1、分类号 单位代码: 学 号: 硕 士 学 位 论 文题 目： BRCA1 基因 rs799917 多态性与肺癌易感性Single-nucleotide polymorphism of BRCA1 rs799917 associated with susceptibility of lung cancer研 究 生： 导 师： 学科专业： 肿瘤放射治疗学 论文课题起止时间： 论文完成时间： 目 录一、摘要中文论著摘要.1英文论著摘要.3二、英文缩略语.6三、论文前言.7实验方法.8结果.11讨论.16结论.19四、本研究创新性的自我评价.20五、参考文献.21六、附录综述.24在学期间科研成绩.
2、31致谢.32个人简介.33中文论著摘要BRCA1 基因 rs799917 多态性与肺癌易感性目 的探讨 BRCA1 基因编码区 rs799917 位点单核苷酸多态性（single nucleotide polymorphism, SNP）与中国辽宁地区汉族人群肺癌易感性的关系。方 法研究对象为辽宁地区汉族人群，并且相互之间无血缘关系。在签署知情同意书后，留取外周血标本，同时完成流行病学调查。采用病例对照研究原则，病例组来自中国医科大学附属第一和第四医院、沈阳军区总医院和辽宁省肿瘤医院的 682 例有明确病理诊断的原发性肺癌患者，其中包括鳞癌 254 例、腺癌226 例、小细胞癌 202 例；
3、对照组来自沈阳军区总医院体检中心的 682 例健康体检人群。所有病例和对照均为 2009 年 9 月至 2012 年 12 月期间收集。参与研究的个体外周血标本采集使用枸橼酸钠抗凝试管，每人采血约5ml，用酚- 氯仿法提取基因组 DNA。采用 Taqman real-time PCR 方法及SDS（美国应用生物系统公司）软件程序进行 SNP 的基因分型。在每个 96 孔检测单元中分别设立阴性对照和重复标本作为质量控制；阴性对照中不加模板DNA，而以蒸馏水代替，用于检验是否存在 PCR 污染。每个 SNP 的信号强度需在两个尺度上满足形成三个明显的簇，随机选择 10%的样本进行盲法重复测量。PC
4、R 反应体系共 5l ，含有 DNA 样本 1.00 l ，2X Taqman Master Mix 2.5l ，20X 引物与探针混合物 0.25l ，蒸馏水 1.25l 。PCR 扩增：95预变性 10min；92变性 30 秒，60退火以及延伸 1 分钟；共 47 个循环。以SDS 软件 Allelic Discrimination 程序进行终点分析，判断待测样本的多态性基因型。 利用 SPSS17.0 软件进行数据整理分析，所有的统计检验均以 p0.05 为有统计学显著性差异。以 2 检验比较病例组与对照组的一般特征；用拟合优度 2检验检验病例组及对照组各基因型是否符合 Hardy-W
5、einberg 分布平衡情况；以单因素和多因素 logistic 回归计算比值比（odds ratio，OR ）及 95%可信区间（confidence interval，CI） 。结 果BRCA1 rs799917 有三种单体型：G/G、A/G、A/A。其三种基因型分布频率在病例组分别是 47.4 40.7 11.9。在对照组分别是 39.8 46.0 14.2。均符合 Handy-Weinberg 遗传平衡定律（p=0.078, 2=3.090; p=0.66, 2=0.194）。两组行 Logistic 回归计算 p 值、OR 和 95%CI 后发现，携带有A/G、A/A 基因型人群较携
6、带有 G/G 基因型人群患肺癌的风险明显降低（p=0.011, OR=0.745; p=0.036 ,OR=0.699），校正性别、年龄及吸烟状态后，差异仍有意义（p=0.021, OR=0.741; p=0.011, OR=0.610）。合并 A/G、A/A 后，病例组及对照组间差异更加明显（p=0.005, OR=0.734），校正性别、年龄及吸烟状态后，仍有差异（p=0.005, OR=0.709）。将病例按病理类型分层分析发现：鳞癌组在校正吸烟、性别、年龄后，A/A 基因型较 G/G 基因型患肺癌风险降低（p=0.012, OR=0.481），A/G 基因型较 G/G 基因型患肺癌风险
7、降低，但差异无统计学意义（p=0.055 ）。合并 A/G、A/A 后，两组间差异仍明显（p=0.012, OR=0.648）。结 论BRCA1 基因 rs799917 位点多态性与肺癌易感性相关，携带 A 等位基因的个体患肺癌发病风险降低，在鳞癌中这种保护作用更为明显。关键词BRCA1； rs799917；单核苷酸多态性；肺癌英文论著摘要Single-nucleotide polymorphisms of BRCA1 associated with susceptibility of lung cancerObjectiveTo evaluate the association betwee
8、n single nucleotide polymorphism (SNP) of BRCA1 rs799917 and susceptibility of lung cancer of ethnic Han Chinese in Liaoning Province. MethodsAll subjects were Han population in Liaoning Province without any relationship to each other. Each patient signed the informed consent in writing to undergo t
9、he diagnostic and therapeutic procedures at the time of hospitalization. The peripheral blood samples were collected right after the signing of the informed consent, and completed epidemiological investigation at the same time. A case control principle was complied in the study. In this study, we re
10、cruited a total of 682 lung cancer patients from The Departments of Radiotherapy of the First Affiliated Hospital of China Medical University, The General Hospital of Shenyang Military Region of Liaoning Province (Shenyang), and The Liaoning Cancer Hospital, from September 2009 to December 2012. All
11、 subjects were histopathologically diagnosed including squamous cell carcinoma (SCC), adenocarcinoma (AD) and small cell lung cancer (SCLC). A total of 694 cancer-free controls were selected from cancer-free population during the same period of The General Hospital of Shenyang Military Region. About
12、 5 mL of blood sample was collected from each patient. DNA was extracted by phenol-chloroform method. We applied the Taqman real-time PCR method and SDS software program to genotype the single nucleotide polymorphism. The TaqMan universal PCR master mix and predesigned SNP-genotyping assay mix which
13、 were purchased from ABI contains PCR primers and probes. In order to ensure the accuracy of genotype results, we contained three positive controls and two negative controls in each 96-well plate. Meanwhile, randomly extracted 10% samples to double-blind repeat test with the meaning of verifying the
14、 results, and obtained the same result. The reaction mix included 2.5 master mix (Applied Biosystems), 1.00 l DNA, 0.25 l probe, and 1.25 l sterile water. PCR conditions consist of an initial denaturing step at 95 for 10 min, then followed by 47 cycles of 92 for 30 sec, 60 for 60 sec eventually with
15、 a final extension at 60 for 60 sec. PCR plates were read on an ABI PRIS 7900 instrument (Applied Biosystems).We used SPSS version 17.0 for data analysis, and the criterion for significance was set at p 0.05.The characteristics between cases and controls were assessed by the 2 test ; Hardy-Weinberg
16、equilibrium (HWE) was tested by a goodness-of-fit 2 test; Associations between genotypes and the risk of lung cancer were estimated with computing odds ratio (OR) and 95 % confidence interval (CI) using logistic regression analysis with adjustments for age, gender, and smoking status.ResultThere wer
17、e three genotypes in the BRCA1 rs799917 of all participants, and they are respectively wild homozygous G/G, heterozygous A/G and mutant homozygous A/A. Genotypes in the two groups were all in accordance with Hardy-Weinberg equilibrium (p=0.078, 2=3.090; p=0.66, 2=0.194). The distribution frequencies
18、 of the BRCA1 rs799917 genotypes were 47.4 for G/G, 40.7for A/G, and 11.9for A/A in the cases, while 39.8for G/G, 46.0 for A/G, and 14.2 for A/A in the controls. Data of the cases and controls were calculated by logistic regression, the frequencies of BRCA1 rs799917 A/G versus G/G and A/A versus G/G
19、 among cases were significantly different from those among controls (p=0.011, OR=0.745; p= 0.036, OR=0.699). After adjustment for gender, age, and smoking status, the differences were also obvious (p=0.021, OR=0.741; p=0.011, OR=0.610). A/G+A/A genotypes group had even much lower risk than those of
20、G/G (p=0.005, OR=0.734). After adjustment for gender, age, and smoking status, the differences were also obvious (p=0.005, OR=0.709). Analyses were stratified according to pathological type, we found that in squamous cell carcinoma subgroup, the genotype of A/A had the lower risk of lung cancer than
21、 those of G/G (p=0.012, OR=0.481), the genotype of A/G also had the lower risk than G/G, but the difference was not statistically significant (p=0.055). A/G+A/A genotypes group also has the lower risk (p=0.012, OR=0.648). ConclusionThe A allele of rs799917 in BRCA1 was associated with the lower risk
22、 of lung cancer of ethnic Han Chinese in Liaoning Province, especially those with squamous cell carcinoma, which identified the A allele as a protective factor for lung cancer.Key wordsBRCA1; rs799917; single-nucleotide polymorphism; lung cancer英文缩略语英文缩写 英文全称 中文全称BRCA1 Breast Cancer Susceptibility G
23、ene 1 人类乳腺癌易感基因 1SNP Single Nucleotide Polymorphism 单核苷酸多态性DNA Deoxyribonucleic acid 脱氧核糖核酸SCC Squamous Cell Carcinoma 鳞状细胞癌AD Adenocarcinoma 腺癌SCLC Small Cell Lung Cancer 小细胞肺癌NSCLC Non-small Cell Lung Cancer 非小细胞肺癌PCR Polymerase Chain Reaction 聚合酶链式反应OR Odds Ratio 比值比CI Confidence Interal 可信区间HRR
24、Homologous Recombination Repair 同源重组修复NHEJ Non-homologous End Joining 非同源末端连接修复NER Nucleotide Excision Repair 核苷酸切除修复BER Base Excision Repair 碱基切除修复MMR Mismatch Repair 错配修复论文BRCA1 基因 rs799917 多态性与肺癌易感性前 言肺癌作为全球头号癌症杀手，严重影响着人类的生命及健康。最新全球癌症统计显示，肺癌的发病率和死亡率均居首位。2012 年，全球肺癌新增病例约180 万，同期肺癌导致的死亡病例超过 160 万 1
25、。我国是肺癌高发国家，其发病率和死亡率一直呈上升趋势，排在癌症死亡病因第一位。已有流行病学研究明确指出，吸烟和空气污染是造成肺癌发生的重要因素 2,3。因个体间存在差异，致使不同个体对烟草和致癌物的敏感性不同，因而造成肺癌易感性的差异，这种遗传易感性主要表现为单核甘酸多态性（Single Nucleotide Polymorphism，SNP）。研究显示 4,5，SNPs 通过改变人体肿瘤细胞代谢过程，参与细胞增殖、细胞周期控制及凋亡，最终导致肿瘤发生。多种基因 SNPs 位点与肺癌发生相关 68，这进一步证实遗传易感性可不同程度的影响肺癌的发生。人类乳腺癌易感基因 1（breast canc
26、er susceptibility gene 1,BRCA1）作为一种抑癌基因，参与包括细胞周期调控、基因转录调节、DNA 损伤修复、诱导细胞凋亡、维持基因和蛋白稳定、核转录活性调节等多种生物学功能 912。早期有研究提示 BRCA1 rs799917 位点与乳腺癌及卵巢癌的易感性相关 13,14，但结果并未完全一致 15。近期关于该基因位点与肿瘤发生的关系涉及到多种肿瘤，但对于其与中国人群肺癌易感性的研究甚少。本研究针对中国辽宁地区人群，采用病例-对照研究设计，探讨 BRCA1 基因编码区 rs799917 位点 SNP 与肺癌发病风险的关联，以期寻找人群中肺癌发病可能的遗传病因，为肺癌的一级预防提供科学依据。
