【作者】 王艳；

【导师】 沈洪兵；

【作者基本信息】 南京医科大学 ， 流行病与卫生统计学， 2008， 硕士

【摘要】 乳腺癌是全世界女性最常见的恶性肿瘤,已成为威胁女性健康的最重要疾病之一。世界范围内乳腺癌发病呈不断上升的趋势,我国虽属女性乳腺癌的低发病区,但近年来乳腺癌的发病率逐年快速增长,尤其在沪、京等地区,乳腺癌的发病率已高居女性恶性肿瘤发病率的第一、第二位。乳腺癌严重威胁着女性的健康和生命,对社会、经济、家庭和妇女心理造成极大的影响。如何识别乳腺癌的高危人群,降低乳腺癌的发病率,是摆在我们面前的重要问题。但乳腺癌病因尚不明确,发病机制也十分复杂,目前认为乳腺癌的发生、发展是环境与遗传因素共同作用的结果。不同遗传背景的个体在相同的危险因素暴露下发生乳腺癌的危险性不同,说明遗传背景的差异影响了个体癌症易感性的高低。因此,识别乳腺癌易感基因是鉴别乳腺癌高危人群的关键。众所周知,乳腺癌的高共显性基因BRCA1、BRCA2突变是乳腺癌的强危险因素,但BRCA1、BRCA2突变在一般人群中对乳腺癌发病的贡献很小,仅占5%以下,故认为大部分的乳腺癌易感性与常见的低共显性基因的遗传变异有关。寻找与乳腺癌遗传易感性相关的常见低共显性基因是目前研究的一个热点。研究表明,在乳腺癌的病理进程中,雌激素起了重要的作用。表皮生长因子(EGF)作为强有力的有丝分裂剂,具有类雌激素作用,同样在乳腺癌的发生发展过程中扮演了重要的角色。EGF在组织中的过表达可持续性激活多条信号传导通路,促进DNA合成,加速细胞的分裂和增殖,抑制细胞的凋亡,从而促进乳腺癌的发生、发展。已有研究发现,EGF基因启动子区的单核苷酸多态性(Single nucleotidepolymorphisms,SNPs)不仅与外周血EGF水平有关,而且与癌症的发病风险相关。同时,由于EGF可以通过启动Ras-MAPK信号通路,激活端粒酶逆转录酶(hTERT),上调hTERT mRNA的表达,而该酶的激活可促使端粒不再随细胞分裂而缩短,细胞得以无限制的增殖下去,最终导致肿瘤的发生。并且,hTERT在正常人类体细胞中不表达,但是在包括乳腺癌在内的多种肿瘤中表达。故推测EGF、hTERT的SNPs及血浆EGF水平可能与乳腺癌遗传易感性有关。本研究旨在通过血清流行病学和分子流行病学相结合的手段,采用病例对照研究方法,对血浆EGF水平以及EGF基因启动子区SNPs和hTERT的功能性SNPs与乳腺癌遗传易感性的关系进行研究,进一步阐明乳腺癌的发病机制和发现与我国人群乳腺癌遗传易感性相关的危险基因型,并将其作为分子标志物用于筛选高危人群,从而为今后乳腺癌的早期诊断和早期治疗提供科学依据。第一部分表皮生长因子基因(EGF)启动子区多态性、血浆EGF水平与乳腺癌易感性的病例对照研究研究发现,EGF启动子对EGF的表达起关键的调控作用,提示该区域的遗传变异可能影响不同个体EGF的表达水平,进而影响疾病的易感性。已有研究表明,EGF启动子区G61A位点的突变可降低外周血EGF的水平,且降低患恶性黑色素瘤的风险。因此,EGF基因启动子区域的多态性改变可能潜在地影响其表达(血浆水平)从而改变个体对肿瘤的易感性。本研究探讨了EGF基因启动子区稀有等位基因频率(MAF,minor allele frequency)高于0.05的多态性改变(G61A、G-1380A和A-1744G)及血浆中EGF水平与乳腺癌发病的关联。采用病例对照研究设计,选择经组织学确诊的629例新发乳腺癌病例和694例年龄和地区匹配的无肿瘤史的健康对照。采用PCR-RFLP(RestrictionFragment Length Polymorphism,限制性片断长度多态性)、PCR-PIRA(Primer Introduced Restriction Analysis,引物引入限制性分析)法设计引物和内切酶进行基因分型。采用ELISA(Enzyme-LinkedImmunosorbnent Assay,酶联免疫吸附试验)法进行血浆EGF水平的检测。此外,基因型检测采用盲法,并随机抽取10%样本重复检测进行实验室质量控制。最后,我们成功获得604例(96.0%)乳腺癌病例和654例(94.2%)对照的MNS16A基因型。结果发现:(1)各基因型在病例组和对照组之间的分布没有显著差异,单因素和多因素logistic回归分析未发现EGF基因启动子区的三个单核苷酸多态性改变(G61A、G-1380A和A-1744G)与乳腺癌发生存在关联。(2)乳腺癌患者中血浆EGF的平均水平(249.06±197.54 pg/ml)显著低于对照组(982.41±375.57 pg/ml,P＜0.001)。血浆中EGF的高水平显著降低乳腺癌的发病风险,且血浆EGF水平与乳腺癌的发病风险呈现显著的剂量反应关系。(3)在对照人群中,携带EGF-1380AA基因型的个体血浆中EGF水平显著高于携带野生型(-1380GG)的个体(1308.87 pg/ml vs 946.87pg/ml,P=0.003)。本研究结果表明,血浆中高水平EGF可能对乳腺癌发病起保护作用,且EGF G-1380A的突变可能是血浆EGF水平的调节因子。第二部分端粒酶逆转录酶基因(hTERT)的功能性多态与乳腺癌易感性及表型的病例对照研究已有研究发现,hTERT的一个串连重复序列多态(MNS16A)可调控该基因的表达,影响端粒酶活性,从而可能潜在地影响个体对肿瘤的易感性。本病例对照研究中,我们探讨了hTERT基因串连重复序列多态(MNS16A)与乳腺癌发病风险及其表型的关联。我们选择经组织学确诊的1029例新发乳腺癌病例和1107例年龄和地区匹配的无肿瘤史的健康对照。采用PCR扩增的方法检测MNS16A的基因型,盲法进行基因型的判读,随机选择10%(213个)的样本进行盲法复测。最后,我们成功获得1006例(97.8%)乳腺癌病例和1095例(99.3%)对照的MNS16A基因型。结果发现:(1)MNS16A多态性改变与乳腺癌的易感性有显著关联,其突变基因型可显著增加乳腺癌的发病风险,并且该作用在年龄≥50岁、绝经后、具有肿瘤家族史、初潮年龄早(＜16岁)、首胎活产年龄晚(≥25岁)的妇女中更为显著。(2)将乳腺癌患者按有/无腋窝淋巴结转移进行分组后发现,MNS16A的302/271基因型在两组间分布存在显著差异(P=0.038)。与携带302/302基因型的乳腺癌患者相比较,携带302/271基因型的乳腺癌患者发生腋窝淋巴结转移的风险为2.13倍(95%CI=1.05-4.33)。本研究结果表明hTERT-MNS16A多态性改变可能与乳腺癌的发生及腋窝淋巴结的转移有关。这一结论尚需要功能学研究或其他种族人群的大样本研究进一步验证。更多还原

【Abstract】 Breast cancer is the most common malignancy among women, accounting for nearly one in three cancers diagnosed among women in the United States. Although the incidence rate of breast cancer in China is about one-third of that in the United States, it has been significantly increasing in the last two decades in both urban and rural areas, especially in big cities as Shanghai and Beijing. However, the exact molecular mechanisms to breast cancer are still unclear. It is well accepted that interaction of environmental factors and genetic factors may contribute to the development of breast cancer. The individuals with different genetic background have different risk of breast cancer. So, identifying the breast cancer susceptibility genes is the most important task in recognizing high risk population. It is well known that the mutations in human family breast cancer susceptibility genes, BRCA1/2, are strong risk factors to breast cancer, but it only account for less than 5% of all breast cancer cases. Thus, it is crucial to investigate other genetic risk factors of breast cancer in general population. Mounting evidence indicates high level of endogenous estrogen is believed to contribute to the etiology of breast cancer. Epidermal growth factor (EGF), a potent mitogenic peptide, is in possession of estrogen-like property and also plays important role in breast carcinogenesis. Over-expression of EGF may increase proliferation, inhibit apoptosis and enhance the invasiveness of breast cancer cells by activating its signal transduction pathways. Studies showed the polymorphisms in the promoter of EGF gene are associated with plasma EGF level and risk of cancer. Meanwhile, EGF up-regulates telomerase reverse transcriptase (hTERT) activity and regulates telomere length maintenance and promotes cell proliferation uncontrolled, eventually leads to cancer. hTERT mRNA is absent in most normal human somatic cells, but is detected in many human cancers, including breast cancer. Thus, we conducted a case-control study aiming to investigate the association of plasma EGF levels and SNPs of EGF and hTERT genes with breast cancer risk in Chinese populations.Part IEGF Promoter SNPs, Plasma EGF levels and Risk of BreastCancer in Chinese WomenEGF expression was suggested to be regulated by EGF promoter activity, suggesting the variants in EGF promoter region may affect the EGF expression in diffrent individuals, eventually influence the susceptibility to cancer. Shahbazi et al. found the variant of G61A in EGF promoter region led to a decreased EGF production in peripheral blood mononuclear cells and decreased susceptibility of malignant melanoma. Thus, the polymorphisms in EGF promoter may influence the plasma EGF level and contribute to inter-individual variability in susceptibility to breast cancer.The aim of this case-control study is to investigate the associations of plasma EGF levels and three EGF promoter SNPs (G61A, G-1380A and A-1744G) with breast cancer risk and the modification effects of EGF promoter SNPs on plasma EGF levels. This case-control study included 629 histologically confirmed incident breast cancer patients and 694 cancer-free controls frequency-matched for age and area. We genotyped the three promoter polymorphisms by PCR-RFLP (PCR-Restriction Fragment Length Polymorphism) and PCR-PIRA (PCR-Primer Introduced Restriction Analysis) method. In this case control study, the plasma EGF concentration was measured with ELISA (Enzyme-Linked Immunosorbnent Assay). In addition, genotyping was performed blindly and 10% of the samples were randomly selected for repeated assays. Finally, a total of 604 (96.0%) breast cancer cases and 654 (94.2%) controls were successfully genotyped.In this case-control study, we did not find any significant associations between the three EGF polymorphisms (G61A, G-1380A and A-1744G) and risk of breast cancer even after adjusting by confounder factors in multifactor logistic analysis. However, we found the mean plasma EGF levels in breast cancer patients (249.06±197.54pg/ml) were significantly lower than those in controls (982.41±375.57pg/ml, P<0.001). According to the quartile levels in the controls, the risks of breast cancer were decreased compared with the lowest quartile of EGF, and the trend of increase was significant (x~2=474.35, P<0.001). In addition, there was a significant difference of plasma EGF levels among different genotypes carriers of the G-1380A locus in controls (P=0.007). The individuals EGF-1380AA had significantly higher plasma EGF levels than -1380GG carriers did (1308.87 pg/ml vs 946.87pg/ml, P=0.003). These findings indicated that plasma EGF levels served as a protective marker against breast cancer in our study and EGF G-1380A variant might be a modifier on it.Part IIA tandem repeat of human telomerase reverse transcriptase(hTERT) and risk of breast cancer development andmetastasis in Chinese womenStudies showed a polymorphic tandem repeat minisatellite (termed MNS16A) in the downstream of hTERT gene was demonstrated to regulate the expression of hTERT rnRNA level, influence telomerase activity, and eventually may contribute to inter-individual variability in susceptibility to breast cancer. So, we hypothesized that the MNS16A variant may be associated with breast cancer risk and phenotypes in Chinese women. This case-control study included 1029 histologically confirmed incident breast cancer patients and 1107 cancer-free controls frequency-matched for age and area. We genotyped the MNS16A variant by PCR method. Genotyping was performed blindly and 10% of the samples were randomly selected for repeated assays. Finally, a total of 1006 (97.8%) cancer cases and 1095 (99.3%) controls were successfully genotyped.In this case-control study, we found a significantly increased risk of breast cancer associated with the MNS16A variant genotypes (302/271, 302/243 and 243/243) and the effects were more evident among the older women (≥50), postmenopausal women, and individuals with family history of cancer, with earlier menarche age (<16), with older age at first live birth (≥25). In breast cancer cases, 302/271 genotype was associated with an increased risk for axillary lymph node metastasis (adjusted OR=2.13, 95%CI=1.05-4.33).These results supported that hTERT-MNS16A variant might be associated with breast cancer development and metastasis in Chinese population.更多还原