COX-2启动子区单核苷酸多态与胰腺癌

　　中国医学科学院肿瘤医院病因及癌变研究室  赵丹

　　【摘要】目的: COX-2与胰腺癌发生发展密切相关，本研究探讨了COX-2启动子区单核苷酸多态与中国人群胰腺癌遗传易感性的关系及其在吸烟与胰腺癌发病中的意义。方法:在393例胰腺癌患者和786例健康对照人群中用PCR-RFLP的方法进行基因分型，并用logistic回归分析其与胰腺癌发生风险及吸烟等危险因素的关系；用报告基因实验研究不同基因型在香烟凝集物刺激时的表达活性，用凝胶电泳迁移率实验(EMSA)分析了不同基因型结合的细胞核蛋白情况，用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)鉴定差异结合的蛋白并用EMSA超迁移实验和染色质免疫共沉淀试验(ChIP)验证COX-2启动子区转录调控情况。结果:吸烟是胰腺癌的危险因素(OR = 1.48；95% CI = 1.26-1.74)。COX-2启动子区-1195G>A和 -765G>C单核苷酸多态位点与胰腺癌发病风险相关，其中–1195AA和–765CG基因型携带者胰腺癌发生风险显著增高( OR = 1.34；95% CI = 1.12-1.60和OR = 1.63；95% CI = 1.252.10)。单体型分析结果显示两个位点之间有协同作用，A-1195-C-765单体型携带者胰腺癌发生风险显著高于G-1195-G-765单体型携带者。765G>C多态和吸烟之间存在着相乘交互作用。-765CG基因型的吸烟者其胰腺癌发生风险(OR = 3.72； 95% CI = 1.70-8.14)高于–765GG基因型的吸烟者(OR = 1.43； 95% CI = 1.21-1.69)和-765GC基因型的不吸烟者(OR = 1.44； 95% CI = 1.04-1.99)OR乘积。报告基因研究结果表明COX-2 –765位点不同基因型的基础表达情况和在香烟凝集物刺激时的表达情况存在显著差异，EMSA结果显示–765位点不同等位基因结合的核蛋白不同。–765位点差异结合的蛋白质经质谱鉴定为核仁磷酸蛋白(nucleophosmin，NPM)，并经过EMSA超迁移实验和ChIP验证，蛋白质免疫印迹实验表明香烟凝集物刺激细胞时NPM水平下降COX-2表达升高。结论: COX-2启动子区单核苷酸多态与中国人群胰腺癌遗传易感性相关，吸烟与765G>C多态对于胰腺癌发病风险之间的生物学交互作用可能由NPM分子介导。

　　Interaction of Cyclooxygenase-2 Variants and Smoking in Pancreatic Cancer: A Possible Role of Nucleophosmin

　　【Abstract】Objective: Over expression of cyclooxygenase-2 (COX-2) is implicated in cancer development. This study examined the functional relevant of genetic polymorphisms in the COX-2 promoter and evaluated their associations with susceptibility to pancreatic cancer. Methods: Genotypes and haplotypes of COX-2 –765G/C, –1195G/A and –1290A/G were analyzed in 393 pancreatic cancer patients and 786 controls. Odds ratios (ORs) and 95% confidence intervals (CIs) were computed by logistic regression. The function of the –765GC polymorphism was examined by a set of biochemical assays. Results: Smoking is a risk factor of pancreatic cancer with the OR 1.48(95% CI = 1.26–1.74). The –1195AA or –765GC genotype carriers had a 1.34-fold (95% CI = 1.12–1.60) or 1.63-fold (95% CI = 1.25–2.10) excess risk for developing pancreatic cancer. These two variants showed a cooperative effect in context of haplotype, with the ORs for the A-1195-C-765-containing haplotypes being significantly greater than those for the G-1195-G-765-containing haplotypes. The –765C allele and smoking displayed a multiplicative joint effect, with an OR being 3.72 (95% CI = 1.70–8.14) for heavy smokers carrying the –765GC genotype. Cigarette smoke remarkably increased COX-2 promoter activity and this effect was more pronounced for the –765C allele compared with the –765G allele. The differential protein binding to –765C allele found in EMSA was identified as nucleophosmin (NPM) by MALDI-TOF-MS and verified by super shift and ChIP assay with anti-NPM antibody. Western blot analysis revealed that cigarette smoke reduced nuclear NPM levels, which was reversely associated with COX-2 expression. Conclusion: Functional COX-2 polymorphisms are associated with susceptibility to pancreatic cancer and tobacco smoke specifically increases –765C promoter activity, which might be mediated by NPM.