had a 1.26-fold and a marginal 1.27-fold risk of developing lung cancer (P=0.013,0.073, respectively), and there was a dose-response effect of the-1271C allele in increasing lung cancer risk (Ptrend=0.024). However, there were no associations between the-1230G>A and-870T>C polymorphisms and lung cancer risk (P>0.05). Another 500 paired cases and controls from Shanghai and Nanjing city were used for replication. Also in this panel, subjects with-1271GC or-1271CC genotypes also had a significantly increased risk of lung cancer (adjusted odds ratio [OR]=1.44,1.65, and P=0.011,0.017, respectively), and there was also a dose-response effect of the-1271C allele in increasing lung cancer risk (Ptrend=0.004).
We further analyzed the association between the 3 tagSNPs and the prognosis of 248 advanced NSCLC patients from Wuhan city. The Kaplain-Meier method and log-rank test showed that, patients with-1271GC or-1271CC genotype have a longer median survival time (MST) than those with-1271GG genotype (Log-rank P=0.004). After adjusted for age, sex, smoking status, histology, TNM stage, surgery, chemotherapy, and radiotherapy status, the multivariate Cox regression model revealed that, compared with the-1271GG genotype, patients with the-1271GC or-1271CC genotypes had a 29% or a 45% decreased death risk (adjusted hazard ratio [HR]=0.71,0.55,P=0.045,0.012, respectively). There was a dose-response effect of the-1271C allele in reducing death risk (Ptrend=0.007). However, there were no associations between the-1230G>A,-870T>C polymorphisms and the survival of advanced NSCLC patients (P>0.05). The effect of-1271C allele was then validated in 335 advanced NSCLC patients from Nanjing city. The Kaplain-Meier method and log-rank test showed that, advanced NSCLC patients with-1271GC or-1271CC genotype have a longer MST than those with-1271 GG genotype (Log-rank P=0.029). The multivariate Cox regression model revealed that, compared with the-1271GG genotype, patients with-1271GC genotype had a 27% decreased death risk (adjusted HR=0.73, P=0.029), and patients with-1271GC+CC genotype have a longer MST and decrease death risk (Log-rank P=0.009, adjusted HR=0.75, P=0.032).
In this part, we constructed the haploblock structure of HSPB1 gene in Han Chinese population, selected 3 tagSNPs, and found the-1271C allele in the-1271G>C polymorphism was associated with increased lung cancer susceptibility and decreased death risk for advanced NSCLC patients in two independent populations respectively. The results indicated that the HSPB1 gene-1271G>C polymorphism may be used as a genetic biomarker for lung cancer risk and prognosis.
PartⅡAssociations of SNPs in the HSPA1L-HSPA1A-HSPA1B gene cluster with lung cancer risk and prognosis.
The HSPA1L-HSPA1A-HSPA1B gene cluster was located on the HLA-III region in chromosome 6p21.3, encoding the inducible Hsp70 protein. Similar with Hsp27, the Hsp70 protein was normally low-expressed in the cytosol while over-expressed in the nucleus and exhibited cyto-protective role for cell DNA and proteins under stress conditions. Researches indicated that Hsp70 was over-expressed in numerous cancer tissues. The serum and/or plasma Hsp70 antigen and antibody may be important biomarkers for cancer diagnosis and prognosis.
According to the Han Chinese Beijing data in the HapMap project and our previous resequencing results of HSPA1L-HSPA1A-HSPA1B gene cluster, we selected and genotyped 6 tagSNPs in the region in 1152 paired Chinese lung cancer cases and controls. The multivariate logistic regression revealed that, when compare with the HSPA1B gene +39CC genotype, subjects with the+39CT and+39CT+TT genotypes had an increased risk of lung cancer development (adjusted OR=1.39, P=0.013,0.073, respectively). There was a dose-response effect of the+39T allele in increasing lung cancer risk (Ptrend=0.026). The HSPAIB gene-912CT polymorphism were located in the same haploblock. The haplotype analysis demonstrated that subjects with T.912-T+39 haplotype had an increased lung cancer risk compared to subjects with the C.912-C+39 haplotype (adjusted OR=1.37, P=0.008). However, there were no associations between the HSPA1L gene+2763C>T, HSPA1L gene+2437A>G, HSPA1A gene-110A>C, HSPA1A gene+190G>C, and HSPAIB gene-912C>T polymorphisms and lung cancer risk (P>0.05). Another 1000 paired cases and controls from Beijing city were used for replication. Also in this panel, multivariate logistic regression analysis indicated that, when compared with the HSPA1B gene+39CC genotype, subjects with+39CT or +39CT+TT genotype had significantly increased risk of lung cancer (adjusted OR=1.30, 1.28, and P=0.038,0.048, respectively).
We further analyzed the associations between the 6 tagSNPs and the prognosis of 281 advanced NSCLC patients from Wuhan city. The Kaplain-Meier method and log-rank test showed that, patients with-912CT or-912TT genotype have shorter MST than those with-912CC genotype (Log-rank P=0.005). Multivariate Cox regression model revealed that, when compared with the-912CC genotype, the-912CT or-912TT genotype was associated with increased death risk for advanced NSCLC patients (adjusted HR=1.62,1.92, P=0.001,0.017, respectively); when compared with the+39CC genotype, advanced NSCLC patients with the+39CT+TT genotypes had a 51% higher death risk (adjusted HR=1.51, P=0.006). The haplotype analyses indicated that,-912C-+39T haplotype was associated with increased death risk for advanced NSCLC patients compared to the.912C-+39C haplotype (adjusted HR=1.51, P=5.9×10-4). In addition, the death risk of advanced NSCLC patients with CC/TC or CC/TT diplotype were significantly increased when compared with patients with the CC/CC diplotype (adjusted HR=1.57,1.78, P=0.004,0.033, respectively).
In this part, we selected 6 tagSNPs in the HSPA1L-HSPA1A-HSPA1B gene cluster, and found consistently that the+39T allele in the HSPA1B gene+39C>T polymorphism was associated with increased lung cancer risk in two independent lung cancer case-control studies. In addition, advanced NSCLC patients with the-912T allele in the promoter of HSPA1B and the+39T allele in the 5'UTR of HSPA1B had increased death risk. These results should be validated in another independent population, and the biological functions of the positive SNPs need further investigations.
Part III Associations of genetic variations in the BCL2, BAG1,APEX1 gene and lung with cancer risk and prognosis.
Hsp70 facilitated its cyto-protection role by interacted with numerous proteins. B cell lymphoma-2 (Bcl-2) and Bcl-2-associated athanogene 1 (BAG-1) were important anti-apoptotic proteins. The BAG-1 protein was initially identified as a binding partner of the anti-cell death protein Bcl-2 and was shown to be involved in the regulation of apoptosis. BAG-1 can bind with Bcl-2 and enhance its anti-apoptotic activity. It is now widely accepted that BAG-1 was the co-chaperone of Hsp70 through binding to the ATP-binding domain of Hsp70 and promoting the interaction between Hsp70 and Bcl-2, and then enhance the structure and activities of Bcl-2. The Hsp70, Bcl-2, and BAG-1 can form a protein complex and took parts in the inhibition of apoptosis pathways. In addition, Hsp70 could interact with DNA repair protein HAP-1 and enhance its endonuclease activities.
According to the Han Chinese Beijing data in the HapMap project, we selected the tagSNPs located in the functional region of BCL2, BAG1, and APEX1 gene. We genotyped the selected tagSNPs in 1152 paired Chinese lung cancer cases and controls, and found that, when compared with the BCL2 gene+21 AA genotype, subjects with the +21AG or+21AG+GG genotypes had a significantly decreased risk of lung cancer development (adjusted OR=0.76,0.77,P=0.025,0.027, respectively). There was a dose-response effect of the+21G allele in decreasing lung cancer risk (Ptrend=0.039). Compared to the BCL2 gene c* 1204GG genotype, the c* 1204GA and AA genotype were associated with marginal decrease risk of lung cancer development (adjusted OR=0.83,0.79, P=0.050,0.087, respectively), while the c.* 1204GA+AA genotype was associated with a significant decrease risk of lung cancer (adjusted OR=0.82, P=0.029). There was also a dose-response effect of the c.*1204A allele in decreasing lung cancer susceptibility (Ptrend=0.039). However, there were no associations between the other tagSNPs (BCL2 gene-938C/A, BAG1 gene c.*1612G/C, and APEX1 gene Ile64Val, Asp148Glu polymorphisms) and lung cancer susceptibility (P>0.05).
Combination analysis of the HSPA1B gene+39CT+TT genotype, the BCL2 gene +21AA genotype and c.*1204GG genotype assessed by logistic regression models revealed that there was a dose-response effect of the risky genotype in increasing the risk of lung cancer development (Ptrend<0.001).
We further analyzed the associations between the selected tagSNPs and the prognosis of 281 advanced NSCLC patients. The Kaplain-Meier method and log-rank test showed that, patients with c* 1204GA or AA genotype have higher median MST than those with the c* 1204GG genotype (Log-rank P=0.027). Multivariate Cox regression model revealed that, when compared with the c.*1204GG genotype, the c.*1204GA or AA genotype genotypes was associated with decreased death risk for advanced NSCLC patients (adjusted HR=0.69,0.67, P=0.013,0.068, respectively). There was a dose-response effect of the c.*1204A allele in reducing death risk (Ptrend=0.018).
Combination analysis of the HSPAIB gene-912CT+TT and+39CT+TT genotype, the BCL2 gene c* 1204GG genotype assessed by multivariate Cox regression models revealed that there was a dose-response effect of the risky genotype in decreasing MST and increasing death risk for advanced NSCLC patients (Ptrend<0.001).
The results of this part demonstrated that the+21G allele of+21 A>G polymorphism and the c.*1204A allele of the c.*1204G>A polymorphism in the BCL2 gene were associated with decreased lung cancer susceptibility and decreased death risk for advanced NSCLC patients. The risky genotypes of positive SNPs in HSPAIB and BCL2 gene combinatorially contributed to increase the risk of lung cancer development and death of advanced NSCLC patients. The epidemiological results of this part warrant validation by larger populations, and the biological functions of the risk SNPs need further investigations.
PartⅣAssociations of risk SNPs in the HSPB1, HSPAIB, and BCL2 genes with DNA damage levels caused by PAHs.
The abundant PAHs in coke-oven emission (COE) may cause DNA damage, which was an important early biological event in carcinogenesis and the main cause for lung cancer of coke oven workers. Determination of the genetic background and DNA damage levels of the occupational PAHs exposed coke-oven workers will provide us with a good model for analyzing the "genotype-phenotype" relationship.
A total of 309 male subjects were recruited from a steel plant in Taiyuan, northern China, of whom 207 workers had been occupationally exposed to COE and were defined as the exposed group. The other 102 workers from the same factories without COE exposure in their workplaces were used as the control group. We genotyped the above positive SNPs (HSPB1 gene-1271G>C, HSPA1B gene-912C>T and+39C>T、BCL2 gene+21A>G and c.*1204G/A polymorphism) and evaluated their associations with the DNA damage levels of 309 workers. The results showed that the Olive Tail Moment (OTM) values in the exposed groups (1.22±1.10) were significantly higher than those in the controls (0.60±0.92) (P<0.001). In the coke-oven works (exposed group), subjects with the-1271GC,-1271CC, or-1271GC+CC genotype had marginally or significantly higher OTM values than subjects with the-1271GG genotype (P=0.033,0.061,0.027, respectively). There was a dose-response effect of the-1271C allele in increasing OTM values in the exposed group (Ptrend=0.031). In the control group, there was also a dose-response effect of the-912T allele in HSPA1B gene in increasing OTM values (Ptrend=0.038). However, there were no relationships between genotypes of the other polymorphisms and the DNA damage levels in both the exposed and control group (all P>0.05).
The results of this part indicated that subjects carrying the-1271C allele of HSPB1 gene had higher DNA damage levels and lower DNA repair capacities (DRC) when exposed to exogenous cytotoxic factors like PAHs. Considering the results of Part I, we can hypothesize that the lower DRC induced by-1271C allele may be an important reason for the increase risk for lung carcinogenesis. Furthermore, the decreased DRC may render the advanced NSCLC patients more sensitive to chemo-and radio-therapy with improved prognosis.
PartⅤFunctional analysis of the HSPB1 gene-1271G>C polymorphism and HSPA1B gene-912C>T and+39C>T polymorphism
The promoter region and 5'untranslated region (5'UTR) were located above the translation start coden. The cis-transcription elements in the promoter can interact with the corresponding transcription factors and then initiate the transcription of target genes, while the secondary structure of the 5'UTR may stabilize the mRNA. Therefore, genetic variations in the above two regions may change the regulation mechanism and affect the expression level of the target gene. We will analyze the biological function of the HSPB1 gene-1271G>C and the HSPA1B gene-912C>T and+39C>T polymorphisms in this part.
Dual luciferase reporter assays were carried out to determinate the effects of the above three SNPs on the relative luciferase activities (RLA). Western blotting was also used to detect the expression levels of Hsp27 protein in the tumor and corresponding normal tissues of lung cancer patients. It was showed that the RLA driven by the promoter containing-1271C allele was significantly lower than that driven by the promoter containing-1271G allele in the 16HBE, H446, A549, and HepG2 cell lines (P < 0.05 for all). Hsp27 protein was over-expressed in lung cancer tissues when compared with that of corresponding normal tissues, and the Hsp27 expression levels in normal tissues of patients carrying the-1271CC genotype were marginally lower than those with the-1271GG genotype (P=0.057). However, the differences among three genotypes of the-1271G>C polymorphism in tumor tissues did not reach the significant level (P>0.05).
The 16HBE, A549, and HepG2 cell lines were transfected with luciferase reporter plasmids that carried four different haplotypes of the HSPA1B gene, including-912C-+39C,-912C-+39T,-912T-+39C, and-912T-+39T haplotype. The results showed that the RLA driven by the promoter containing-912C-+39T,-912T-+39C, and-912T-+39T haplotype was significantly lower than that driven by the promoter containing-912C-+39C haplotype (all P<0.05). The RLA driven by the promoter containing-912T-+39T haplotype was significantly lower than that driven by the promoter containing-912C-+39T or-912T-+39C haplotype (all P<0.05); The RLA driven by the promoter containing-912C-+39T or-912T-+39C haplotype was significantly lower than that driven by the promoter containing-912C-+39C haplotype (all P<0.05). However, the RLA driven by the promoter containing-912C-+39T or-912T-+39C haplotype was not significantly different (P>0.05). The two reporter plasmids containing only the HSPAIB gene+39C>T polymorphism+39C or+39T allele were also transfected onto the 16HBE, A549, and HepG2 cell lines, and showed that the RLA driven by the promoter containing+39T allele was significantly lower than that driven by the promoter containing+39C allele (all P<0.05).
The results of this part suggests that the-1271C allele may lead to a lower Hsp27 protein synthesis level than the-1271G allele, while lung cancer patients carried the-1271CC genotype had a marginally decreased Hsp27 expression level in the normal lung tissues. In addition, the-912T and+39T allele may also lead to a lower Hsp70 protein synthesis level than the-912C and+39C allele
In summary, we selected tagSNPs in the HSPB1 genes and HSPA1L-HSPA1A-HSPAIB gene cluster and detected their associations with lung cancer risk and prognosis. The biological functions of the positive SNPs were also evaluated by the genotype-phenotype correlation study in occupationally PAHs exposed coke-oven workers as well as by luciferase reporter assay and western blotting analysis. This study has some advantages, first, to our knowledge, this study provided the first evidence that genetic variants in the HSPB1 and HSPA1B gene were associated with lung cancer risk and prognosis in Chinese populations; second, the epidemiological studies on the early events of lung cancer (DNA damage), lung cancer development and prognosis together with the biological functional studies make a plausible explanations for the functions of the positive SNPs; finally, the correlation between genetic variations of occupational PAHs exposed coke-oven workers and their DNA damage level will give a good study model for analyzing the genotype-phenotype relationship.
There were several limitations in our study that needed to be addressed. First, the association between SNPs in the HSPA1B and BCL2 gene and lung cancer risk and prognosis should be validated by additional large population-based prospective studies and well-designed clinical investigations. Second, we should evaluate the biological mechanisms for COE induced DNA damage in the further studies. Finally, it is a complicate process from DNA damage to lung carcinogenesis. The lung cancer patients enrolled in our study can not represent the development of lung cancer of coke-oven workers. Thus associations between environmental risk factors and genetic variations lung cancer susceptibility in coke oven workers also need to be detected.
大多数肺癌患者在发病时己处于晚期,5年生存率仅约15%。肺癌患者的TNM分期和治疗方式是影响其预后的最主要临床特征,研究表明,位于同一TNM分期的不同个体接受同一化疗方案或药物的疗效存在明显差异,提示个体的基本特征及遗传背景决定了个体对放化疗的敏感性及患者的预后。
热休克蛋白(heat shock proteins, HSPs)细胞内重要的机体应激保护性蛋白质,在受到热、氧化应激、毒物或其他应激反应之后迅速大量表达。HSPs作为分子伴侣,可参与新生蛋白质的合成、折叠、装配和转运至特定的细胞结构发挥其生物学作用,还可帮助变性或受损蛋白质的复性或降解。Hsp27和Hsp70是研究最为广泛的HSPs家族成员,两者可能通过发挥下列作用参与肿瘤的发生、发展和预后：(1)保护细胞或生物免遭各种有害应激因素的损害,从而赋予细胞从各种应激中恢复的能力、提高细胞的存活率；(2)作为分子伴侣维持细胞内蛋白质的正确结构和生物学活性,防止变性蛋白的不可逆性聚集,维持细胞内环境稳态；(3)可通过与重要的凋亡相关蛋白质相互作用参与抑制内源性或外源性细胞凋亡通路；(4)可通过与DNA修复酶相互作用而参与DNA损伤的修复过程。
肺癌发生和预后的个体差异性可能与关键致病基因的遗传变异有关,我们推测位于Hsp27和Hsp70基因功能区域的单核苷酸多态性(single nucleotide po lymorphism, SNP)位点可能通过调节基因的表达量而影响其功能的发挥,进而影响个体的肺癌发病风险及肺癌患者的预后。本研究结合人群流行病学和体内外生物学研究,从“基因再测序_标签SNPs的选择→人群流行病学研究→基因型-表型研究SNPs功能研究”等多个层面系统地探讨了影响Hsp27和Hsp70表达的功能性SNPs对焦炉作业人群DNA损伤、肺癌易感性和预后的影响,以期为探索职业人群的个体防护措施和寻找肺癌发病及预后的生物学标志物提供科学依据。本研究共分为五部分：
第一部分HSPB1基因多态性与肺癌易感性、预后的关联性研究Hsp27是小分子量热休克蛋白家族中的重要成员,在机体受到体内外应激因素刺激后迅速大量表达。研究表明Hsp27具有抑制细胞内源性和外源性凋亡通路的作用,且可作为许多肿瘤(如胃癌、肝癌、前列腺癌等)的预后标志物。本部分首先通过对60例健康的中国汉族人群的HSPB1基因进行再测序,根据测序结果构建出该基因的单体域(Haploblock),选择出3个tagSNPs,分别为-1271G>C、-1230G>A和-870T>C位点。在武汉1152对肺癌病例对照人群中对上述3个tagSNPs进行基因分析,多元logistic回归分析发现,-1271GC和-1271CC基因型与-1271GG基因型相比能提高肺癌发病风险(校正后OR分别为1.26和1.27,P值分别为0.013和0.073),且随着-1271C等位基因个数的增加,肺癌发病风险逐渐提高(Rrend=0.024)。其他2个tagSNPs位点与肺癌发病风险无明显差异(P>0.05)。我们继而在上海南京的500对肺癌病例对照人群中验证了-1271G>C多态性与肺癌发病风险的关系,多元logistic回归分析发现,在此人群中,-1271GC和-1271CC基因型与-1271GG基因型相比均能显著增加肺癌发病风险(校正后OR分别为1.44和1.65,P值分别为0.011和0.017),且有明显的等位基因剂量效应关系(Ptrend=0.004)。
我们进一步分析上述3个HSPB1基因tagSNPs位点与武汉248例晚期NSCLC患者预后的关系。采用Kaplain-Meier方法分析发现,与-1271GG基因型相比,携带-1271GC和-1271CC基因型患者的中位生存期(median survival time, MST)显著延长(Log-rank P=0.004)。采用多元Cox回归模型校正年龄、性别、吸烟状态、病理学分型、TNM分期、手术治疗、化疗和放疗等因素后发现,与-1271 GG基因型相比,-1271GC和-1271CC基因型能分别降低29%和45%的患者死亡危险度(校正后HR分别为0.71和0.55,P值分别为0.045和0.012)；随着携带-1271C等位基因数目的增加,患者死亡危险度逐渐降低(Ptrend=0.007)。未发现另外2个tagSNP位点与晚期NSCLC患者预后的关系(P>0.05)。我们继而在335例南京晚期NSCLC患者人群中验证了上述结果,采用Kaplain-Meier方法分析发现,与-1271GG基因型相比,携带-1271GC基因型或-1271CC基因型患者的MST显著延长(Log-rankP=0.029)。多元Cox回归分析发现,与携带-1271GG基因型相比,携带-1271GC基因型的患者能降低27%的死亡危险度(校正后HR=0.73,P=0.029)；将-1271GC和-1271CC基因型合并后分析发现,与-1271GG基因型相比,携带-1271GC+CC基因型患者的MST明显增加(Log-rank P=0.009),死亡危险度显著降低(校正后HR=0.75,P=0.032)。
本部分我们构建了中国汉族人群HSPB1基因的单体域并挑选出3个tagSNPs进行后续研究,分别在2个独立的肺癌病例对照研究人群和晚期NSCLC患者预后人群中分析发现：-1271G>C多态性的-1271C等位基因能提高中国汉族人群肺癌的发病风险、降低晚期NSCLC患者的死亡危险度。该研究结果提示HSPB1基因-1271G>C多态可能作为预测肺癌发病和预后的遗传标志物。
第二部分HSPAIL-HSPAIA-HSPAIB基因簇多态性与肺癌易感性、预后的关联性研究
HSPA1L-HSPA1A-HSPA1B基因簇位于人6p21.33的HLA-III区域,均编码产生诱导性的Hsp70蛋白。Hsp70蛋白在静息状态下低表达于胞浆,而在应激状态下大量表达于细胞核中,发挥对细胞内遗传物质和蛋白质的保护作用。研究发现在大多数肿瘤组织中均有Hsp70的高表达,肿瘤病人循环系统中的Hsp70抗原和抗体也可能作为肿瘤诊断和预后的重要生物学标志。
本部分研究结合HapMap计划公布的此区域的中国汉族人群SNPs数据信息和本实验室前期对60例中国汉族人群HSPA1L-HSPA1A-HSPA1B基因簇的再测序结果,共同挑选出该区域的6个tagSNPs位点进行后续研究。在武汉1152对肺癌病例对照人群中进行上述位点的基因分型,多元logistic回归分析发现,以HSPA1B基因+39CC基因型为对照,+39CT和+39CT+TT基因型均能显著增加肺癌发病风险(校正后OR均为1.39,P值分别为0.013和0.010)；随着+39T等位基因个数的增加,肺癌发病风险提高(Ptrend=0.026)。HSPA1B基因-912C>T和+39C>T多态位于同一个单体域中。对该基因的单体型结果发现,携带-912T-+39T单体型个体与携带-912C-+39C单体型个体比较,肺癌发病风险明显增加(校正后OR=1.37,P=0.008)。未发现其他5个tagSNPs位点(HSPA L_+2763C>T、HSPA1L_+2437A>G、HSPA1A_-110A>C、HSPA1A_+190G>C、及HSPA1B_-912C>T位点)与肺癌发病风险的关系(P>0.05)。我们继而在北京1000对肺癌病例对照人群中验证了HSPA1B基因+39C>T多态性与肺癌发病风险的关系,多元logistic回归分析发现,以HSPA1B基因+39CC基因型为对照,+39CT和+39CT+TT基因型均能显著增加肺癌发病风险(校正后OR分别为1.30和1.28,P值分别为0.038和0.048)。
我们进一步研究了上述6个tagSNPs位点与武汉281例晚期NSCLC患者预后的关系,采用Kaplain-Meier方法分析发现,与HSPA1B基因-912CC基因型相比,携带-912CT或-912TT基因型患者的MST显著缩短(Log-rank P=0.005);与HSPA1B基因+39CC基因型相比,携带+39CT基因型患者的MST显著缩短(Log-rankP=0.028)。多元Cox回归分析发现,与-912CC基因型相比,-912CT和-912TT基因型能分别增加62%和92%的患者死亡危险度(校正后HR分别为1.62和1.92,P值分别问0.001和0.017)；与+39CC基因型相比,携带+39CT+TT基因型能临界增加51%的患者死亡危险度(校正后HR=1.51,P=0.066)。对HSPA1B基因单体型和单体型对的分析结果发现,以-912C-+39C单体型为参照,携带-912C-+39T单体型的晚期NSCLC患者的死亡危险度险明显增加(校正后HR=1.51,P=5.9x10-4)；以CC／CC单体型对为参照,携带CC/TC和CC/TT单体型的晚期NSCLC患者的死亡危险度明显增加(校正后HR分别为1.57、1.78,校正后P分别为0.004、0.033)。
本部分我们挑选了HSPA1L-HSPAIA-HSPA1B基因簇的6个tagSNPs,分别在两个独立肺癌病例对照研究人群分析发现,HSPA1B基因+39C>T位点的+39T等位基因能提高肺癌发病风险。HSPA1B基因启动子区-912T等位基因和HSPA1B基因5'UTR区+39T等位基因还能增加晚期NSCLC患者的死亡危险度,此结果还需要在另一独立晚期NSCLC患者中进行验证,并分析此2个多态性位点的生物学功能。
第三部分BCL2、BAG1、APEX1基因多态性与肺癌易感性、预后的关联性研究
Hsp70主要通过与细胞内多种蛋白质结合而发挥细胞保护作用。BAG-1(Bcl-2-associated athanogene 1, Bcl-2关联永生蛋白1)是细胞内重要的凋亡抑制蛋白,其可以作为共伴侣(co-chaperone)结合在Hsp70蛋白的N端,并促进Hsp70蛋白的C端底物结合区与相应蛋白如Bcl-2对结合。Bcl-2也是细胞内重要的凋亡抑制蛋白,BAG-1可结合Bcl-2蛋白并增强其抑制凋亡的作用。上述三种蛋白(Hsp70、Bcl-2和BAG-1)相互作用,共同发挥抗凋亡的作用。此外,Hsp70还可与DNA修复蛋白HAP-1 (Human AP endonuclease 1,人无嘌呤无嘧啶核酸内切酶)相互作用增强其核酸内切酶活性。
本部分根据HapMap数据库公布的中国汉族人群SNPs数据信息选择与Hsp70相互作用的BCL2、BAG1、APEX1基因功能区域的tagSNPs位点进行研究。在武汉1152对肺癌病例对照人群中分析发现,以BCL2基因+21AA基因型为对照,+21AG和+21AG+GG基因型能显著降低肺癌发病风险(校正后OR分别为0.76和0.77,P值分别为0.025和0.027)；随着+21G等位基因个数的增加,肺癌发病风险逐渐降低(Ptrend=0.039)。以BCL2基因c.*1204GG基因型为对照,c.*1204GA和AA基因型均能临界降低肺癌发病风险(校正后OR分别为0.83和0.79,P值分别为0.050和0.087),携带c.*1204GA+AA基因型个体肺癌的发病风险显著降低(校正后OR=0.82,P=0.029)；随着c.*1204A等位基因个数的增加,肺癌发病风险逐渐降低(Ptrend=0.035)。未发现其他4个SNPs位点(如BCL2基因-938C／A位点、BAG1基因c.车1612G／C位点、APEX1基因Ile64Val和Asp148Glu位点)与肺癌发病风险的关系。将HSPA1B基因+39C>T位点与BCL2基因+21A>G、c.*1204G>A位点联合分析发现,随着携带危险性基因型数目的增多,肺癌的发病风险逐渐增加(Ptrend<0.001)。
我们进一步研究了上述三个基因的tagSNPs位点与武汉281例晚期NSCLC患者预后的关系,采用Kaplain-Meier方法分析发现,与BCL2基因c.*1204GG基因型相比,携带c.*1204GA或AA基因型患者的MST显著延长(Log-rank P=0.027)。采用多元Cox回归分析发现,与携带c.*1204GG基因型相比,c.*1204GA和c.*1204AA基因型能分别降低31%和33%的患者死亡危险度(校正后HR分别为0.69和0.67,P值分别为0.013和0.068)；随着携带c.*1204A等位基因数目的增加,患者死亡危险度逐渐降低(Ptrend=0.018)。将HSPA1B基因-912C>T和+39C>T位点与BCL2基因c.*1204G>A位点联合分析发现,随着携带危险性基因型数目的增多,晚期NSCLC患者的MST逐渐减少(Log-rank P<0.001),死亡危险度逐渐增加(Ptrend<0.001)。
本部分结果提示BCL2基因+21A>G多态的+21G等位基因和c.*1204G>A多态的c.*1204A等位基因能减低肺癌的发病风险,后者还能延长晚期NSCLC患者的MST,减低其死亡危险度。HSPA1B与BCL2基因阳性位点在提高肺癌发病风险和晚期NSCLC患者死亡危险度方面具有联合作用。本部分的人群结果还有待于在更大样本的独立人群中进行验证,并利用体内外生物学实验中分析阳性位点的功能。
第四部分HSPB1、HSPA1B、BCL2基因阳性SNPs位点与PAHs所致的DNA损伤的关联性研究
炼焦过程中产生的焦炉逸散物中含有大量致癌性PAHs,可导致机体的DNA损伤,后者是肿瘤发生的早期生物学事件,也是职业性焦炉工肺癌发病的主要原因。由于时间的限制,本课题组尚未收集到足够的焦炉工肺癌人群,但对这一特定职业PAHs暴露人群的遗传背景和外周血DNA损伤程度进行检测,将作为一个良好的反映“基因型-表型”关系的分析模型。
本部分研究共收集了某焦化厂309例工人,其中包括207例直接暴露于焦炉逸散物至少半年以上的焦炉作业工人(暴露组)和102无职业PAHs暴露的工人(对照组),采用血浆BPDE-白蛋白加合物水平作为内暴露水平对所有人群的环境暴露剂量进行校正。对本研究前三部分中发现的阳性SNPs位点(HSPB1基因-1271G>C、HSPA1B基因-912C>T和+39C>T、BCL2基因+21A>G和c.*1204G／A位点)进行基因分析,探讨与机体DNA损伤程度的关系。研究发现,焦炉作业工人外周血淋巴细胞Olive尾矩(Olive Tail Moment, OTM) (1.22±1.10)显著高于对照组(0.60±0.92)(P<0.001)；多元协方差分析校正年龄、工龄、吸烟和BPDE白蛋白加合物水平后发现,在暴露组中,与携带HSPB1基因-1271GG基因型相比,携带-1271GC、-1271CC、-1271GC+CC基因型的职业焦炉作业人群的OTM值明显增加(校正后P值分别为0.033、0.061、0.027)；随着携带-1271C等位基因数目的增加,暴露组个体外周血淋巴细胞OTM值逐渐增加(Ptrend=0.031)。随着携带HSPA1B基因-912T等位基因数目的增加,对照组个体外周血淋巴细胞OTM值逐渐增加(Ptrend=0.038)。未发现HSPA1B基因-912C>T和+39C>T位点及BCL2基因+21A>G和c.*1204G>A位点各基因型与暴露组和对照组外周血淋巴细胞OTM值的关系(P值均>0.05)。
本部分的研究结果提示,在机体受到外源性有害物质(如PAHs暴露)时,携带HSPB1基因-1271C等位基因个体的DNA损伤程度明显增加,DNA修复能力降低。结合第一部分的结果可以推测这可能是-1271C等位基因增加肺癌发病风险的重要原因,还可能是使携带-1271C等位基因的晚期NSCLC肿瘤患者提高了对放化疗的敏感性及其预后的重要原因。
第五部分HSPB1、HSPA1B基因调节区阳性SNPs位点的功能研究
位于基因翻译起始点上游含有启动子区和5’端非翻译区(5'untranslated region,5'UTR),前者的DNA转录元件可以和相应的转录因子结合而启动基因的转录,后者的二级结构具有维持mRNA稳定性的作用。因此,位于这2个区域的SNPs位点可能通过影响上述调节机制而改变基因的表达水平。本部分主要对第一部分研究发现的位于HSPB1基因启动子区-1271G>C位点和第二部分发现的位于HSPA1B基因启动子区-912C>T位点和5'UTR区+39C>T位点对相应蛋白表达水平的影响进行功能研究。
我们采用报告基因试验检测了上述三个位点对报告基因荧光素酶活性的影响,采用Western blotting方法检测了-1271G>C基因型对肺癌患者肺癌组织及癌旁组织中Hsp27蛋白表达的影响。结果发现在16HBE、H446、A549和HepG2四种细胞株中,含-1271C等位基因的HSPB1基因启动子驱动的荧光素酶活性均明显低于含-1271G等位基因的HSPB1基因启动子(P值均<0.05)。Western blotting检测发现Hsp27蛋白在肺癌组织的表达量明显高于在癌旁组织中的表达；在肺癌组织中,携带上述三种不同-1271G>C基因型个体的Hsp27蛋白表达没有显著性差异；在癌旁组织中,携带-1271CC基因型个体的Hsp27蛋白表达临界低于携带-1271GG基因型个体(P=0.057)。
将含4种不同HSPA1B基因-912C>T、+39C>T位点组成的单体型(即-912C-+39C、-912C-+39T、-912T-+39C、-912T-+39T单体型)的报告基因质粒转染16HBE、A549和HepG2三种细胞株后发现,-912C-+39T、-912T-+39C、-912T-+39T单体型驱动的驱动的荧光素酶活性均明显低于含-912C-+39C单体型的报告基因质粒(P均<0.05)；-912T-+39T单体型驱动的荧光素酶活性均明显低于含-912C-+39T、-912T-+39C单体型的报告基因质粒(P均<0.05)；-912T-+39C单体型驱动的荧光素酶活性均明显低于含-912C-+39C单体型的报告基因质粒(P均<0.05)；含-912C-+39T、-912T-+39C单体型的报告基因质粒驱动的荧光素酶活性无显著性差异(P均>0.05)。荧光素酶活性高低依次为：-912C-+39C>-912C-+39T=-912T-+39C>-912T-+39T。将只含有HSPA1B基因+39C>T多态性位点(分别携带+39C和+39T等位基因)的报告基因质粒转染上述三种细胞株后发现,携带+39T等位基因驱动的荧光素酶活性均明显低于+39C等位基因(P均<0.05)。
本部分的结果提示含-1271C等位基因的HSPB1基因启动子驱动的荧光素酶的表达和活性明显降低,且在癌旁组织中,携带-1271CC基因型个体的Hsp27蛋白表达临界低于携带-1271GG基因型个体；含-912T和+39T等位基因的HSPA1B基因启动子驱动的荧光素酶的表达和活性明显降低。
综上所述,本论文以肺癌病例对照人群和晚期NSCLC患者为研究对象,探讨HSPB1基因(表达Hsp27蛋白)和HSPA1L-HSPA1A-HSPA1B基因簇(表达Hsp70蛋白)及Hsp70相互作用基因(BCL2、BAG1、APEX1)的tagSNPs位点与肺癌发病风险和晚期NSCLC患者预后的关系；进一步从基因型-表型上分析了阳性位点与焦炉工DNA损伤的关系,并对HSPB1和HSPA1B基因5’端调节区阳性SNPs进行功能实验,探索了阳性位点对目基因表达的影响。本研究的创新之处在于：(1)首次发现HSPB1及HSPA1B基因多态性对肺癌发病和预后的影响(2)从肺癌的早期生物学效应(DNA损伤)、肺癌发生、肺癌预后三个方面出发,比较完整的阐明功能性SNPs的作用；(3)在多中心的独立样本中验证了流行病学研究的结果；(4)将职业性PAHs暴露的焦炉作业工人的遗传背景和DNA损伤水平联系起来,建立了一个良好的反映基因-环境相互作用的“基因型-表型”研究模型。
本研究的不足之处及有待深入研究的地方：(1)本研究发现HSPA1B和BCL2基因多态性均与肺癌发病风险和预后有关,此结果还需要在其他人群中进行验证,对HSPA1B和BCL2位点的生物学功能和基因-基因相互作用的机制还有待于进一步研究和证实；(2)本研究还需要进一步研究焦炉逸散物致焦炉工DNA损伤的生物学机制和作用靶点,为焦炉工人的个体防护措施提供依据；(3)从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,需进一步采用队列研究证实研究影响焦炉工肺癌易感性的环境和遗传生物学标志,为职业人群筛检方法的建立和肺癌发生发展机制的进一步阐明提供科学依据。
Lung cancer is the most frequently occurring malignancy with increasing incidence and is the leading cause of mortality in cancer-related deaths in China and world-wide. Individual's genetic background and the environmental pollutant both contribute to lung carcinogenesis, which is a multiple factors related multi-step process. The abundant polycyclic-aromatic-hydrocarbons (PAHs) in coke-oven emission (COE) may cause DNA damage for coke-oven workers. DNA damage is a crucial early biological event for lung carcinogenesis and the main cause for lung cancer of coke oven workers. Numerous occupational epidemiological studies have revealed that, coke oven workers with a similar environmental PAHs exposure level had dramatically different DNA damage levels. Furthermore, only a small number of those exposed develop lung cancer with distinct latency periods, suggesting that genetic heterogeneity may play a role in determining the individual's genetic toxicity and lung cancer susceptibility when exposing to carcinogenic PAHs.
Most lung cancer patients are often diagnosed at an advanced stage with only a 15% five-year survival rate. The TNM (tumor node metastasis) stage and therapy strategies are the most important clinical prognostic factors for lung cancer patients. However, lung cancer patients with similar therapy strategies and the same stages had dramatically different responses to anticancer therapies and had distinct survival outcomes, likely due to the genetic heterogeneity of gene/protein expression profiles.
Heat shock proteins (HSPs) are a group of stress inducible cyto-protective proteins that can be stimulated by heat shock, oxidative stress, cytotoxic pollutant and etc, which can initiate the cellular programmed heat shock response and follow with the synthesis of HSPs. HSPs function as molecular chaperones, binding to the nascent polypeptides to assist them proper refolding, assembling and translocation, and facilitating the misfolding peptides to repair or targeting damaged polypeptides for degradation. Hsp27 and Hsp70 were the widely studied and crucial members of the HSPs super-family, and can both participate in cancer development, proliferation and prognosis through the following pathways. First, Hsp27 and Hsp70 can protect and enable the cell to recover and survive from harmful stress stimili. Second, Hsp27 and Hsp70 can keep the correct structure and biological activities of proteins and prohibit the irreversible aggregation of degenerate proteins, which help to maintain the cellular homeostasis. Third, Hsp27 and Hsp70 can interact with many key apoptosis-related proteins and exhibit anti-apoptotic functions in both intrinsic and extrinsic apoptosis pathways. Finally, Hsp27 and Hsp70 may also participate in the DNA repair pathways by interacting with Uracil-DNA glycosylase (UDG), Human AP endonuclease 1 (HAP1), human mutL homolog 1 (hMLH1) and human mutL homolog 2 (hMLH2) proteins.
The heterogeneity of lung cancer susceptibility and prognosis may be due to the genetic variants of the key process genes. As a result, we hypothesized that the single nucleotide polymorphisms (SNPs) which exist in the HSPB1 genes and HSPA1A-HSPA1B-HSPA1L gene cluster may affect the expression levels and/or functional activities of Hsp27 and Hsp70 proteins and contribute to lung cancer susceptibility and survival. In the present study, we carried out the population based epidemiological studies and both the in vivo and in vivo biological researches, including gene resequencing, tagSNPs identification, case-control and case-only survival studies, genotype-phenotype correlation study, and the functional analyses. This study aimed to identify the associations between functional genetic variants of the hsp27 and hsp70 genes and the DNA damage levels of coke-oven workers, lung cancer susceptibility and survival, which would provide scientific research methods and rules for identifying genetic biomarkers for lung cancer risk and prognosis.
PartⅠAssociations of genetic variations in the HSPB1 gene with lung cancer risk and prognosis.
Heat shock protein 27 (Hsp27) is the key member of the small HSPs family, which can be elevated by various environmental and pathophysiologic stumili. Previous studies have confirmed the anti-apoptotic properties of Hsp27 in both intrinsic and extrinsic apoptosis pathways. Hsp27 has been reported to be an independent prognosis biomarker for many cancers, such as gastric, liver, and prostate carcinoma. In this part, we resequenced the full-length HSPB1 gene in 60 randomly selected healthy Han Chinese, constructed the haploblock structure of the HSPB1 gene, and selected 3 tagSNPs (-1271G>C,-1230G>A,-870T>C) by the Haploview4.0 software. We genotyped the three selected tagSNPs in 1152 paired Chinese lung cancer cases and controls from Wuhan city. The multivariate logistic regression analysis, after adjusted for age, sex, smoking status, pack-years and family history of cancer, revealed that, when compare with the-1271GG genotype, subjects with the-1271GC and-1271CC genotypes had a 1.26-fold and a marginal 1.27-fold risk of developing lung cancer (P=0.013,0.073, respectively), and there was a dose-response effect of the-1271C allele in increasing lung cancer risk (Ptrend=0.024). However, there were no associations between the-1230G>A and-870T>C polymorphisms and lung cancer risk (P>0.05). Another 500 paired cases and controls from Shanghai and Nanjing city were used for replication. Also in this panel, subjects with-1271GC or-1271CC genotypes also had a significantly increased risk of lung cancer (adjusted odds ratio [OR]=1.44,1.65, and P=0.011,0.017, respectively), and there was also a dose-response effect of the-1271C allele in increasing lung cancer risk (Ptrend=0.004).
We further analyzed the association between the 3 tagSNPs and the prognosis of 248 advanced NSCLC patients from Wuhan city. The Kaplain-Meier method and log-rank test showed that, patients with-1271GC or-1271CC genotype have a longer median survival time (MST) than those with-1271GG genotype (Log-rank P=0.004). After adjusted for age, sex, smoking status, histology, TNM stage, surgery, chemotherapy, and radiotherapy status, the multivariate Cox regression model revealed that, compared with the-1271GG genotype, patients with the-1271GC or-1271CC genotypes had a 29% or a 45% decreased death risk (adjusted hazard ratio [HR]=0.71,0.55,P=0.045,0.012, respectively). There was a dose-response effect of the-1271C allele in reducing death risk (Ptrend=0.007). However, there were no associations between the-1230G>A,-870T>C polymorphisms and the survival of advanced NSCLC patients (P>0.05). The effect of-1271C allele was then validated in 335 advanced NSCLC patients from Nanjing city. The Kaplain-Meier method and log-rank test showed that, advanced NSCLC patients with-1271GC or-1271CC genotype have a longer MST than those with-1271 GG genotype (Log-rank P=0.029). The multivariate Cox regression model revealed that, compared with the-1271GG genotype, patients with-1271GC genotype had a 27% decreased death risk (adjusted HR=0.73, P=0.029), and patients with-1271GC+CC genotype have a longer MST and decrease death risk (Log-rank P=0.009, adjusted HR=0.75, P=0.032).
In this part, we constructed the haploblock structure of HSPB1 gene in Han Chinese population, selected 3 tagSNPs, and found the-1271C allele in the-1271G>C polymorphism was associated with increased lung cancer susceptibility and decreased death risk for advanced NSCLC patients in two independent populations respectively. The results indicated that the HSPB1 gene-1271G>C polymorphism may be used as a genetic biomarker for lung cancer risk and prognosis.
PartⅡAssociations of SNPs in the HSPA1L-HSPA1A-HSPA1B gene cluster with lung cancer risk and prognosis.
The HSPA1L-HSPA1A-HSPA1B gene cluster was located on the HLA-III region in chromosome 6p21.3, encoding the inducible Hsp70 protein. Similar with Hsp27, the Hsp70 protein was normally low-expressed in the cytosol while over-expressed in the nucleus and exhibited cyto-protective role for cell DNA and proteins under stress conditions. Researches indicated that Hsp70 was over-expressed in numerous cancer tissues. The serum and/or plasma Hsp70 antigen and antibody may be important biomarkers for cancer diagnosis and prognosis.
According to the Han Chinese Beijing data in the HapMap project and our previous resequencing results of HSPA1L-HSPA1A-HSPA1B gene cluster, we selected and genotyped 6 tagSNPs in the region in 1152 paired Chinese lung cancer cases and controls. The multivariate logistic regression revealed that, when compare with the HSPA1B gene +39CC genotype, subjects with the+39CT and+39CT+TT genotypes had an increased risk of lung cancer development (adjusted OR=1.39, P=0.013,0.073, respectively). There was a dose-response effect of the+39T allele in increasing lung cancer risk (Ptrend=0.026). The HSPAIB gene-912C
We further analyzed the associations between the 6 tagSNPs and the prognosis of 281 advanced NSCLC patients from Wuhan city. The Kaplain-Meier method and log-rank test showed that, patients with-912CT or-912TT genotype have shorter MST than those with-912CC genotype (Log-rank P=0.005). Multivariate Cox regression model revealed that, when compared with the-912CC genotype, the-912CT or-912TT genotype was associated with increased death risk for advanced NSCLC patients (adjusted HR=1.62,1.92, P=0.001,0.017, respectively); when compared with the+39CC genotype, advanced NSCLC patients with the+39CT+TT genotypes had a 51% higher death risk (adjusted HR=1.51, P=0.006). The haplotype analyses indicated that,-912C-+39T haplotype was associated with increased death risk for advanced NSCLC patients compared to the.912C-+39C haplotype (adjusted HR=1.51, P=5.9×10-4). In addition, the death risk of advanced NSCLC patients with CC/TC or CC/TT diplotype were significantly increased when compared with patients with the CC/CC diplotype (adjusted HR=1.57,1.78, P=0.004,0.033, respectively).
In this part, we selected 6 tagSNPs in the HSPA1L-HSPA1A-HSPA1B gene cluster, and found consistently that the+39T allele in the HSPA1B gene+39C>T polymorphism was associated with increased lung cancer risk in two independent lung cancer case-control studies. In addition, advanced NSCLC patients with the-912T allele in the promoter of HSPA1B and the+39T allele in the 5'UTR of HSPA1B had increased death risk. These results should be validated in another independent population, and the biological functions of the positive SNPs need further investigations.
Part III Associations of genetic variations in the BCL2, BAG1,APEX1 gene and lung with cancer risk and prognosis.
Hsp70 facilitated its cyto-protection role by interacted with numerous proteins. B cell lymphoma-2 (Bcl-2) and Bcl-2-associated athanogene 1 (BAG-1) were important anti-apoptotic proteins. The BAG-1 protein was initially identified as a binding partner of the anti-cell death protein Bcl-2 and was shown to be involved in the regulation of apoptosis. BAG-1 can bind with Bcl-2 and enhance its anti-apoptotic activity. It is now widely accepted that BAG-1 was the co-chaperone of Hsp70 through binding to the ATP-binding domain of Hsp70 and promoting the interaction between Hsp70 and Bcl-2, and then enhance the structure and activities of Bcl-2. The Hsp70, Bcl-2, and BAG-1 can form a protein complex and took parts in the inhibition of apoptosis pathways. In addition, Hsp70 could interact with DNA repair protein HAP-1 and enhance its endonuclease activities.
According to the Han Chinese Beijing data in the HapMap project, we selected the tagSNPs located in the functional region of BCL2, BAG1, and APEX1 gene. We genotyped the selected tagSNPs in 1152 paired Chinese lung cancer cases and controls, and found that, when compared with the BCL2 gene+21 AA genotype, subjects with the +21AG or+21AG+GG genotypes had a significantly decreased risk of lung cancer development (adjusted OR=0.76,0.77,P=0.025,0.027, respectively). There was a dose-response effect of the+21G allele in decreasing lung cancer risk (Ptrend=0.039). Compared to the BCL2 gene c* 1204GG genotype, the c* 1204GA and AA genotype were associated with marginal decrease risk of lung cancer development (adjusted OR=0.83,0.79, P=0.050,0.087, respectively), while the c.* 1204GA+AA genotype was associated with a significant decrease risk of lung cancer (adjusted OR=0.82, P=0.029). There was also a dose-response effect of the c.*1204A allele in decreasing lung cancer susceptibility (Ptrend=0.039). However, there were no associations between the other tagSNPs (BCL2 gene-938C/A, BAG1 gene c.*1612G/C, and APEX1 gene Ile64Val, Asp148Glu polymorphisms) and lung cancer susceptibility (P>0.05).
Combination analysis of the HSPA1B gene+39CT+TT genotype, the BCL2 gene +21AA genotype and c.*1204GG genotype assessed by logistic regression models revealed that there was a dose-response effect of the risky genotype in increasing the risk of lung cancer development (Ptrend<0.001).
We further analyzed the associations between the selected tagSNPs and the prognosis of 281 advanced NSCLC patients. The Kaplain-Meier method and log-rank test showed that, patients with c* 1204GA or AA genotype have higher median MST than those with the c* 1204GG genotype (Log-rank P=0.027). Multivariate Cox regression model revealed that, when compared with the c.*1204GG genotype, the c.*1204GA or AA genotype genotypes was associated with decreased death risk for advanced NSCLC patients (adjusted HR=0.69,0.67, P=0.013,0.068, respectively). There was a dose-response effect of the c.*1204A allele in reducing death risk (Ptrend=0.018).
Combination analysis of the HSPAIB gene-912CT+TT and+39CT+TT genotype, the BCL2 gene c* 1204GG genotype assessed by multivariate Cox regression models revealed that there was a dose-response effect of the risky genotype in decreasing MST and increasing death risk for advanced NSCLC patients (Ptrend<0.001).
The results of this part demonstrated that the+21G allele of+21 A>G polymorphism and the c.*1204A allele of the c.*1204G>A polymorphism in the BCL2 gene were associated with decreased lung cancer susceptibility and decreased death risk for advanced NSCLC patients. The risky genotypes of positive SNPs in HSPAIB and BCL2 gene combinatorially contributed to increase the risk of lung cancer development and death of advanced NSCLC patients. The epidemiological results of this part warrant validation by larger populations, and the biological functions of the risk SNPs need further investigations.
PartⅣAssociations of risk SNPs in the HSPB1, HSPAIB, and BCL2 genes with DNA damage levels caused by PAHs.
The abundant PAHs in coke-oven emission (COE) may cause DNA damage, which was an important early biological event in carcinogenesis and the main cause for lung cancer of coke oven workers. Determination of the genetic background and DNA damage levels of the occupational PAHs exposed coke-oven workers will provide us with a good model for analyzing the "genotype-phenotype" relationship.
A total of 309 male subjects were recruited from a steel plant in Taiyuan, northern China, of whom 207 workers had been occupationally exposed to COE and were defined as the exposed group. The other 102 workers from the same factories without COE exposure in their workplaces were used as the control group. We genotyped the above positive SNPs (HSPB1 gene-1271G>C, HSPA1B gene-912C>T and+39C>T、BCL2 gene+21A>G and c.*1204G/A polymorphism) and evaluated their associations with the DNA damage levels of 309 workers. The results showed that the Olive Tail Moment (OTM) values in the exposed groups (1.22±1.10) were significantly higher than those in the controls (0.60±0.92) (P<0.001). In the coke-oven works (exposed group), subjects with the-1271GC,-1271CC, or-1271GC+CC genotype had marginally or significantly higher OTM values than subjects with the-1271GG genotype (P=0.033,0.061,0.027, respectively). There was a dose-response effect of the-1271C allele in increasing OTM values in the exposed group (Ptrend=0.031). In the control group, there was also a dose-response effect of the-912T allele in HSPA1B gene in increasing OTM values (Ptrend=0.038). However, there were no relationships between genotypes of the other polymorphisms and the DNA damage levels in both the exposed and control group (all P>0.05).
The results of this part indicated that subjects carrying the-1271C allele of HSPB1 gene had higher DNA damage levels and lower DNA repair capacities (DRC) when exposed to exogenous cytotoxic factors like PAHs. Considering the results of Part I, we can hypothesize that the lower DRC induced by-1271C allele may be an important reason for the increase risk for lung carcinogenesis. Furthermore, the decreased DRC may render the advanced NSCLC patients more sensitive to chemo-and radio-therapy with improved prognosis.
PartⅤFunctional analysis of the HSPB1 gene-1271G>C polymorphism and HSPA1B gene-912C>T and+39C>T polymorphism
The promoter region and 5'untranslated region (5'UTR) were located above the translation start coden. The cis-transcription elements in the promoter can interact with the corresponding transcription factors and then initiate the transcription of target genes, while the secondary structure of the 5'UTR may stabilize the mRNA. Therefore, genetic variations in the above two regions may change the regulation mechanism and affect the expression level of the target gene. We will analyze the biological function of the HSPB1 gene-1271G>C and the HSPA1B gene-912C>T and+39C>T polymorphisms in this part.
Dual luciferase reporter assays were carried out to determinate the effects of the above three SNPs on the relative luciferase activities (RLA). Western blotting was also used to detect the expression levels of Hsp27 protein in the tumor and corresponding normal tissues of lung cancer patients. It was showed that the RLA driven by the promoter containing-1271C allele was significantly lower than that driven by the promoter containing-1271G allele in the 16HBE, H446, A549, and HepG2 cell lines (P < 0.05 for all). Hsp27 protein was over-expressed in lung cancer tissues when compared with that of corresponding normal tissues, and the Hsp27 expression levels in normal tissues of patients carrying the-1271CC genotype were marginally lower than those with the-1271GG genotype (P=0.057). However, the differences among three genotypes of the-1271G>C polymorphism in tumor tissues did not reach the significant level (P>0.05).
The 16HBE, A549, and HepG2 cell lines were transfected with luciferase reporter plasmids that carried four different haplotypes of the HSPA1B gene, including-912C-+39C,-912C-+39T,-912T-+39C, and-912T-+39T haplotype. The results showed that the RLA driven by the promoter containing-912C-+39T,-912T-+39C, and-912T-+39T haplotype was significantly lower than that driven by the promoter containing-912C-+39C haplotype (all P<0.05). The RLA driven by the promoter containing-912T-+39T haplotype was significantly lower than that driven by the promoter containing-912C-+39T or-912T-+39C haplotype (all P<0.05); The RLA driven by the promoter containing-912C-+39T or-912T-+39C haplotype was significantly lower than that driven by the promoter containing-912C-+39C haplotype (all P<0.05). However, the RLA driven by the promoter containing-912C-+39T or-912T-+39C haplotype was not significantly different (P>0.05). The two reporter plasmids containing only the HSPAIB gene+39C>T polymorphism+39C or+39T allele were also transfected onto the 16HBE, A549, and HepG2 cell lines, and showed that the RLA driven by the promoter containing+39T allele was significantly lower than that driven by the promoter containing+39C allele (all P<0.05).
The results of this part suggests that the-1271C allele may lead to a lower Hsp27 protein synthesis level than the-1271G allele, while lung cancer patients carried the-1271CC genotype had a marginally decreased Hsp27 expression level in the normal lung tissues. In addition, the-912T and+39T allele may also lead to a lower Hsp70 protein synthesis level than the-912C and+39C allele
In summary, we selected tagSNPs in the HSPB1 genes and HSPA1L-HSPA1A-HSPAIB gene cluster and detected their associations with lung cancer risk and prognosis. The biological functions of the positive SNPs were also evaluated by the genotype-phenotype correlation study in occupationally PAHs exposed coke-oven workers as well as by luciferase reporter assay and western blotting analysis. This study has some advantages, first, to our knowledge, this study provided the first evidence that genetic variants in the HSPB1 and HSPA1B gene were associated with lung cancer risk and prognosis in Chinese populations; second, the epidemiological studies on the early events of lung cancer (DNA damage), lung cancer development and prognosis together with the biological functional studies make a plausible explanations for the functions of the positive SNPs; finally, the correlation between genetic variations of occupational PAHs exposed coke-oven workers and their DNA damage level will give a good study model for analyzing the genotype-phenotype relationship.
There were several limitations in our study that needed to be addressed. First, the association between SNPs in the HSPA1B and BCL2 gene and lung cancer risk and prognosis should be validated by additional large population-based prospective studies and well-designed clinical investigations. Second, we should evaluate the biological mechanisms for COE induced DNA damage in the further studies. Finally, it is a complicate process from DNA damage to lung carcinogenesis. The lung cancer patients enrolled in our study can not represent the development of lung cancer of coke-oven workers. Thus associations between environmental risk factors and genetic variations lung cancer susceptibility in coke oven workers also need to be detected.
引文
1. Boyle P and Levin B World Cancer Report 2008. IARC Publications,2008.
2. Jemal A, Siegel R, Ward E, et al. Cancer statistics,2008. CA Cancer J Clin,2008, 58(2):71-96.
3. Yang L, Parkin DM, Li LD, et al. Estimation and projection of the national profile of cancer mortality in China:1991-2005. Br J Cancer,2004,90(11):2157-2166.
4. Shields PG and Harris CC Cancer risk and low-penetrance susceptibility genes in gene-environment interactions. J Clin Oncol,2000,18(11):2309-2315.
5. IARC Polynuclear Aromatic Compounds, Part 3, Industrial Exposures in Aluminium Production, Coal Gasification, Coke Production, and Iron and Steel Founding. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans,1984,34:65-131.
6. Bosetti C, Boffetta P and La Vecchia C Occupational exposures to polycyclic aromatic hydrocarbons, and respiratory and urinary tract cancers:a quantitative review to 2005. Ann Oncol,2007,18(3):431-446.
7. Chen Y, Bai Y, Yuan J, et al. Association of polymorphisms in AhR, CYP1A1, GSTM1, and GSTT1 genes with levels of DNA damage in peripheral blood lymphocytes among coke-oven workers. Cancer Epidemiol Biomarkers Prev,2006, 15(9):1703-1707.
8. Wang F, He Y, Guo H, et al. Genetic variants of nucleotide excision repair genes are associated with DNA damage in coke oven workers. Cancer Epidemiol Biomarkers Prev,2010,19(1):211-218.
9. Herbst RS, Heymach JV and Lippman SM Lung cancer. N Engl J Med,2008, 359(13):1367-1380.
10. Efferth T and Volm M Pharmacogenetics for individualized cancer chemotherapy. Pharmacol Ther,2005,107(2):155-176.
11. Gething MJ and Sambrook J Protein folding in the cell. Nature,1992,355(6355): 33-45.
12. Hightower LE Heat shock, stress proteins, chaperones, and proteotoxicity. Cell, 1991,66(2):191-197.
13. Macario AJ and Conway de Macario E Sick chaperones, cellular stress, and disease. N Engl J Med,2005,353(14):1489-1501.
14. Hartl FU Molecular chaperones in cellular protein folding. Nature,1996,381(6583): 571-579.
15. Lindquist S The heat-shock response. Annu Rev Biochem,1986,55:1151-1191.
16. Garrido C, Bruey JM, Fromentin A, et al. HSP27 inhibits cytochrome c-dependent activation of procaspase-9. FASEB J,1999,13(14):2061-2070.
17. Beere HM, Wolf BB, Cain K, et al. Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome. Nat Cell Biol, 2000,2(8):469-475.
18. Concannon CG, Gorman AM and Samali A On the role of Hsp27 in regulating apoptosis. Apoptosis,2003,8(1):61-70.
19. Mendez F, Sandigursky M, Franklin WA, et al. Heat-shock proteins associated with base excision repair enzymes in HeLa cells. Radiat Res,2000,153(2):186-195.
20. Nadin SB, Vargas-Roig LM, Drago G, et al. Hsp27, Hsp70 and mismatch repair proteins hMLHl and hMSH2 expression in peripheral blood lymphocytes from healthy subjects and cancer patients. Cancer Lett,2007,252(1):131-146.
21. Jolly C and Morimoto RI Role of the heat shock response and molecular chaperones in oncogenesis and cell death. J Natl Cancer Inst,2000,92(19):1564-1572.
22. Arrigo AP The cellular "networking" of mammalian Hsp27 and its functions in the control of protein folding, redox state and apoptosis. Adv Exp Med Biol,2007,594: 14-26.
23. Ciocca DR, Oesterreich S, Chamness GC, et al. Biological and clinical implications of heat shock protein 27,000 (Hsp27):a review. J Natl Cancer Inst,1993,85(19): 1558-1570.
24. Merendino AM, Paul C, Vignola AM, et al. Heat shock protein-27 protects human bronchial epithelial cells against oxidative stress-mediated apoptosis:possible implication in asthma Cell Stress Chaperones,2002,7(3):269-280.
25. Pivovarova AV, Chebotareva NA, Chernik IS, et al. Small heat shock protein Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actin. FEBS J,2007,274(22):5937-5948.
26. Mosser DD and Morimoto RI Molecular chaperones and the stress of oncogenesis. Oncogene,2004,23(16):2907-2918.
27. Ciocca DR and Calderwood SK Heat shock proteins in cancer:diagnostic, prognostic, predictive, and treatment implications. Cell Stress Chaperones,2005, 10(2):86-103.
28. Calderwood SK, Khaleque MA, Sawyer DB, et al. Heat shock proteins in cancer: chaperones of tumorigenesis. Trends Biochem Sci,2006,31(3):164-172.
29. Garrido C, Fromentin A, Bonnotte B, et al. Heat shock protein 27 enhances the tumorigenicity of immunogenic rat colon carcinoma cell clones. Cancer Res,1998, 58(23):5495-5499.
30. Kamada M, So A, Muramaki M, et al. Hsp27 knockdown using nucleotide-based
therapies inhibit tumor growth and enhance chemotherapy in human bladder cancer cells. Mol Cancer Ther,2007,6(1):299-308.
31. Bruey JM, Ducasse C, Bonniaud P, et al. Hsp27 negatively regulates cell death by interacting with cytochrome c. Nat Cell Biol,2000,2(9):645-652.
32. Paul C, Manero F, Gonin S, et al. Hsp27 as a negative regulator of cytochrome C release. Mol Cell Biol,2002,22(3):816-834.
33. Havasi A, Li Z, Wang Z, et al. Hsp27 inhibits Bax activation and apoptosis via a phosphatidylinositol 3-kinase-dependent mechanism. J Biol Chem,2008,283(18): 12305-12313.
34. Chauhan D, Li G, Hideshima T, et al. Hsp27 inhibits release of mitochondrial protein Smac in multiple myeloma cells and confers dexamethasone resistance. Blood,2003,102(9):3379-3386.
35. Charette SJ, Lavoie JN, Lambert H, et al. Inhibition of Daxx-mediated apoptosis by heat shock protein 27. Mol Cell Biol,2000,20(20):7602-7612.
36. He M, Guo H, Yang X, et al. Functional SNPs in HSPA1A gene predict risk of coronary heart disease. PLoS ONE,2009,4(3):e4851.
37. Barrett JC, Fry B, Maller J, et al. Haploview:analysis and visualization of LD and haplotype maps. Bioinformatics,2005,21(2):263-265.
38. Tregouet DA and Garelle V A new JAVA interface implementation of THESIAS: testing haplotype effects in association studies. Bioinformatics,2007,23(8): 1038-1039.
39. Stephens M and Donnelly P A comparison of bayesian methods for haplotype reconstruction from population genotype data Am J Hum Genet,2003,73(5): 1162-1169.
40. Mehlen P, Kretz-Remy C, Preville X, et al. Human hsp27, Drosophila hsp27 and human alphaB-crystallin expression-mediated increase in glutathione is essential for the protective activity of these proteins against TNFalpha-induced cell death. EMBO J,1996,15(11):2695-2706.
41. Arrigo AP, Virot S, Chaufour S, et al. Hsp27 consolidates intracellular redox homeostasis by upholding glutathione in its reduced form and by decreasing iron intracellular levels. Antioxid Redox Signal,2005,7(3-4):414-422.
42. Feng JT, Liu YK, Song HY, et al. Heat-shock protein 27:a potential biomarker for hepatocellular carcinoma identified by serum proteome analysis. Proteomics,2005, 5(17):4581-4588.
43. Korneeva I, Bongiovanni AM, Girotra M, et al. Serum antibodies to the 27-kd heat shock protein in women with gynecologic cancers. Am J Obstet Gynecol,2000, 183(1):18-21.
44. Korneeva I, Bongiovanni AM, Girotra M, et al. IgA antibodies to the 27-kDa heat-shock protein in the genital tracts of women with gynecologic cancers. Int J Cancer,2000,87(6):824-828.
45. Evgrafov OV, Mersiyanova I, Irobi J, et al. Mutant small heat-shock protein 27 causes axonal Charcot-Marie-Tooth disease and distal hereditary motor neuropathy. Nat Genet,2004,36(6):602-606.
46. Dierick I, Irobi J, Janssens S, et al. Genetic variant in the HSPB1 promoter region impairs the HSP27 stress response. Hum Mutat,2007,28(8):830-840.
47. Kim EH, Lee HJ, Lee DH, et al. Inhibition of heat shock protein 27-mediated resistance to DNA damaging agents by a novel PKC delta-V5 heptapeptide. Cancer Res,2007,67(13):6333-6341.
48. Lee YJ, Lee DH, Cho CK, et al. HSP25 inhibits radiation-induced apoptosis through reduction of PKCdelta-mediated ROS production. Oncogene,2005,24(23): 3715-3725.
49. Zhang Y and Shen X Heat shock protein 27 protects L929 cells from cisplatin-induced apoptosis by enhancing Akt activation and abating suppression of thioredoxin reductase activity. Clin Cancer Res,2007,13(10):2855-2864.
50. O'Callaghan-Sunol C, Gabai VL and Sherman MY Hsp27 modulates p53 signaling and suppresses cellular senescence. Cancer Res,2007,67(24):11779-11788.
51. Sobin LH and Wittekind CH. TNM classification of malignant tumors (6th edition). 2002, Wiley-Liss:New York.
52. Mori-Iwamoto S, Kuramitsu Y, Ryozawa S, et al. Proteomics finding heat shock protein 27 as a biomarker for resistance of pancreatic cancer cells to gemcitabine. Int J Oncol,2007,31(6):1345-1350.
53. Ciocca DR, Rozados VR, Cuello Carrion FD, et al. Hsp25 and Hsp70 in rodent tumors treated with doxorubicin and lovastatin. Cell Stress Chaperones,2003,8(1): 26-36.
54. Calderwood SK and Ciocca DR Heat shock proteins:stress proteins with Janus-like properties in cancer. Int J Hyperthermia,2008,24(1):31-39.
55. Milner CM and Campbell RD Structure and expression of the three MHC-linked HSP70 genes. Immunogenetics,1990,32(4):242-251.
56. Favatier F, Bornman L, Hightower LE, et al. Variation in hsp gene expression and Hsp polymorphism:do they contribute to differential disease susceptibility and stress tolerance? Cell Stress Chaperones,1997,2(3):141-155.
57. Vargas-Alarcon G, Londono JD, Hernandez-Pacheco G, et al. Heat shock protein 70 gene polymorphisms in Mexican patients with spondyloarthropathies. Ann Rheum Dis,2002,61(1):48-51.
58. Aron Y, Busson M, Polla BS, et al. Analysis of hsp70 gene polymorphism in allergic asthma Allergy,1999,54(2):165-170.
59. Rajalingam R, Mehra NK and Singal DP Polymorphism in heat-shock protein 70-1 (HSP70-1) gene promoter region and susceptibility to tuberculoid leprosy and pulmonary tuberculosis in Asian Indians. Indian J Exp Biol,2000,38(7):658-662.
60. Pae CU, Kim TS, Kwon OJ, et al. Polymorphisms of heat shock protein 70 gene (HSPA1A, HSPA1B and HSPA1L) and schizophrenia Neurosci Res,2005,53(1): 8-13.
61. Wu YR, Wang CK, Chen CM, et al. Analysis of heat-shock protein 70 gene polymorphisms and the risk of Parkinson's disease. Hum Genet,2004,114(3): 236-241.
62. Wang Y, Broderick P, Webb E, et al. Common 5p15.33 and 6p21.33 variants influence lung cancer risk. Nat Genet,2008,40(12):1407-1409.
63.杨晓波.博士毕业论文.华中科技大学图书馆,2007.
64. Jalbout M, Bouaouina N, Gargouri J, et al. Polymorphism of the stress protein HSP70-2 gene is associated with the susceptibility to the nasopharyngeal carcinoma Cancer Lett,2003,193(1):75-81.
65. Mestiri S, Bouaouina N, Ahmed SB, et al. Genetic variation in the tumor necrosis factor-alpha promoter region and in the stress protein hsp70-2:susceptibility and prognostic implications in breast carcinoma .Cancer,2001,91(4):672-678.
66. Debler J, Schiemann U, Seybold U, et al. Heat-shock protein HSP70-2 genotypes in patients with Crohn's disease:a more severe clinical course with intestinal
complications in presence of the PstI-polymorphism. Eur J Med Res,2003,8(3): 120-124.
67. Zhou F, Wang F, Li F, et al. Association of hsp70-2 and hsp-hom gene polymorphisms with risk of acute high-altitude illness in a Chinese population. Cell Stress Chaperones,2005,10(4):349-356.
68. Zhong L, Peng X, Hidalgo GE, et al. Antibodies to HSP70 and HSP90 in serum in non-small cell lung cancer patients. Cancer Detect Prev,2003,27(4):285-290.
69. Suzuki K, Ito Y, Wakai K, et al. Serum heat shock protein 70 levels and lung cancer risk:a case-control study nested in a large cohort study. Cancer Epidemiol Biomarkers Prev,2006,15(9):1733-1737.
70. Temple SE, Cheong KY, Ardlie KG, et al. The septic shock associated HSPA1B1267 polymorphism influences production of HSPA1A and HSPA1B. Intensive Care Med,2004,30(9):1761-1767.
71. Gress TM, Muller-Pillasch F, Weber C, et al. Differential expression of heat shock proteins in pancreatic carcinoma. Cancer Res,1994,54(2):547-551.
72. Gotz R, Kramer BW, Camarero G, et al. BAG-1 haplo-insufficiency impairs lung tumorigenesis. BMC Cancer,2004,4:85.
73. Takayama S and Reed JC Molecular chaperone targeting and regulation by BAG family proteins. Nat Cell Biol,2001,3(10):E237-E241.
74. Takayama S, Sato T, Krajewski S, et al. Cloning and functional analysis of BAG-1: a novel Bcl-2-binding protein with anti-cell death activity. Cell,1995,80(2): 279-284.
75. Cutress RI, Townsend PA, Brimmell M, et al. BAG-1 expression and function in human cancer. Br J Cancer,2002,87(8):834-839.
76. Gehring U Biological activities of HAP46/BAG-1. The HAP46/BAG-1 protein: regulator of HSP70 chaperones, DNA-binding protein and stimulator of transcription. EMBO Rep,2004,5(2):148-153.
77. Frenzel A, Grespi F, Chmelewskij W, et al. Bcl2 family proteins in carcinogenesis and the treatment of cancer. Apoptosis,2009,14(4):584-596.
78. Cory S and Adams JM The Bcl2 family:regulators of the cellular life-or-death switch. Nat Rev Cancer,2002,2(9):647-656.
79. Kim DH, Xu W, Ma C, et al. Genetic variants in the candidate genes of the apoptosis pathway and susceptibility to chronic myeloid leukemia Blood,2009, 113(11):2517-2525.
80. Faderl S, Keating MJ, Do KA, et al. Expression profile of 11 proteins and their prognostic significance in patients with chronic lymphocytic leukemia (CLL). Leukemia,2002,16(6):1045-1052.
81. Keshgegian AA, Johnston E and Cnaan A Bcl-2 oncoprotein positivity and high MIB-1 (Ki-67) proliferative rate are independent predictive markers for recurrence in prostate carcinoma Am J Clin Pathol,1998,110(4):443-449.
82. Ofner D, Riehemann K, Maier H, et al. Immunohistochemically detectable bcl-2 expression in colorectal carcinoma:correlation with tumour stage and patient survival. Br J Cancer,1995,72(4):981-985.
83. Zhang GJ, Kimijima I, Tsuchiya A, et al. The role of bcl-2 expression in breast carcinomas (Review). Oncol Rep,1998,5(5):1211-1216.
84. Bankfalvi A, Tory K, Kemper M, et al. Clinical relevance of immunohistochemical expression of p53-targeted gene products mdm-2, p21 and bcl-2 in breast carcinoma Pathol Res Pract,2000,196(7):489-501.
85. Martin B, Paesmans M, Berghmans T, et al. Role of Bcl-2 as a prognostic factor for survival in lung cancer:a systematic review of the literature with meta-analysis. Br J Cancer,2003,89(1):55-64.
86. Renouf DJ, Wood-Baker R, Ionescu DN, et al. BCL-2 expression is prognostic for improved survival in non-small cell lung cancer. J Thorac Oncol,2009,4(4): 486-491.
87. Heubner M, Wimberger P, Otterbach F, et al. Association of the AA genotype of the BCL2 (-938C>A) promoter polymorphism with better survival in ovarian cancer. Int J Biol Markers,2009,24(4):223-229.
88. Bachmann HS, Otterbach F, Callies R, et al. The AA genotype of the regulatory BCL2 promoter polymorphism (938C>A) is associated with a favorable outcome in lymph node negative invasive breast cancer patients. Clin Cancer Res,2007,13(19): 5790-5797.
89. Lehnerdt GF, Franz P, Bankfalvi A, et al. The regulatory BCL2 promoter polymorphism (-938C>A) is associated with relapse and survival of patients with oropharyngeal squamous cell carcinoma Ann Oncol,2009,20(6):1094-1099.
90. Nuckel H, Frey UH, Bau M, et al. Association of a novel regulatory polymorphism (-938C>A) in the BCL2 gene promoter with disease progression and survival in chronic lymphocytic leukemia Blood,2007,109(1):290-297.
91. Moon JH, Sohn SK, Lee MH, et al. BCL2 gene polymorphism could predict the treatment outcomes in acute myeloid leukemia patients. Leuk Res,2010,34(2): 166-172.
92. Singh NP, McCoy MT, Tice RR, et al. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res,1988,175(1):184-191.
93. Cheng J, Leng S, Dai Y, et al. Association between nucleotide excision repair gene polymorphisms and chromosomal damage in coke-oven workers. Biomarkers,2007, 12(1):76-86.
94. Wang H, Chen W, Zheng H, et al. Association between plasma BPDE-Alb adduct concentrations and DNA damage of peripheral blood lymphocytes among coke oven workers. Occup Environ Med,2007,64(11):753-758.
95.朱志良,庄志雄,黄钰,等.单细胞凝胶电泳图像分析系统的研制及应用.中华劳动卫生职业病杂志,2001,19(4)：298-300.
96. Olive PL, Banath JP and Durand RE Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the "comet" assay. Radiat Res,1990,122(1):86-94.
97.潘秋萍,孙恩林,陈胜,等.焦炉工人血清中p21蛋白和HSPs水平的研究中国职业医学,1999,26(5)：4-6.
98. Yao H, Zhang Z, Xiao Z, et al. Identification of metastasis associated proteins in human lung squamous carcinoma using two-dimensional difference gel electrophoresis and laser capture microdissection. Lung Cancer,2009,65(1):41-48.
99. httn://www.genomatix.de/nroducts/MatInsnectnr/
100. Hanai J, Dhanabal M, Karumanchi SA, et al. Endostatin causes G1 arrest of endothelial cells through inhibition of cyclin D1. J Biol Chem,2002,277(19): 16464-16469.
101. Abravaya K, Phillips B and Morimoto RI Heat shock-induced interactions of heat shock transcription factor and the human hsp70 promoter examined by in vivo footprinting. Mol Cell Biol,1991,11(1):586-592.
102. Vivinus S, Baulande S, van Zanten M, et al. An element within the 5'untranslated region of human Hsp70 mRNA which acts as a general enhancer of mRNA translation. Eur J Biochem,2001,268(7):1908-1917.
103. Rubtsova MP, Sizova DV, Dmitriev SE, et al. Distinctive properties of the 5'-untranslated region of human hsp70 mRNA. J Biol Chem,2003,278(25): 22350-22356.
1. Klein RJ, Zeiss C, Chew EY, et al. Complement factor H polymorphism in age-related macular degeneration. Science,2005,308(5720):385-389.
2. International Human Genome Sequencing Consortium. Finishing the euchromatic sequence of the human genome. Nature,2004,431(7011):931-945.
3. http://www.ncbi.nlm.nih.gov/projects/SNP.
4. The International HapMap Project. Nature,2003,426(6968):789-796.
5. http://hapmap.ncbi.nlm.nih.gov/.
6. A haplotype map of the human genome. Nature,2005,437(7063):1299-1320.
7. Matsuzaki H, Loi H, Dong S, et al. Parallel genotyping of over 10,000 SNPs using a one-primer assay on a high-density oligonucleotide array. Genome Res,2004,14(3): 414-425.
8. Teo YY, Small KS and Kwiatkowski DP Methodological challenges of genome-wide association analysis in Africa Nat Rev Genet,2010,11(2):149-160.
9. Risch N and Merikangas K The future of genetic studies of complex human diseases. Science,1996,273(5281):1516-1517.
10. Thomas DC, Haile RW and Duggan D Recent developments in genomewide association scans:a workshop summary and review. Am J Hum Genet,2005,77(3): 337-345.
11. Chen GK and Witte JS Enriching the analysis of genomewide association studies with hierarchical modeling. Am J Hum Genet,2007,81(2):397-404.
12. Clark AG Finding genes underlying risk of complex disease by linkage disequilibrium mapping. Curr Opin Genet Dev,2003,13(3):296-302.
13. Wellcome Trust Case Control Consortium. Genome-wide association study of
14,000 cases of seven common diseases and 3,000 shared controls. Nature,2007, 447(7145):661-678.
14. Chanock SJ, Manolio T, Boehnke M, et al. Replicating genotype-phenotype associations. Nature,2007,447(7145):655-660.
15. Dudbridge F and Gusnanto A Estimation of significance thresholds for genomewide association scans. Genet Epidemiol,2008,32(3):227-234.
16. Pe'er I, Yelensky R, Altshuler D, et al. Estimation of the multiple testing burden for genomewide association studies of nearly all common variants. Genet Epidemiol, 2008,32(4):381-385.
17. Hoggart CJ, Clark TG, De Iorio M, et al. Genome-wide significance for dense SNP and resequencing data Genet Epidemiol,2008,32(2):179-185.
18. Teo YY, Small KS, Fry AE, et al. Power consequences of linkage disequilibrium variation between populations. Genet Epidemiol,2009,33(2):128-135.
19. Amos CI Successful design and conduct of genome-wide association studies. Hum Mol Genet,2007,16 Spec No.2:R220-R225.
20. Donnelly P Progress and challenges in genome-wide association studies in humans. Nature,2008,456(7223):728-731.
21. Hindorff LA, Sethupathy P, Junkins HA, et al. A Catalog of Published Genome-Wide Association Studies. Available at:www.genome.gov/gwastudies. Accessed [12/04/10].
22. Manolio TA, Brooks LD and Collins FS A HapMap harvest of insights into the genetics of common disease. J Clin Invest,2008,118(5):1590-1605.
23. Scott LJ, Mohlke KL, Bonnycastle LL, et al. A genome-wide association study of type 2 diabetes in Finns detects multiple susceptibility variants. Science,2007,
316(5829):1341-1345.
24. Saxena R, Voight BF, Lyssenko V, et al. Genome-wide association analysis identifies loci for type 2 diabetes and triglyceride levels. Science,2007,316(5829): 1331-1336.
25. Zeggini E, Weedon MN, Lindgren CM, et al. Replication of genome-wide association signals in UK samples reveals risk loci for type 2 diabetes. Science, 2007,316(5829):1336-1341.
26. Zeggini E, Scott LJ, Saxena R, et al. Meta-analysis of genome-wide association data and large-scale replication identifies additional susceptibility loci for type 2 diabetes. Nat Genet,2008,40(5):638-645.
27. Weedon MN, Lango H, Lindgren CM, et al. Genome-wide association analysis identifies 20 loci that influence adult height Nat Genet,2008,40(5):575-583.
28. Sanna S, Jackson AU, Nagaraja R, et al. Common variants in the GDF5-UQCC region are associated with variation in human height. Nat Genet,2008,40(2): 198-203.
29. http://www.wtccc.org.uk/.
30. Manolio TA Collaborative genome-wide association studies of diverse diseases: programs of the NHGRI's office of population genomics. Pharmacogenomics,2009, 10(2):235-241.
31. Hung RJ, McKay JD, Gaborieau V, et al. A susceptibility locus for lung cancer maps to nicotinic acetylcholine receptor subunit genes on 15q25. Nature,2008,452(7187): 633-637.
32. Amos CI, Wu X, Broderick P, et al. Genome-wide association scan of tag SNPs identifies a susceptibility locus for lung cancer at 15q25.1. Nat Genet,2008,40(5): 616-622.
33. Liu P, Vikis HG, Wang D, et al. Familial aggregation of common sequence variants on 15q24-25.1 in lung cancer. J Natl Cancer Inst,2008,100(18):1326-1330.
34. Saccone SF, Hinrichs AL, Saccone NL, et al. Cholinergic nicotinic receptor genes implicated in a nicotine dependence association study targeting 348 candidate genes with 3713 SNPs. Hum Mol Genet,2007,16(1):36-49.
35. Wang Y, Broderick P, Webb E, et al. Common 5p15.33 and 6p21.33 variants influence lung cancer risk. Nat Genet,2008,40(12):1407-1409.
36. McKay JD, Hung RJ, Gaborieau V, et al. Lung cancer susceptibility locus at 5p15.33. Nat Genet,2008,40(12):1404-1406.
37. Broderick P, Wang Y, Vijayakrishnan J, et al. Deciphering the impact of common genetic variation on lung cancer risk:a genome-wide association study. Cancer Res, 2009,69(16):6633-6641.
38. Li Y, Sheu CC, Ye Y, et al. Genetic variants and risk of lung cancer in never smokers: a genome-wide association study. Lancet Oncol,2010,11(4):321-330.
39. Moffatt MF, Kabesch M, Liang L, et al. Genetic variants regulating ORMDL3 expression contribute to the risk of childhood asthma Nature,2007,448(7152): 470-473.
40. Sleiman PM, Annaiah K, Imielinski M, et al. ORMDL3 variants associated with asthma susceptibility in North Americans of European ancestry. J Allergy Clin Immunol,2008,122(6):1225-1227.
41. Galanter J, Choudhry S, Eng C, et al. ORMDL3 gene is associated with asthma in three ethnically diverse populations. Am J Respir Crit Care Med,2008,177(11): 1194-1200.
42. Tavendale R, Macgregor DF, Mukhopadhyay S, et al. A polymorphism controlling ORMDL3 expression is associated with asthma that is poorly controlled by current medications. J Allergy Clin Immunol,2008,121(4):860-863.
43. Leung TF, Sy HY, Ng MC, et al. Asthma and atopy are associated with chromosome 17q21 markers in Chinese children. Allergy,2009,64(4):621-628.
44. Hirota T, Harada M, Sakashita M, et al. Genetic polymorphism regulating ORM1-like 3 (Saccharomyces cerevisiae) expression is associated with childhood atopic asthma in a Japanese population. J Allergy Clin Immunol,2008,121(3): 769-770.
45. Himes BE, Hunninghake GM, Baurley JW, et al. Genome-wide association analysis identifies PDE4D as an asthma-susceptibility gene. Am J Hum Genet,2009,84(5): 581-593.
46. Sleiman PM, Flory J, Imielinski M, et al. Variants of DENND1B associated with asthma in children. N Engl J Med,2010,362(1):36-44.
47. Li X, Howard TD, Zheng SL, et al. Genome-wide association study of asthma identifies RAD50-IL13 and HLA-DR/DQ regions. J Allergy Clin Immunol,2010, 125(2):328-335.
48. Pillai SG, Ge D, Zhu G, et al. A genome-wide association study in chronic obstructive pulmonary disease (COPD):identification of two major susceptibility loci. PLoS Genet,2009,5(3):e1000421.
49. Cho MH, Boutaoui N, Klanderman BJ, et al. Variants in FAM13A are associated with chronic obstructive pulmonary disease. Nat Genet,2010,42(3):200-202.
50. de Bakker PI, Yelensky R, Pe'er I, et al. Efficiency and power in genetic association studies. Nat Genet,2005,37(11):1217-1223.
51. Pe'er I, de Bakker PI, Maller J, et al. Evaluating and improving power in whole-genome association studies using fixed marker sets. Nat Genet,2006,38(6): 663-667.
52. Thomas D Gene-environment-wide association studies:emerging approaches. Nat Rev Genet,2010,11(4):259-272.
53. Chakravarti A and Little P Nature, nurture and human disease. Nature,2003, 421(6921):412-414.
54. Guttmacher AE, Collins FS and Carmona RH The family history--more important than ever. N Engl J Med,2004,351(22):2333-2336.
55. Feuk L, Marshall CR, Wintle RF, et al. Structural variants:changing the landscape of chromosomes and design of disease studies. Hum Mol Genet,2006,15 Spec No 1: R57-R66.
56. Wain LV, Armour JA and Tobin MD Genomic copy number variation, human health, and disease. Lancet,2009,374(9686):340-350.
57. Marshall CR, Noor A, Vincent JB, et al. Structural variation of chromosomes in autism spectrum disorder. Am J Hum Genet,2008,82(2):477-488.
58. Szatmari P, Paterson AD, Zwaigenbaum L, et al. Mapping autism risk loci using genetic linkage and chromosomal rearrangements. Nat Genet,2007,39(3):319-328.
59. Xu B, Roos JL, Levy S, et al. Strong association of de novo copy number mutations with sporadic schizophrenia Nat Genet,2008,40(7):880-885.
60. McCarroll SA, Huett A, Kuballa P, et al. Deletion polymorphism upstream of IRGM associated with altered IRGM expression and Crohn's disease. Nat Genet,2008, 40(9):1107-1112.
61. Ionita-Laza I, Rogers AJ, Lange C, et al. Genetic association analysis of copy-number variation (CNV) in human disease pathogenesis. Genomics,2009, 93(1):22-26.
62. McCarthy MI, Abecasis GR, Cardon LR, et al. Genome-wide association studies for complex traits:consensus, uncertainty and challenges. Nat Rev Genet,2008,9(5): 356-369.
63. McCarroll SA Extending genome-wide association studies to copy-number variation. Hum Mol Genet,2008,17(R2):R135-R142.
64. Cooper RS, Tayo B and Zhu X Genome-wide association studies:implications for multiethnic samples. Hum Mol Genet,2008,17(R2):R151-R155.
65. Wu C, Hu Z, Yu D, et al. Genetic variants on chromosome 15q25 associated with lung cancer risk in Chinese populations. Cancer Res,2009,69(12):5065-5072.
66. Pleasance ED, Stephens PJ, O'Meara S, et al. A small-cell lung cancer genome with complex signatures of tobacco exposure. Nature,2010,463(7278):184-190.
67. Pleasance ED, Cheetham RK, Stephens PJ, et al. A comprehensive catalogue of somatic mutations from a human cancer genome. Nature,2010,463(7278): 191-196.
68. Sun T, Gao Y, Tan W, et al. A six-nucleotide insertion-deletion polymorphism in the CASP8 promoter is associated with susceptibility to multiple cancers. Nat Genet, 2007,39(5):605-613.
69. Miao X, Zhang X, Zhang L, et al. Adenosine diphosphate ribosyl transferase and x-ray repair cross-complementing 1 polymorphisms in gastric cardia cancer. Gastroenterology,2006,131(2):420-427.
70. Visel A, Zhu Y, May D, et al. Targeted deletion of the 9p21 non-coding coronary artery disease risk interval in mice. Nature,2010,464(7287):409-412.
71. Chen Y, Bai Y, Yuan J, et al. Association of polymorphisms in AhR, CYP1A1, GSTM1, and GSTT1 genes with levels of DNA damage in peripheral blood lymphocytes among coke-oven workers. Cancer Epidemiol Biomarkers Prev,2006, 15(9):1703-1707.
72. Wang F, He Y, Guo H, et al. Genetic variants of nucleotide excision repair genes are associated with DNA damage in coke oven workers. Cancer Epidemiol Biomarkers Prev,2010,19(1):211-218.
2. Jemal A, Siegel R, Ward E, et al. Cancer statistics,2008. CA Cancer J Clin,2008, 58(2):71-96.
3. Yang L, Parkin DM, Li LD, et al. Estimation and projection of the national profile of cancer mortality in China:1991-2005. Br J Cancer,2004,90(11):2157-2166.
4. Shields PG and Harris CC Cancer risk and low-penetrance susceptibility genes in gene-environment interactions. J Clin Oncol,2000,18(11):2309-2315.
5. IARC Polynuclear Aromatic Compounds, Part 3, Industrial Exposures in Aluminium Production, Coal Gasification, Coke Production, and Iron and Steel Founding. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans,1984,34:65-131.
6. Bosetti C, Boffetta P and La Vecchia C Occupational exposures to polycyclic aromatic hydrocarbons, and respiratory and urinary tract cancers:a quantitative review to 2005. Ann Oncol,2007,18(3):431-446.
7. Chen Y, Bai Y, Yuan J, et al. Association of polymorphisms in AhR, CYP1A1, GSTM1, and GSTT1 genes with levels of DNA damage in peripheral blood lymphocytes among coke-oven workers. Cancer Epidemiol Biomarkers Prev,2006, 15(9):1703-1707.
8. Wang F, He Y, Guo H, et al. Genetic variants of nucleotide excision repair genes are associated with DNA damage in coke oven workers. Cancer Epidemiol Biomarkers Prev,2010,19(1):211-218.
9. Herbst RS, Heymach JV and Lippman SM Lung cancer. N Engl J Med,2008, 359(13):1367-1380.
10. Efferth T and Volm M Pharmacogenetics for individualized cancer chemotherapy. Pharmacol Ther,2005,107(2):155-176.
11. Gething MJ and Sambrook J Protein folding in the cell. Nature,1992,355(6355): 33-45.
12. Hightower LE Heat shock, stress proteins, chaperones, and proteotoxicity. Cell, 1991,66(2):191-197.
13. Macario AJ and Conway de Macario E Sick chaperones, cellular stress, and disease. N Engl J Med,2005,353(14):1489-1501.
14. Hartl FU Molecular chaperones in cellular protein folding. Nature,1996,381(6583): 571-579.
15. Lindquist S The heat-shock response. Annu Rev Biochem,1986,55:1151-1191.
16. Garrido C, Bruey JM, Fromentin A, et al. HSP27 inhibits cytochrome c-dependent activation of procaspase-9. FASEB J,1999,13(14):2061-2070.
17. Beere HM, Wolf BB, Cain K, et al. Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome. Nat Cell Biol, 2000,2(8):469-475.
18. Concannon CG, Gorman AM and Samali A On the role of Hsp27 in regulating apoptosis. Apoptosis,2003,8(1):61-70.
19. Mendez F, Sandigursky M, Franklin WA, et al. Heat-shock proteins associated with base excision repair enzymes in HeLa cells. Radiat Res,2000,153(2):186-195.
20. Nadin SB, Vargas-Roig LM, Drago G, et al. Hsp27, Hsp70 and mismatch repair proteins hMLHl and hMSH2 expression in peripheral blood lymphocytes from healthy subjects and cancer patients. Cancer Lett,2007,252(1):131-146.
21. Jolly C and Morimoto RI Role of the heat shock response and molecular chaperones in oncogenesis and cell death. J Natl Cancer Inst,2000,92(19):1564-1572.
22. Arrigo AP The cellular "networking" of mammalian Hsp27 and its functions in the control of protein folding, redox state and apoptosis. Adv Exp Med Biol,2007,594: 14-26.
23. Ciocca DR, Oesterreich S, Chamness GC, et al. Biological and clinical implications of heat shock protein 27,000 (Hsp27):a review. J Natl Cancer Inst,1993,85(19): 1558-1570.
24. Merendino AM, Paul C, Vignola AM, et al. Heat shock protein-27 protects human bronchial epithelial cells against oxidative stress-mediated apoptosis:possible implication in asthma Cell Stress Chaperones,2002,7(3):269-280.
25. Pivovarova AV, Chebotareva NA, Chernik IS, et al. Small heat shock protein Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actin. FEBS J,2007,274(22):5937-5948.
26. Mosser DD and Morimoto RI Molecular chaperones and the stress of oncogenesis. Oncogene,2004,23(16):2907-2918.
27. Ciocca DR and Calderwood SK Heat shock proteins in cancer:diagnostic, prognostic, predictive, and treatment implications. Cell Stress Chaperones,2005, 10(2):86-103.
28. Calderwood SK, Khaleque MA, Sawyer DB, et al. Heat shock proteins in cancer: chaperones of tumorigenesis. Trends Biochem Sci,2006,31(3):164-172.
29. Garrido C, Fromentin A, Bonnotte B, et al. Heat shock protein 27 enhances the tumorigenicity of immunogenic rat colon carcinoma cell clones. Cancer Res,1998, 58(23):5495-5499.
30. Kamada M, So A, Muramaki M, et al. Hsp27 knockdown using nucleotide-based
therapies inhibit tumor growth and enhance chemotherapy in human bladder cancer cells. Mol Cancer Ther,2007,6(1):299-308.
31. Bruey JM, Ducasse C, Bonniaud P, et al. Hsp27 negatively regulates cell death by interacting with cytochrome c. Nat Cell Biol,2000,2(9):645-652.
32. Paul C, Manero F, Gonin S, et al. Hsp27 as a negative regulator of cytochrome C release. Mol Cell Biol,2002,22(3):816-834.
33. Havasi A, Li Z, Wang Z, et al. Hsp27 inhibits Bax activation and apoptosis via a phosphatidylinositol 3-kinase-dependent mechanism. J Biol Chem,2008,283(18): 12305-12313.
34. Chauhan D, Li G, Hideshima T, et al. Hsp27 inhibits release of mitochondrial protein Smac in multiple myeloma cells and confers dexamethasone resistance. Blood,2003,102(9):3379-3386.
35. Charette SJ, Lavoie JN, Lambert H, et al. Inhibition of Daxx-mediated apoptosis by heat shock protein 27. Mol Cell Biol,2000,20(20):7602-7612.
36. He M, Guo H, Yang X, et al. Functional SNPs in HSPA1A gene predict risk of coronary heart disease. PLoS ONE,2009,4(3):e4851.
37. Barrett JC, Fry B, Maller J, et al. Haploview:analysis and visualization of LD and haplotype maps. Bioinformatics,2005,21(2):263-265.
38. Tregouet DA and Garelle V A new JAVA interface implementation of THESIAS: testing haplotype effects in association studies. Bioinformatics,2007,23(8): 1038-1039.
39. Stephens M and Donnelly P A comparison of bayesian methods for haplotype reconstruction from population genotype data Am J Hum Genet,2003,73(5): 1162-1169.
40. Mehlen P, Kretz-Remy C, Preville X, et al. Human hsp27, Drosophila hsp27 and human alphaB-crystallin expression-mediated increase in glutathione is essential for the protective activity of these proteins against TNFalpha-induced cell death. EMBO J,1996,15(11):2695-2706.
41. Arrigo AP, Virot S, Chaufour S, et al. Hsp27 consolidates intracellular redox homeostasis by upholding glutathione in its reduced form and by decreasing iron intracellular levels. Antioxid Redox Signal,2005,7(3-4):414-422.
42. Feng JT, Liu YK, Song HY, et al. Heat-shock protein 27:a potential biomarker for hepatocellular carcinoma identified by serum proteome analysis. Proteomics,2005, 5(17):4581-4588.
43. Korneeva I, Bongiovanni AM, Girotra M, et al. Serum antibodies to the 27-kd heat shock protein in women with gynecologic cancers. Am J Obstet Gynecol,2000, 183(1):18-21.
44. Korneeva I, Bongiovanni AM, Girotra M, et al. IgA antibodies to the 27-kDa heat-shock protein in the genital tracts of women with gynecologic cancers. Int J Cancer,2000,87(6):824-828.
45. Evgrafov OV, Mersiyanova I, Irobi J, et al. Mutant small heat-shock protein 27 causes axonal Charcot-Marie-Tooth disease and distal hereditary motor neuropathy. Nat Genet,2004,36(6):602-606.
46. Dierick I, Irobi J, Janssens S, et al. Genetic variant in the HSPB1 promoter region impairs the HSP27 stress response. Hum Mutat,2007,28(8):830-840.
47. Kim EH, Lee HJ, Lee DH, et al. Inhibition of heat shock protein 27-mediated resistance to DNA damaging agents by a novel PKC delta-V5 heptapeptide. Cancer Res,2007,67(13):6333-6341.
48. Lee YJ, Lee DH, Cho CK, et al. HSP25 inhibits radiation-induced apoptosis through reduction of PKCdelta-mediated ROS production. Oncogene,2005,24(23): 3715-3725.
49. Zhang Y and Shen X Heat shock protein 27 protects L929 cells from cisplatin-induced apoptosis by enhancing Akt activation and abating suppression of thioredoxin reductase activity. Clin Cancer Res,2007,13(10):2855-2864.
50. O'Callaghan-Sunol C, Gabai VL and Sherman MY Hsp27 modulates p53 signaling and suppresses cellular senescence. Cancer Res,2007,67(24):11779-11788.
51. Sobin LH and Wittekind CH. TNM classification of malignant tumors (6th edition). 2002, Wiley-Liss:New York.
52. Mori-Iwamoto S, Kuramitsu Y, Ryozawa S, et al. Proteomics finding heat shock protein 27 as a biomarker for resistance of pancreatic cancer cells to gemcitabine. Int J Oncol,2007,31(6):1345-1350.
53. Ciocca DR, Rozados VR, Cuello Carrion FD, et al. Hsp25 and Hsp70 in rodent tumors treated with doxorubicin and lovastatin. Cell Stress Chaperones,2003,8(1): 26-36.
54. Calderwood SK and Ciocca DR Heat shock proteins:stress proteins with Janus-like properties in cancer. Int J Hyperthermia,2008,24(1):31-39.
55. Milner CM and Campbell RD Structure and expression of the three MHC-linked HSP70 genes. Immunogenetics,1990,32(4):242-251.
56. Favatier F, Bornman L, Hightower LE, et al. Variation in hsp gene expression and Hsp polymorphism:do they contribute to differential disease susceptibility and stress tolerance? Cell Stress Chaperones,1997,2(3):141-155.
57. Vargas-Alarcon G, Londono JD, Hernandez-Pacheco G, et al. Heat shock protein 70 gene polymorphisms in Mexican patients with spondyloarthropathies. Ann Rheum Dis,2002,61(1):48-51.
58. Aron Y, Busson M, Polla BS, et al. Analysis of hsp70 gene polymorphism in allergic asthma Allergy,1999,54(2):165-170.
59. Rajalingam R, Mehra NK and Singal DP Polymorphism in heat-shock protein 70-1 (HSP70-1) gene promoter region and susceptibility to tuberculoid leprosy and pulmonary tuberculosis in Asian Indians. Indian J Exp Biol,2000,38(7):658-662.
60. Pae CU, Kim TS, Kwon OJ, et al. Polymorphisms of heat shock protein 70 gene (HSPA1A, HSPA1B and HSPA1L) and schizophrenia Neurosci Res,2005,53(1): 8-13.
61. Wu YR, Wang CK, Chen CM, et al. Analysis of heat-shock protein 70 gene polymorphisms and the risk of Parkinson's disease. Hum Genet,2004,114(3): 236-241.
62. Wang Y, Broderick P, Webb E, et al. Common 5p15.33 and 6p21.33 variants influence lung cancer risk. Nat Genet,2008,40(12):1407-1409.
63.杨晓波.博士毕业论文.华中科技大学图书馆,2007.
64. Jalbout M, Bouaouina N, Gargouri J, et al. Polymorphism of the stress protein HSP70-2 gene is associated with the susceptibility to the nasopharyngeal carcinoma Cancer Lett,2003,193(1):75-81.
65. Mestiri S, Bouaouina N, Ahmed SB, et al. Genetic variation in the tumor necrosis factor-alpha promoter region and in the stress protein hsp70-2:susceptibility and prognostic implications in breast carcinoma .Cancer,2001,91(4):672-678.
66. Debler J, Schiemann U, Seybold U, et al. Heat-shock protein HSP70-2 genotypes in patients with Crohn's disease:a more severe clinical course with intestinal
complications in presence of the PstI-polymorphism. Eur J Med Res,2003,8(3): 120-124.
67. Zhou F, Wang F, Li F, et al. Association of hsp70-2 and hsp-hom gene polymorphisms with risk of acute high-altitude illness in a Chinese population. Cell Stress Chaperones,2005,10(4):349-356.
68. Zhong L, Peng X, Hidalgo GE, et al. Antibodies to HSP70 and HSP90 in serum in non-small cell lung cancer patients. Cancer Detect Prev,2003,27(4):285-290.
69. Suzuki K, Ito Y, Wakai K, et al. Serum heat shock protein 70 levels and lung cancer risk:a case-control study nested in a large cohort study. Cancer Epidemiol Biomarkers Prev,2006,15(9):1733-1737.
70. Temple SE, Cheong KY, Ardlie KG, et al. The septic shock associated HSPA1B1267 polymorphism influences production of HSPA1A and HSPA1B. Intensive Care Med,2004,30(9):1761-1767.
71. Gress TM, Muller-Pillasch F, Weber C, et al. Differential expression of heat shock proteins in pancreatic carcinoma. Cancer Res,1994,54(2):547-551.
72. Gotz R, Kramer BW, Camarero G, et al. BAG-1 haplo-insufficiency impairs lung tumorigenesis. BMC Cancer,2004,4:85.
73. Takayama S and Reed JC Molecular chaperone targeting and regulation by BAG family proteins. Nat Cell Biol,2001,3(10):E237-E241.
74. Takayama S, Sato T, Krajewski S, et al. Cloning and functional analysis of BAG-1: a novel Bcl-2-binding protein with anti-cell death activity. Cell,1995,80(2): 279-284.
75. Cutress RI, Townsend PA, Brimmell M, et al. BAG-1 expression and function in human cancer. Br J Cancer,2002,87(8):834-839.
76. Gehring U Biological activities of HAP46/BAG-1. The HAP46/BAG-1 protein: regulator of HSP70 chaperones, DNA-binding protein and stimulator of transcription. EMBO Rep,2004,5(2):148-153.
77. Frenzel A, Grespi F, Chmelewskij W, et al. Bcl2 family proteins in carcinogenesis and the treatment of cancer. Apoptosis,2009,14(4):584-596.
78. Cory S and Adams JM The Bcl2 family:regulators of the cellular life-or-death switch. Nat Rev Cancer,2002,2(9):647-656.
79. Kim DH, Xu W, Ma C, et al. Genetic variants in the candidate genes of the apoptosis pathway and susceptibility to chronic myeloid leukemia Blood,2009, 113(11):2517-2525.
80. Faderl S, Keating MJ, Do KA, et al. Expression profile of 11 proteins and their prognostic significance in patients with chronic lymphocytic leukemia (CLL). Leukemia,2002,16(6):1045-1052.
81. Keshgegian AA, Johnston E and Cnaan A Bcl-2 oncoprotein positivity and high MIB-1 (Ki-67) proliferative rate are independent predictive markers for recurrence in prostate carcinoma Am J Clin Pathol,1998,110(4):443-449.
82. Ofner D, Riehemann K, Maier H, et al. Immunohistochemically detectable bcl-2 expression in colorectal carcinoma:correlation with tumour stage and patient survival. Br J Cancer,1995,72(4):981-985.
83. Zhang GJ, Kimijima I, Tsuchiya A, et al. The role of bcl-2 expression in breast carcinomas (Review). Oncol Rep,1998,5(5):1211-1216.
84. Bankfalvi A, Tory K, Kemper M, et al. Clinical relevance of immunohistochemical expression of p53-targeted gene products mdm-2, p21 and bcl-2 in breast carcinoma Pathol Res Pract,2000,196(7):489-501.
85. Martin B, Paesmans M, Berghmans T, et al. Role of Bcl-2 as a prognostic factor for survival in lung cancer:a systematic review of the literature with meta-analysis. Br J Cancer,2003,89(1):55-64.
86. Renouf DJ, Wood-Baker R, Ionescu DN, et al. BCL-2 expression is prognostic for improved survival in non-small cell lung cancer. J Thorac Oncol,2009,4(4): 486-491.
87. Heubner M, Wimberger P, Otterbach F, et al. Association of the AA genotype of the BCL2 (-938C>A) promoter polymorphism with better survival in ovarian cancer. Int J Biol Markers,2009,24(4):223-229.
88. Bachmann HS, Otterbach F, Callies R, et al. The AA genotype of the regulatory BCL2 promoter polymorphism (938C>A) is associated with a favorable outcome in lymph node negative invasive breast cancer patients. Clin Cancer Res,2007,13(19): 5790-5797.
89. Lehnerdt GF, Franz P, Bankfalvi A, et al. The regulatory BCL2 promoter polymorphism (-938C>A) is associated with relapse and survival of patients with oropharyngeal squamous cell carcinoma Ann Oncol,2009,20(6):1094-1099.
90. Nuckel H, Frey UH, Bau M, et al. Association of a novel regulatory polymorphism (-938C>A) in the BCL2 gene promoter with disease progression and survival in chronic lymphocytic leukemia Blood,2007,109(1):290-297.
91. Moon JH, Sohn SK, Lee MH, et al. BCL2 gene polymorphism could predict the treatment outcomes in acute myeloid leukemia patients. Leuk Res,2010,34(2): 166-172.
92. Singh NP, McCoy MT, Tice RR, et al. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res,1988,175(1):184-191.
93. Cheng J, Leng S, Dai Y, et al. Association between nucleotide excision repair gene polymorphisms and chromosomal damage in coke-oven workers. Biomarkers,2007, 12(1):76-86.
94. Wang H, Chen W, Zheng H, et al. Association between plasma BPDE-Alb adduct concentrations and DNA damage of peripheral blood lymphocytes among coke oven workers. Occup Environ Med,2007,64(11):753-758.
95.朱志良,庄志雄,黄钰,等.单细胞凝胶电泳图像分析系统的研制及应用.中华劳动卫生职业病杂志,2001,19(4)：298-300.
96. Olive PL, Banath JP and Durand RE Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the "comet" assay. Radiat Res,1990,122(1):86-94.
97.潘秋萍,孙恩林,陈胜,等.焦炉工人血清中p21蛋白和HSPs水平的研究中国职业医学,1999,26(5)：4-6.
98. Yao H, Zhang Z, Xiao Z, et al. Identification of metastasis associated proteins in human lung squamous carcinoma using two-dimensional difference gel electrophoresis and laser capture microdissection. Lung Cancer,2009,65(1):41-48.
99. httn://www.genomatix.de/nroducts/MatInsnectnr/
100. Hanai J, Dhanabal M, Karumanchi SA, et al. Endostatin causes G1 arrest of endothelial cells through inhibition of cyclin D1. J Biol Chem,2002,277(19): 16464-16469.
101. Abravaya K, Phillips B and Morimoto RI Heat shock-induced interactions of heat shock transcription factor and the human hsp70 promoter examined by in vivo footprinting. Mol Cell Biol,1991,11(1):586-592.
102. Vivinus S, Baulande S, van Zanten M, et al. An element within the 5'untranslated region of human Hsp70 mRNA which acts as a general enhancer of mRNA translation. Eur J Biochem,2001,268(7):1908-1917.
103. Rubtsova MP, Sizova DV, Dmitriev SE, et al. Distinctive properties of the 5'-untranslated region of human hsp70 mRNA. J Biol Chem,2003,278(25): 22350-22356.
1. Klein RJ, Zeiss C, Chew EY, et al. Complement factor H polymorphism in age-related macular degeneration. Science,2005,308(5720):385-389.
2. International Human Genome Sequencing Consortium. Finishing the euchromatic sequence of the human genome. Nature,2004,431(7011):931-945.
3. http://www.ncbi.nlm.nih.gov/projects/SNP.
4. The International HapMap Project. Nature,2003,426(6968):789-796.
5. http://hapmap.ncbi.nlm.nih.gov/.
6. A haplotype map of the human genome. Nature,2005,437(7063):1299-1320.
7. Matsuzaki H, Loi H, Dong S, et al. Parallel genotyping of over 10,000 SNPs using a one-primer assay on a high-density oligonucleotide array. Genome Res,2004,14(3): 414-425.
8. Teo YY, Small KS and Kwiatkowski DP Methodological challenges of genome-wide association analysis in Africa Nat Rev Genet,2010,11(2):149-160.
9. Risch N and Merikangas K The future of genetic studies of complex human diseases. Science,1996,273(5281):1516-1517.
10. Thomas DC, Haile RW and Duggan D Recent developments in genomewide association scans:a workshop summary and review. Am J Hum Genet,2005,77(3): 337-345.
11. Chen GK and Witte JS Enriching the analysis of genomewide association studies with hierarchical modeling. Am J Hum Genet,2007,81(2):397-404.
12. Clark AG Finding genes underlying risk of complex disease by linkage disequilibrium mapping. Curr Opin Genet Dev,2003,13(3):296-302.
13. Wellcome Trust Case Control Consortium. Genome-wide association study of
14,000 cases of seven common diseases and 3,000 shared controls. Nature,2007, 447(7145):661-678.
14. Chanock SJ, Manolio T, Boehnke M, et al. Replicating genotype-phenotype associations. Nature,2007,447(7145):655-660.
15. Dudbridge F and Gusnanto A Estimation of significance thresholds for genomewide association scans. Genet Epidemiol,2008,32(3):227-234.
16. Pe'er I, Yelensky R, Altshuler D, et al. Estimation of the multiple testing burden for genomewide association studies of nearly all common variants. Genet Epidemiol, 2008,32(4):381-385.
17. Hoggart CJ, Clark TG, De Iorio M, et al. Genome-wide significance for dense SNP and resequencing data Genet Epidemiol,2008,32(2):179-185.
18. Teo YY, Small KS, Fry AE, et al. Power consequences of linkage disequilibrium variation between populations. Genet Epidemiol,2009,33(2):128-135.
19. Amos CI Successful design and conduct of genome-wide association studies. Hum Mol Genet,2007,16 Spec No.2:R220-R225.
20. Donnelly P Progress and challenges in genome-wide association studies in humans. Nature,2008,456(7223):728-731.
21. Hindorff LA, Sethupathy P, Junkins HA, et al. A Catalog of Published Genome-Wide Association Studies. Available at:www.genome.gov/gwastudies. Accessed [12/04/10].
22. Manolio TA, Brooks LD and Collins FS A HapMap harvest of insights into the genetics of common disease. J Clin Invest,2008,118(5):1590-1605.
23. Scott LJ, Mohlke KL, Bonnycastle LL, et al. A genome-wide association study of type 2 diabetes in Finns detects multiple susceptibility variants. Science,2007,
316(5829):1341-1345.
24. Saxena R, Voight BF, Lyssenko V, et al. Genome-wide association analysis identifies loci for type 2 diabetes and triglyceride levels. Science,2007,316(5829): 1331-1336.
25. Zeggini E, Weedon MN, Lindgren CM, et al. Replication of genome-wide association signals in UK samples reveals risk loci for type 2 diabetes. Science, 2007,316(5829):1336-1341.
26. Zeggini E, Scott LJ, Saxena R, et al. Meta-analysis of genome-wide association data and large-scale replication identifies additional susceptibility loci for type 2 diabetes. Nat Genet,2008,40(5):638-645.
27. Weedon MN, Lango H, Lindgren CM, et al. Genome-wide association analysis identifies 20 loci that influence adult height Nat Genet,2008,40(5):575-583.
28. Sanna S, Jackson AU, Nagaraja R, et al. Common variants in the GDF5-UQCC region are associated with variation in human height. Nat Genet,2008,40(2): 198-203.
29. http://www.wtccc.org.uk/.
30. Manolio TA Collaborative genome-wide association studies of diverse diseases: programs of the NHGRI's office of population genomics. Pharmacogenomics,2009, 10(2):235-241.
31. Hung RJ, McKay JD, Gaborieau V, et al. A susceptibility locus for lung cancer maps to nicotinic acetylcholine receptor subunit genes on 15q25. Nature,2008,452(7187): 633-637.
32. Amos CI, Wu X, Broderick P, et al. Genome-wide association scan of tag SNPs identifies a susceptibility locus for lung cancer at 15q25.1. Nat Genet,2008,40(5): 616-622.
33. Liu P, Vikis HG, Wang D, et al. Familial aggregation of common sequence variants on 15q24-25.1 in lung cancer. J Natl Cancer Inst,2008,100(18):1326-1330.
34. Saccone SF, Hinrichs AL, Saccone NL, et al. Cholinergic nicotinic receptor genes implicated in a nicotine dependence association study targeting 348 candidate genes with 3713 SNPs. Hum Mol Genet,2007,16(1):36-49.
35. Wang Y, Broderick P, Webb E, et al. Common 5p15.33 and 6p21.33 variants influence lung cancer risk. Nat Genet,2008,40(12):1407-1409.
36. McKay JD, Hung RJ, Gaborieau V, et al. Lung cancer susceptibility locus at 5p15.33. Nat Genet,2008,40(12):1404-1406.
37. Broderick P, Wang Y, Vijayakrishnan J, et al. Deciphering the impact of common genetic variation on lung cancer risk:a genome-wide association study. Cancer Res, 2009,69(16):6633-6641.
38. Li Y, Sheu CC, Ye Y, et al. Genetic variants and risk of lung cancer in never smokers: a genome-wide association study. Lancet Oncol,2010,11(4):321-330.
39. Moffatt MF, Kabesch M, Liang L, et al. Genetic variants regulating ORMDL3 expression contribute to the risk of childhood asthma Nature,2007,448(7152): 470-473.
40. Sleiman PM, Annaiah K, Imielinski M, et al. ORMDL3 variants associated with asthma susceptibility in North Americans of European ancestry. J Allergy Clin Immunol,2008,122(6):1225-1227.
41. Galanter J, Choudhry S, Eng C, et al. ORMDL3 gene is associated with asthma in three ethnically diverse populations. Am J Respir Crit Care Med,2008,177(11): 1194-1200.
42. Tavendale R, Macgregor DF, Mukhopadhyay S, et al. A polymorphism controlling ORMDL3 expression is associated with asthma that is poorly controlled by current medications. J Allergy Clin Immunol,2008,121(4):860-863.
43. Leung TF, Sy HY, Ng MC, et al. Asthma and atopy are associated with chromosome 17q21 markers in Chinese children. Allergy,2009,64(4):621-628.
44. Hirota T, Harada M, Sakashita M, et al. Genetic polymorphism regulating ORM1-like 3 (Saccharomyces cerevisiae) expression is associated with childhood atopic asthma in a Japanese population. J Allergy Clin Immunol,2008,121(3): 769-770.
45. Himes BE, Hunninghake GM, Baurley JW, et al. Genome-wide association analysis identifies PDE4D as an asthma-susceptibility gene. Am J Hum Genet,2009,84(5): 581-593.
46. Sleiman PM, Flory J, Imielinski M, et al. Variants of DENND1B associated with asthma in children. N Engl J Med,2010,362(1):36-44.
47. Li X, Howard TD, Zheng SL, et al. Genome-wide association study of asthma identifies RAD50-IL13 and HLA-DR/DQ regions. J Allergy Clin Immunol,2010, 125(2):328-335.
48. Pillai SG, Ge D, Zhu G, et al. A genome-wide association study in chronic obstructive pulmonary disease (COPD):identification of two major susceptibility loci. PLoS Genet,2009,5(3):e1000421.
49. Cho MH, Boutaoui N, Klanderman BJ, et al. Variants in FAM13A are associated with chronic obstructive pulmonary disease. Nat Genet,2010,42(3):200-202.
50. de Bakker PI, Yelensky R, Pe'er I, et al. Efficiency and power in genetic association studies. Nat Genet,2005,37(11):1217-1223.
51. Pe'er I, de Bakker PI, Maller J, et al. Evaluating and improving power in whole-genome association studies using fixed marker sets. Nat Genet,2006,38(6): 663-667.
52. Thomas D Gene-environment-wide association studies:emerging approaches. Nat Rev Genet,2010,11(4):259-272.
53. Chakravarti A and Little P Nature, nurture and human disease. Nature,2003, 421(6921):412-414.
54. Guttmacher AE, Collins FS and Carmona RH The family history--more important than ever. N Engl J Med,2004,351(22):2333-2336.
55. Feuk L, Marshall CR, Wintle RF, et al. Structural variants:changing the landscape of chromosomes and design of disease studies. Hum Mol Genet,2006,15 Spec No 1: R57-R66.
56. Wain LV, Armour JA and Tobin MD Genomic copy number variation, human health, and disease. Lancet,2009,374(9686):340-350.
57. Marshall CR, Noor A, Vincent JB, et al. Structural variation of chromosomes in autism spectrum disorder. Am J Hum Genet,2008,82(2):477-488.
58. Szatmari P, Paterson AD, Zwaigenbaum L, et al. Mapping autism risk loci using genetic linkage and chromosomal rearrangements. Nat Genet,2007,39(3):319-328.
59. Xu B, Roos JL, Levy S, et al. Strong association of de novo copy number mutations with sporadic schizophrenia Nat Genet,2008,40(7):880-885.
60. McCarroll SA, Huett A, Kuballa P, et al. Deletion polymorphism upstream of IRGM associated with altered IRGM expression and Crohn's disease. Nat Genet,2008, 40(9):1107-1112.
61. Ionita-Laza I, Rogers AJ, Lange C, et al. Genetic association analysis of copy-number variation (CNV) in human disease pathogenesis. Genomics,2009, 93(1):22-26.
62. McCarthy MI, Abecasis GR, Cardon LR, et al. Genome-wide association studies for complex traits:consensus, uncertainty and challenges. Nat Rev Genet,2008,9(5): 356-369.
63. McCarroll SA Extending genome-wide association studies to copy-number variation. Hum Mol Genet,2008,17(R2):R135-R142.
64. Cooper RS, Tayo B and Zhu X Genome-wide association studies:implications for multiethnic samples. Hum Mol Genet,2008,17(R2):R151-R155.
65. Wu C, Hu Z, Yu D, et al. Genetic variants on chromosome 15q25 associated with lung cancer risk in Chinese populations. Cancer Res,2009,69(12):5065-5072.
66. Pleasance ED, Stephens PJ, O'Meara S, et al. A small-cell lung cancer genome with complex signatures of tobacco exposure. Nature,2010,463(7278):184-190.
67. Pleasance ED, Cheetham RK, Stephens PJ, et al. A comprehensive catalogue of somatic mutations from a human cancer genome. Nature,2010,463(7278): 191-196.
68. Sun T, Gao Y, Tan W, et al. A six-nucleotide insertion-deletion polymorphism in the CASP8 promoter is associated with susceptibility to multiple cancers. Nat Genet, 2007,39(5):605-613.
69. Miao X, Zhang X, Zhang L, et al. Adenosine diphosphate ribosyl transferase and x-ray repair cross-complementing 1 polymorphisms in gastric cardia cancer. Gastroenterology,2006,131(2):420-427.
70. Visel A, Zhu Y, May D, et al. Targeted deletion of the 9p21 non-coding coronary artery disease risk interval in mice. Nature,2010,464(7287):409-412.
71. Chen Y, Bai Y, Yuan J, et al. Association of polymorphisms in AhR, CYP1A1, GSTM1, and GSTT1 genes with levels of DNA damage in peripheral blood lymphocytes among coke-oven workers. Cancer Epidemiol Biomarkers Prev,2006, 15(9):1703-1707.
72. Wang F, He Y, Guo H, et al. Genetic variants of nucleotide excision repair genes are associated with DNA damage in coke oven workers. Cancer Epidemiol Biomarkers Prev,2010,19(1):211-218.
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