【作者】 刘秀芳；

【导师】 王亚平；

【作者基本信息】 南京大学 ， 内科学， 2011， 博士

【摘要】 氧气是所有需氧生物生命活动所必需的,但机体在外界环境刺激下或是内部代谢过程中均能产生大量的活性氧(Reactive oxygen species, ROS)。ROS是需氧生物,包括哺乳动物体内最多的内源性有毒物质。ROS可以通过直接的氧化作用损伤蛋白质,脂质和核酸等重要的生物大分子,进而使细胞功能失调,造成多种生物学应答,如炎症反应和细胞凋亡。ROS的攻击可造成氧化损伤,是导致癌症和年龄相关性疾病的重要病因。在多种DNA氧化损伤中,8-羟基鸟嘌呤(8-hydroxy-2’-deoxyguanosine,8-OHdG)是最为常见的氧化损伤产物之一,它对了解突变相关性疾病,尤其是肿瘤的发生具有重要提示作用。8-OHdG是一种稳定的DNA氧化损伤产物,研究中经常测定该产物水平作为氧化损伤程度的指标。在DNA复制过程中8-OHdG易与A错配,导致G：C→A：T的突变,是肿瘤、糖尿病等多种年龄相关性疾病的发病因素之一。高水平的DNA氧化损伤产物(8-OHdG)与多种肿瘤相关,如乳腺癌,结直肠癌,子宫颈癌,肝肾肿瘤,卵巢癌,胃癌和肺癌等,被认为在肿瘤发生、发展中起到了重要作用。DNA修复系统是机体的防御屏障,在多种不同的修复通路中,碱基切除修复系统(Base Excision Repair, BER)对DNA氧化造成的损伤起到修复作用。BER是个多步骤、涉及多种酶的过程,BER通路最关键的步骤是由DNA糖基化酶识别并切除受损伤的碱基。在人类,hOGG1(human8-OHdGuanine glycosylase)、hMYH (human MutY homolog)两种糖基化酶是与修复8-OHdG相关的两种主要糖基化酶,分别在DNA复制过程中识别并切除8-OHdG和与之错配的A。hOGG1基因和hMYH基因分别编码hOGG1和hMYH这两种蛋白。这两个基因均具有多个与疾病相关的突变位点,这些疾病相关性变异大多能影响基因修复8-OHdG的能力。而DNA修复能力的缺陷是多种肿瘤的风险因素。在世界范围内,胃癌和结直肠都是常见的肿瘤及导致死亡的主要原因。根据胃癌和结直肠癌的发病原因分析,以8-OHdG为代表的氧化损伤产物的增加可能是胃肠道肿瘤一个常见的危险因素,DNA修复能力的下降可能会使胃肠道肿瘤的易感性增加。因此,对碱基切除修复基因在胃癌和结直肠癌中进行突变筛查,探讨修复基因与胃肠道肿瘤发病易感性的关系可能会对疾病的预防提供新的途径。在本研究中,我们对主要运用高分辨率熔解曲线方法对hMYH阳hOGG1两个基因的变异在胃肠道肿瘤病人和对照中进行了筛查,分析这些变异与胃肠道肿瘤的发病易感性之间的关系。同时分析了突变与胃癌临床病理特征之间的关系,并对启动子活性进行了检测,探讨这些变异对基因功能影响的作用机制。本研究分为两个部分进行：第一部分：碱基切除修复基因hMYH和hOGG1变异与结直肠癌发病易感性的关系在世界范围内,结直肠癌是发病率较高的癌症之一,同时也是死亡率很高的癌症之一。氧化损伤可能与结直肠癌的发病相关,因此我们在结直肠癌患者和对照中筛查了碱基切除修复基因hMYH和hOGG1的变异,并分析其与中国人群结直肠癌发生的关系。我们收集了381例结直肠癌患者和554例作为正常对照体检人群的外周血样本,提取基因组DNA。筛查碱基切除修复基因hMYH基因第15内含子Alu插入变异,hMYH位于第12外显子c.972位点C>G的变异造成的错义突变p.Gln324His, hOGG1基因5’UTR变异和位于第7外显子c.977C>G变异造成的错义突变p.Ser326Cys。用琼脂糖凝胶电泳方法区分AluYb8hMYH插入基因型,用高分辨率熔解曲线(HRM)方法及随后的测序确定hOGG15’UTR基因型,用非标记探针HRM方法确定hMYH基因p.Gln324His突变和hOGG1基因p.Ser326Cys突变基因型。病例对照研究结果提示,AluYb8MYH携带者在结直肠患者中的频率显著增高(P=0.011)。根据HRM结果,结合随后的测序,在hOGG1基因5’UTR发现了四种杂合突变,c.-53G>C, c.-45G>A, c.-23A>G和c.-18G>T,未发现纯合突变。而hOGG1基因5’UTR突变,p.Ser326Cys和hMYH基因p.Gln324His突变在结直肠中分布与正常人群没有差异。总之,AluYb8hMYH可能是结直肠癌的发病风险因素之一,本突变的筛查可能对中国人群的结直肠癌的早期预防有意义。第二部分：碱基切除修复基因hMYH和hOGG1变异与胃癌易感性及其功能机制研究在我国胃癌的发生率和死亡率都高于世界平均水平,DNA氧化损伤与胃癌的发生存在一定的关系,因此我们在胃癌患者和正常对照中筛查了两个DNA氧化损伤修复基因的突变并分析其与中国人群胃癌发生的关系。我们收集了622例胃癌患者和932例正常人外周血样,提取DNA,通过高分辨率熔解曲线(HRM)分析技术筛查hOGG1基因5’UTR,第7外显子和hMYH基因第12外显子进行了筛查。利用生物信息学工具对这些突变进行功能预测,发现位于5’-UTR区域的突变均能改变转录因子的结合位点。对除c.-45G>A之外的突变进行了胃癌病例-对照分析。结果显示c.-53G>C在胃癌患者中的分布频率显著增高(P=0.008,OR=2.304,95%CI,1.258-4.221),提示该变异增加了胃癌的发病风险。c.-23A>G,c.-18G>T和p.Ser326Cys与胃癌未见相关性。hMYH基因p.Gln324His在胃癌和对照中分布频率差异显著(P<0.001)双荧光素酶报告基因系统检测显示c.-53G>C突变型的启动子活性比野生型的活性下降了约10%到17.8%。因此,我们推测,hOGG1基因5’-UTR区域的突变c.-53G>C可能是通过影响转录因子的结合,使基因转录减少、基因功能下降、机体的氧化损伤修复能力减弱,导致携带者胃癌发病风险增高。综上所述,本研究发现了AluYb8hMYH插入与中国人群结直肠癌发病相关,并且首次报道了hOGG1基因c.-53G>C变异与胃癌发病相关,功能研究显示该变异可导致基因启动子活性下降。而未发现hOGG1基因5’UTR突变与结直肠癌发病相关,这些研究结果提示,碱基切割修复基因不同的突变增高疾病风险的种类存在差异,在表型正常个体的突变筛查、发病风险评估,对开展针对性的疾病预防具有重要的借鉴意义。更多还原

【Abstract】 Part One:Effects of Base Excision Repair Gene hOGG1and hMYH genes Polymorphisms on colorectal cancer in the Chinese populationColorectal cancer is one of the most common cancers in the world and among the leading causes of death. The aim of this study is to investigate whether AluYb8insertion and p.Gln324His variations of human MutY homolog(hMYH) gene and p.Ser326Cys variation and5’UTR of the human oxoguanine glycosylase1(hOGG1) gene are related to the risk of colorectal cancer in the Chinese population.381colorectal cancer patients,554healthy controls were recruited to screen for these variations. Agarose gels assay was employed to discriminate genotypes of AluYb8hMYH polymorphism. High resolution melting curve analysis and sequencing were used to decide the genotype fo hOGGl5’UTR. Unlabeled probe high resolution melting curve was employed to screen for p.Gln324His variations of hMYH gene and p.Ser326Cys variation of hOGG1geneThe case-control study indicated that AluYb8MYH carriers were markedly associated with colorectal cancer (P=0.011). Four variants c.-53G>C, c.-45G>A, c.-23A>G and c.-18G>T, were detected in the5’-UTR of the hOGG1gene. The hOGG15’UTR variations, p.Ser326Cys and hMYH gene p.Gln324His didn’t show a correspondence with colorectal cancer. In summary the variant AluYb8hMYH is a risk factor for colorectal cancer.Part Two:Effects of hOGG1and hMYH gene polymorphisms on gastric cancer in the Chinese population.The incidence and mortality of gastric cancer is among the highest malignant tumors in China. The aim of this study is to investigate whether variations of the hOGG1gene and hMYH gene are related to the risk of gastric cancers in the Chinese population.There were622gastric cancer patients and932healthy controls recruited to screen for variations in the5’UTR and to screen for the missense mutation (p.Ser326Cys) in exon7of the hOGG1gene and p. Gln324His using high resolution melting curve analysis (HRM) and subsequent sequencing. The promoter luciferase activity assay was applied to assess the potential influence of the detected variants on gene function.The case-control study indicated that the c.-53G/C herterozygous genotype was markedly associated with gastric cancer (P=0.008, OR=2.304,95%CI,1.258-4.221). The clinicopathological association analysis showed that the variant of c.-53G>C in the hOGG1gene was prevalent in low differentiation patients (P=0.012, OR=3.174,95%CI:1.352-7.448). This variant decreased the gene promoter activity by approximately17.8%(P=0.041) and exhibited a synergistic effect with the missense mutation p.Ser326Cys of hOGG1by enhancing susceptibility to gastric cancers.The variant c.-53G>C in the5’-UTR of the hOGGl gene is a risk factor for gastric cancer and is potentially associated with low differentiation degree in the Chinese population.In summary, our work found that AluYb8hMYH insertion was related to colorectal cancer, and the work also indicated that c.-53G>C mutation was related to gastric cancer in the Chinese population. The different result showed that the mutation of hOGG1gene and hMYH gene can suggest different disease.更多还原