李玉涛, 王 一, 徐月娟, 张艳红, 郭士成,4, 赵振宏, 李 强, 卢大儒,6, 金 力,6,王久存,6, 钱 成, 吴俊杰,*
1.复旦大学现代人类学教育部重点实验室,上海 200433 2.东南大学第二人民医院肿瘤内科,南京 210003 3.复旦大学校医院,上海 200433 4.Department of Bioengineering, University of California at San Diego, CA 92093, USA 5.第二军医大学长海医院呼吸内科,上海 200433 6.复旦大学(泰州)健康科学研究院,泰州 225300 7.复旦大学附属中山医院胸外科,上海 200032
·论著·
C反应蛋白基因rs2808630、吸烟与非小细胞肺癌易感性的相关性
李玉涛1, 王一1, 徐月娟2, 张艳红3, 郭士成1,4, 赵振宏1, 李强5, 卢大儒1,6, 金力1,6,王久存1,6, 钱成7*, 吴俊杰1,5*
1.复旦大学现代人类学教育部重点实验室,上海200433 2.东南大学第二人民医院肿瘤内科,南京210003 3.复旦大学校医院,上海200433 4.Department of Bioengineering, University of California at San Diego, CA 92093, USA 5.第二军医大学长海医院呼吸内科,上海200433 6.复旦大学(泰州)健康科学研究院,泰州225300 7.复旦大学附属中山医院胸外科,上海200032
目的: 探讨C反应蛋白(CRP)基因rs2808630多态性与非小细胞肺癌(NSCLC)的相关性。方法: 根据连接酶连接反应原理,对上海及江苏泰州地区974例NSCLC患者和1 005例健康人进行CRP基因rs2808630基因分型,分析该位点与NSCLC的相关性。结果: CRP基因rs2808630位点TC较TT患NSCLC风险增加(P=0.034),但校正后不相关。吸烟人群中该位点携带TC 基因型者较携带 TT基因型者患NSCLC风险增加 [比值比(OR)=1.329,95% 可信区间(CI) 1.017~1.736,P=0.037],携带TC基因型者患鳞癌的风险是携带TT基因型者的1.383倍(95% CI 1.023~1.870,P=0.035);进一步分层分析发现,吸烟人群中携带TC基因型者患NSCLC的风险是携带TT基因型者的1.479倍(95% CI 1.058~2.067,P= 0.022)。 结论: CRP基因rs2808630 T>C可能与吸烟协同导致NSCLC,尤其是鳞癌的发生。
C反应蛋白;rs2808630位点;病例-对照研究;吸烟;非小细胞肺癌
肺癌是全球患病率和病死率最高的恶性肿瘤之一,起病较为隐匿,大部分患者发现时已为晚期,且5年生存率低于20%[1-2]。吸烟是肺癌发生的危险因素,与肺癌患者预后相关[2-3]。肺癌分为小细胞肺癌(small cell lung cancer,SCLC)和非小细胞肺癌(non-small cell lung cancer,NSCLC)两大类,其中NSCLC发病率占肺癌的85%~90%[2]。NSCLC主要分为腺癌、鳞癌两种病理类型,其中鳞癌与吸烟密切相关[4]。
肺癌、肝癌、食管癌等多种癌症的发生与炎症反应密切相关[5-6]。炎症微环境有利于诱导基因突变和表观遗传学特征的改变,促进细胞增殖、抗凋亡、促进癌细胞扩散[7-8]。C反应蛋白(Creactive protein,CRP)是一种敏感的非特异性炎症标志物,最初从急性肺炎球菌性肺炎患者血浆中分离得到,因其对肺炎链球菌C多糖有高亲合性,故命名为CRP。CRP属于pentraxin(PTX)蛋白家族,参与机体防御,能识别外来病原体和宿主的受损细胞,并通过体液和细胞免疫将其清除。巢式病例对照研究[9]显示,CRP水平升高与肺癌风险增加有关。在恶性肿瘤如肺癌、肝癌、大肠癌、食管癌等患者血清中CRP水平升高,且与预后较差相关[10-13]。CRP基因rs2808630的单核苷酸多态性(single nucleotide polymorphisms,SNP)与非西班牙裔黑人血清中CRP水平降低相关[14]。欧洲人群的全基因组关联分析显示,rs2808630增加肺癌风险[15]。因此,探索rs2808630及吸烟与中国人群肺癌发生的相关性至关重要。
1.1一般资料2009年1月—11月在第二军医大学长海医院、江苏省泰州市第一人民医院等医院收集NSCLC患者的血液样本。纳入标准:经病理组织学检查确诊的新发NSCLC患者,无其他器官恶性肿瘤史,无年龄、性别限制。对照组按年龄、性别和地区进行频数匹配,选择同一时期相应地区或社区健康体检者的血液样本。本研究通过复旦大学生命科学学院伦理委员会审核批准;流行病学调查资料收集及血液样本采集均获得研究对象知情同意。1.2DNA提取及基因分型检测采用Qiagen血液DNA提取试剂盒(德国Qiagen公司),提取全血基因组DNA。基因分型检测参照Zhang等[16]的方法进行。基因分型质量控制按照质量控制(QC)流程进行,包括>95%的基因分型检出率、重复检出的基因型、内部阳性对照样品检测。
1.3统计学处理采用SPSS 19.0软件进行处理。人口统计变异值、人群吸烟状况、癌症家族史、对照组与病例组等位基因频率差异及哈迪-温伯格平衡(Hardy-Weinberg equilibrium,HWE)检验采用χ2检验分析。分别对等位基因模型、基因型模型、显性模型和隐性模型进行非条件Logistic回归分析,计算比值比(odds ratios,OR)和95%可信区间(95%CI),校正年龄、性别、吸烟状况和癌症家族史后进一步计算OR和95%CI。按年龄、性别、吸烟情况、家族史和肺癌组织学类型对各种模型分别进行分析。检验水准(α)为0.05。
2.1研究对象的一般情况纳入NSCLC病例974例,其中第二军医大学长海医院536例,复旦大学(泰州)健康科学研究院352例,上海其他医院86例;对照组1 005例。结果(表1)显示:病例组和对照组研究对象的年龄分布和性别构成差异无统计学意义;病例组吸烟者占70.03%,对照组吸烟者占49.95%,差异有统计学意义(P<0.001);病例组中有癌症家族史者占34.60%,对照组中有癌症家族史者占14.63%,差异有统计学意义(P<0.001)。
2.2rs2808630位点多态性与NSCLC易感性相关性CRP基因rs2808630位点TT基因型1 395个、TC基因型528个、CC基因型56个,基因型检出率为100%。CRP基因rs2808630 T/C位点基因型频率符合HWE定律(P=0.457),说明调查群体达到遗传平衡,即本次群体调查数据可信。
表1 研究对象的一般情况
*部分患者的年龄和吸烟情况缺失,故未能纳入统计
结果(表2)显示:rs2808630位点等位基因T和C频率在病例组中分别是82.9%和17.1%,在对照组中分别为84.7%和15.3%,两组间等位基因频率差异无统计学意义。TC基因型个体较TT基因型个体患NSCLC的风险明显增加(P=0.034),但经性别、年龄、吸烟情况和癌症家族史校正后两者差异无统计学意义(P=0.082)。显性模型中,TC+CC基因型个体患NSCLC的风险较野生纯合TT基因型携带者增加,但差异无统计学意义(校正后P=0.095)。隐性模型中,CC基因型个体与TT+TC基因型个体患NSCLC的风险差异无统计学意义(校正后P=0.976)。上述结果说明,rs2808630位点T>C突变与NSCLC患病风险关系不明显。2.3rs2808630位点多态性与NSCLC易感性的分层分析校正后分层分析显示,等位基因T>C突变吸烟者患NSCLC风险增加(P=0.036),且鳞癌风险也增加(P=0.039,表3)。基因型模型中,与TT基因型吸烟者相比,TC基因型吸烟者患NSCLC的风险增加1.329倍(OR=1.329,95%CI 1.017~1.736,P= 0.037),且携带TC基因型者患鳞癌风险增加1.383倍(OR=1.383,95%CI 1.023~1.870,P= 0.035);携带CC基因型者与携带野生型TT基因型者的各因素差异均无统计学意义,表明TC是rs2808630位点的NSCLC风险基因型(表4)。显性模型中,TC+CC基因型吸烟者较TT基因型吸烟者患NSCLC风险显著升高1.335倍(P=0.030),且携带TC+CC基因型者鳞癌风险是携带TT基因型者的1.381倍(P=0.030,表5)。隐性模型中未见阳性结果(表6)。
表2 CRP基因rs2808630多态性与NSCLC易感性相关性分析
a校正性别、年龄、吸烟情况和癌症家族史
进一步分层分析,吸烟者等位基因T>C的突变使患鳞癌风险显著上升(P=0.010),TC基因型吸烟者患鳞癌风险是TT基因型吸烟者的1.479倍(OR=1.479,95%CI 1.058~2.067,P=0.022;表7)。
表3 等位基因模型中CRP基因 rs2808630多态性与NSCLC易感性的相关性
a校正性别、年龄、吸烟情况和癌症家族史中其他3种. ADC:腺癌;SCC:鳞癌
表4 基因型模型中CRP基因 rs2808630多态性与NSCLC易感性的相关性
a校正性别、年龄、吸烟状况和癌症家族史中其他3种. ADC:腺癌;SCC:鳞癌
表5 显性模型中CRP 基因rs2808630多态性与NSCLC易感性的相关性
a校正性别、年龄、吸烟情况和癌症家族史中其他3种. ADC:腺癌;SCC:鳞癌
表6 隐性模型中CRP 基因rs2808630多态性与NSCLC易感性的相关性
a校正性别、年龄、吸烟情况和癌症家族史中其他3种. ADC:腺癌;SCC:鳞癌
表7 吸烟分层各模型中CRP基因 rs2808630多态性与鳞癌易感性的相关性
a校正性别、年龄和癌症家族史
CRP能抑制MCA-38小鼠结肠腺癌细胞上皮间充质转化[17];人头颈部鳞癌细胞系BHY、PCI 1和PCI 13等细胞系分泌的CRP能降低髓样树突状细胞的迁移能力[18];胶质母细胞瘤细胞通过分泌白细胞介素-6(IL-6)诱导肝细胞产生CRP,后者可促进小胶质细胞释放IL-1β,进而促进肿瘤血管生成[19]。因此认为,CRP参与癌症的发生发展。
小鼠慢性阻塞性肺疾病(COPD)模型中,香烟烟雾提取物能促进炎症因子释放[20]。吸烟可导致人肺部炎症和氧化应激,增加COPD风险[21]。CRP、纤维蛋白原和白细胞水平的同时上升与COPD 急性加重风险增加相关[22]。COPD患者气道出现过度炎症及基质破坏,一些蛋白家族如解毒酶、蛋白酶、抗蛋白酶和细胞因子的SNP被认为参与COPD和肺癌的发生[23]。CRP基因rs2808630位点位于3′非翻译区(3′untranslated region,3′UTR)远端,处于非进化保守区域[14]。中国人群中,rs2808630与rs1205强连锁(D’=1.0),而rs1205 CC+CT 与COPD风险增加相关[24]。结果提示,CRP rs2808630可能在中国人群中参与COPD、吸烟相关肿瘤的发生。
陕西省[25]、湖北省[26]人群的病例-对照研究中,CRP T>C突变与肺癌的患病风险无显著相关性,但这些研究中样本量较小,且未考虑该位点在不同致病因素(如吸烟等)暴露下与肺癌易感性的关系,结果可能存在偏倚。本研究扩大样本量,通过吸烟、家族史、癌症类型等分层分析,发现rs2808630位点TC基因型在吸烟史者中患NSCLC的风险增加,TC基因型携带者患鳞癌风险也明显增加;进一步分层分析显示,携带TC基因型的吸烟者更易患鳞癌。此位点突变可能通过影响血清CRP蛋白水平[14],并与吸烟共同导致气道炎症如COPD等[20-22]的发生,最终导致肺癌尤其是鳞癌[4]的发生。
综上所述,CRP基因rs2808630位点TC基因型与中国人群吸烟者NSCLC发生风险升高有关,而且,携带该基因型的吸烟者罹患鳞癌的风险更显著,提示该位点与吸烟协同参与肺癌尤其是鳞癌的发生发展,但机制有待进一步研究。
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[本文编辑]姬静芳
C-reactive protein rs2808630 T>C in smokers increases non-small cell lung cancer susceptibility
LI Yu-tao1, WANG Yi1, XU Yue-juan2, ZHANG Yan-hong3, GUO Shi-cheng1,4, ZHAO Zhen-hong1, LI Qiang5, LU Da-ru1,6, JIN Li1,6, WANG Jiu-cun1,6, QIAN Cheng7*, WU Jun-jie1,5*
1. Ministry of Education Key Laboratory of Contemporary Anthropology, Fudan University, Shanghai 200433, China 2. Department of Oncology, The Second People’s Hospital of Nanjing, Southeast University, Nanjing 210003, Jiangsu, China 3. Hospital of Fudan University, Shanghai 200433, China 4. Department of Bioengineering, University of California at San Diego, CA 92093, USA 5. Department of Respiratory Medicine, Changhai Hospital, Second Military Medical University, Shanghai 200433, China 6. Fudan Institute of Health Sciences, Taizhou 225300, Jiangsu, China 7. Department of Thoracic Surgery, Zhongshan Hospital, Fudan University, Shanghai 200032, China
Objective: To explore the association between C reactive protein (CRP) rs2808630 and non-small cell lung cancer (NSCLC). Methods: CRP rs2808630 Genotyping of 974 NSCLC patients and 1 005 healthy controls from Shanghai and Taizhou of Jiangsu Province based on double ligation and multiplex fluorescence PCR. The effect of rs2808630 on NSCLC susceptibility was analyzed. Results: Compared with TT genotype, rs2808630 TC genotype significantly increased NSCLC risk (P=0.034), but this association became insignificant after adjusting with age, gender, smoking status, and family history. However, in the subgroup of smokers, TC genotype was significantly associated with increased NSCLC risk after adjustment (OR=1.329, 95% CI 1.017-1.736,P=0.037). In addition, TC genotype exhibited a significantly elevated risk of squamous cell carcinoma (OR=1.383, 95% CI 1.023-1.870,P=0.035). Further stratified analysis revealed that TC genotype posed a much higher risk of squamous cell carcinoma among smokers (OR=1.479, 95% CI 1.058-2.067,P=0.022). Conclusions: The interaction between rs2808630 T>C and smoking behavior might play an important role in leading to NSCLC especially squamous cell carcinoma.
C-reactive protein; rs2808630; case-control study; smoking; non-small cell lung cancer
2015-12-31[接受日期]2016-03-28
国家自然科学基金(81372236),国家科技支撑计划重点项目(2011BAI09B00),上海博士后科学基金(12R21411500),上海市科学技术委员会科研计划项目(12nm0500900),上海国际合作基金项目(14430712002),复旦大学高端计算中心支持项目. Supported by National Natural Science Foundation of China (81372236), Key Projects in the National Science & Technology Pillar Program (2011BAI09B00), Shanghai Postdoctoral Science Foundation (12R21411500), Shanghai Science and Technology Research Program (12nm0500900), Shanghai Foundation for International Cooperation (14430712002) and Fudan University High-End Computing Center Fund.
李玉涛,硕士. E-mail:18721714989@163.com
Corresponding authors). Tel: 021-64041990, E-mail: qian.cheng@zs-hospital.sh.cn; Tel: 021-31162477, E-mail: wjjxcc@126.com
10.12025/j.issn.1008-6358.2016.20151059
R 734.2
A