【作者】 牛玉明；

【导师】 陈宁；

【作者基本信息】 南京医科大学 ， 口腔颌面外科学， 2011， 博士

【摘要】 恶性肿瘤是目前导致人类死亡的最主要的致病因素之一,头颈部鳞状细胞癌(Head and Neck Squamous Cell Carcinoma,HNSCC)泛指发生于口腔颌面部,咽喉及上呼吸消化道等部位的鳞状细胞癌,在恶性肿瘤的死亡因素中列第八位,严重威胁着人类的健康和生命。头颈部鳞状细胞癌的发病原因尚不明确,随着流行病学和分子生物学的发展,目前认为头颈部鳞状细胞癌的发生发展是外界环境与个体遗传因素共同作用的结果。遗传生物学的研究结果表明,个体遗传背景的差异往往导致了患者罹患头颈部鳞状细胞癌易感性的不同,因此,对于易感基因的鉴别可能是筛查患头颈部鳞状细胞癌高危人群和预防其发生的关键。环氧化酶(cyclooxygenase,COX)是一类前列腺素合成过程中重要的限速酶,COX有两个亚单位:COX-1和COX-2。COX-1在正常组织中稳定表达并且合成前列腺素以维持正常的生理功能。而COX-2在大多数组织中不表达或低表达,可被生长因子、内毒素及肿瘤促进因子诱导产生。研究已经证实COX-2在多种恶性肿瘤(包括食管癌、乳腺癌、卵巢癌、结肠癌、喉癌、胰腺癌、前列腺癌、肺癌、肝癌等)中高表达。此外,我们前期的研究发现COX-2的高表达与头颈部鳞状细胞癌( head and neck squamous cell carcinoma,HNSCC)的发生发展密切相关。单核苷酸多态性(single nucleotide polymorphism,SNP),是一种常见的遗传变异(genetic variation),发生在DNA编码区(coding region)的SNP,可引起所翻译蛋白质的一级结构的变异,进而可能改变蛋白质结构域的功能。如果SNP发生在DNA的非编码区(non-coding region),特别是基因上游的启动子区域(promoter region)及5’和3’端的非翻译区域(untranslated region),则可能影响该基因的转录过程,而改变mRNA的转录及表达,进而影响下游蛋白质的表达和功能,从而可能造成个体对肿瘤易感性的差异。本课题采用分子流行病学“病例—对照”的研究方法,运用分子生物学实验技术,进行COX-2基因遗传变异与头颈部鳞状细胞癌发生易感性的流行病学研究。第一部分COX-2基因单核苷酸多态性与头颈部鳞状细胞癌易感性的相关性研究环氧化酶2(COX-2)是前列腺素(PGs)合成过程中一个主要的限速酶,可将花生四烯酸(AA)代谢成各种前列腺素产物,从而参与机体的多种病理生理过程。COX-2过度表达能够促进细胞增殖、抑制凋亡和促进血管新生,从而导致肿瘤的形成。许多癌前病变和恶性肿瘤中均有COX-2基因的扩增及其蛋白质的高表达。COX-2的表达及其稳定性受基因启动子区及5’和3’非翻译区的多种转录因子调控,在这些区域内的多态性改变可能会影响基因的表达,从而改变个体对肿瘤的易感性。研究表明,在中国人群中COX-2基因启动子区-1195A>G(rs689466),-765G>C(rs20417)位点等位基因的变异可以导致发生食管癌的危险性显著性升高,1759G>A(rs3218625)位点SNP改变将导致COX-2蛋白质中587位的Gly被Arg取代,从而使COX-2催化花生四烯酸的活性增加约1.5倍;而3’端非翻译区8473T>C(rs5275)位点的SNP改变与则可能肺癌及乳腺癌等疾病的发病发生有关。本研究通过病例—对照研究(260例新发头颈部鳞状细胞癌患者:包括169位口腔癌患者、91位喉癌患者和1047例健康对照),探讨①COX-2启动子区域SNP变异位点:-1195A>G,-765G>C;②COX-2编码区SNP变异位点:1759G>A;③3’端非翻译区(3’UTR)SNP变异位点:8473T>C四个变异位点与头颈部鳞状细胞癌易感性的关系。采用Taqman-MGB及PCR-RFLP(Restriction Fragment Length Polymorphism,限制性片断长度多态性)技术进行基因分型,运用多因素Logistic回归,分析基因型及烟酒等危险因素与头颈部鳞状细胞癌发生风险的关系。结果表明:(1)-1195A>G位点SNP改变与头颈部鳞状细胞癌的发生不存在显著统计学关联,与携带野生型纯合子AA的个体相比,携带杂合子AG的个体发病风险:调整OR= 0.87(95%CI=0.63-1.20);携带变异型纯合子GG的个体发病风险:调整OR=1.02(95%CI=0.69-1.50)。(2)-765G>C位点SNP改变与头颈部鳞状细胞癌的发生没有显著的相关性,与携带野生纯合型GG的个体相比,携带杂合子GA的个体发病风险:调整OR=1.01(95%CI=0.61-1.67)。(3)1759G>A位点SNP改变未能发现与头颈部鳞状细胞癌的发生有显著的相关性,与携带野生纯合型GG的个体相比,携带杂合子GA的个体发病风险:调整OR=0.42(95%CI=0.16-1.07)。(4)8473T>C位点SNP改变与头颈部鳞状细胞癌的发生没有显著的统计学关联,与携带野生纯合型TT的个体相比,携带杂合子TC的个体发病风险:调整OR=0.90(95%CI=0.66-1.22);携带变异型纯合子CC的个体发病风险:调整OR=1.48(95%CI=0.68-3.25)。本研究结果提示,-1195A>G,-765G>C,1759G>A,8473T>C位点的SNP改变与中国汉族人群中头颈部鳞状细胞癌发生的易感性之间不存在显著的统计学关联。第二部分COX-2基因启动子区域单核苷酸多态性与启动子活性改变的功能性研究COX-2基因启动子区域含有与多种转录因子相互结合的位点,比如PEA3、IL6、NFKB、SP1和c-MYB等。在COX-2基因的转录过程中,这些转录因子与启动子区域密切结合。COX-2基因启动子区域SNP改变将直接影响其与特定的转录因子的结合能力,导致基因的转录能力的改变,进而引起蛋白质表达水平发生变化。本研究通过PCR技术扩增从-1940核苷酸到+201核苷酸的COX-2基因启动子序列,片段长度共2141bp,产物纯化后直接进行测序确认,然后利用定点突变技术获得含有-1195A>G,-765G>C位点等位基因突变的DNA片段。通过双酶切消化及连接反应与pGL3-Basic载体相结合,成功构建含有COX-2基因启动子区域-1195A>G和-765G>C SNP位点的3种含有COX-2基因启动子的报告基因质粒:pGL3-A_-1195-G_-765,pGL3-G_-1195-G_-765,pGL3-A_-1195-C_-765。通过瞬时转染Tca-8113,Hela和MG-63细胞系并结合双荧光素酶检测发现:pGL3-A-1195-G-765质粒的荧光素酶活性均显著高于pGL3-G-1195-G-765质粒(P<0.001);而pGL3-A_-1195-G_-765质粒与pGL3-A_-1195-C_-765质粒的荧光素酶活性无显著性差异。本研究结果表明,-1195A→G的SNP改变可导致启动子区域转录活性的改变,降低启动子的转录活性;而-765G→C的SNP改变未能导致启动子区域转录活性的变化。更多还原

【Abstract】 Head and neck squamous cell carcinoma (HNSCC) cancer is one of the most frequent malignant tumors in the world. In recent years, it shows a significantly increasing trend in the last two decades. However, the exact molecular mechanisms to develop head and neck squamous cell carcinoma are still unclear. It is well accepted that interaction of environmental factors and genetic factors may contribute to the etiology of head and neck squamous cell carcinoma. The individuals with different genetic background have different risk of head and neck squamous cell carcinoma. So, it is important to find the head and neck squamous cell carcinoma susceptibility genes in a high-risk population.Overexpressing of cyclooxygenase-2(COX-2) has been implicated in the development of many type of human cancer. Single nucleotide polymorphism (SNP) in the COX-2 gene might contribute to diffenertial COX-2 expressiong and enzymatic activity. The SNP located in the non-coding region, especially in promoter region or 3’untranslated region(3’UTR) of COX-2 may affect the biological functions of COX-2 and risk of developing head and neck squamous cell carcinoma. Thus, we conducted a case-control study to investigate the association of COX-2 genes polymorphisms with head and neck squamous cell carcinoma susceptibility in Chinese populations.Part I: COX-2 gene polymorphism and the susceptibility of head and neck squamous cell carcinomaCOX-2 is the critical enzymes that convert arachidonic acid into prostaglandins and thromboxanes. COX-2 is always involved in inflammation angiogenesis, anti-apoptosis and carcinogenesis. COX-2 overexpression was found in a large proportion of tumor tissues and was significantly associated with advanced tumor stage and lymph node metastasis, such as colon, breast, gland, stomach, prostate and head and neck cancer.The expression of COX-2 is regulated by a complex signal transduction pathway in which many nuclear proteins interact with the COX-2 promoter region and play a decisive role in gene transcription. Therefore, single nucleotide polymorphisms (SNP) in the COX-2 promoter may have a great impact on gene transcriptional activity by altering the binding capability with certain nuclear proteins, resulting in inter-individual variability in susceptibility to cancer. In research of Chinese population, two SNP-1195A>G(rs689466), -765G>C(rs20417) in the promoter region are associated with risk of developing gastroesophageal cancer. The SNP 1759G>A(rs3218625) will induce 587Gly/Arg change had significant impact on COX-2 activity; the COX-2-Arg587 isoform showed 1.5 fold elevated enzymatic activity than the COX-2-Gly587 isoform in converting arachidonic acid into prostaglandins. The SNP 8473T>C (rs5275) in 3’UTR region are associated with risk of lung cancer.The aim of this case-control study including 260 head and neck squamous cell carcinoma cases and 1047 controls is to investigate the associations of the SNP -1195A>G, -765G>C in the cox-2 promoter regiong, the SNP 1759G>A in the coding region and the SNP 8473T>C in the 3’UTR regiong with head and neck squamous cell carcinoma risk in Chinese populations.We genotyped the four polymorphisms by PCR-RFLP (PCR-Restriction Fragment Length Polymorphism) and TaqMan-MGB method. In addition, genotyping was performed blindly and 10% of the samples were randomly selected for repeated assays. Finally, a total of 259 head and neck squamous cell carcinoma cases and 1035 controls were successfully genotyped.In the present case-control study, the genotype of -1195AG and -1195GG, were associated with no significantly increased risk of head and neck squamous cell carcinoma (adjusted OR=0.87, 95% CI=0.63-1.20; OR=1.02, 95% CI=0.69-1.50 ); compared with the wild-type genotype; the genotype of -765GC was not associated with the risk for developing head and neck squamous cell carcinoma (adjusted OR=1.01,95% CI=0.61-1.67); compared with the wild-type genotype, the genotype of 1759GA was not a risk factor for susceptibility to head and neck squamous cell carcinoma (adjusted OR=0.42, 95% CI=0.16-1.07 ); the genotype of 8473TC and 8473CC may be not the risk factors for developing head and neck squamous cell carcinoma (adjusted OR=0.90, 95% CI=0.66-1.22; OR=1.48, 95%CI= 0.68-3.25).These findings suggested that the four SNP may not associate with head and neck squamous cell carcinoma risk in Chinese population.Part II: Functional relationgship between SNP in the COX-2 promoter regiong and the changes of promote activityThe COX-2 promoter region has identified several key cis-acting regulatory elements to bind these transcription factors (such as PEA3, AP1. interleukin-6, nuclear factor KB, Sp1 and c-MYB), which may play a decisive role in the regulation of COX-2 transcription. The mutations within the consensus sequences of certain cis-acting elements significantly altered gene expression.In our research we constructed a reporter plasmid encompassing -1940 to +201 base pairs of human COX-2 promoter region. The constructs used in this study were restriction mapped and sequenced to confirm their authenticity. The PCR product was digested, site-directed mutated and ligated, respectively, into an appropriately digested pGL3-Basic vector (Promega) containing the firefly luciferase gene as a reporter. Then we transient transfected the Tca-8113, Hela and MG-63 cells with these re-construct plasmids .Accroding to the Luciferase Assays, we found -1195A-containing COX2 promoters were higher than those driven by the -1195G-containing counterparts(P<0.001). No significant effect of the -765G>C polymorphism on the promoter activity was observed under our experimental conditions. These results suggest that the -1195G>A SNP can influences COX-2 promoter activity, but the -765G/C containing haplotypes no associated with the promoter activity .更多还原