论文题目 CASP8基因启动子区六碱基插入缺失多态与多种肿瘤

1、论文题目论文题目：CASP8 基因启动子区六碱基插入/缺失多态与多种肿瘤易感性相关作者简介作者简介：孙瞳，女，1978 年 7 月出生，2002 年 9 月师从于北京协和医学院林东昕教授，于 2007 年 7 月获博士学位。中中 文文 摘摘 要要背景背景：人类免疫系统的重要职能之一是消灭机体内潜在的恶性细胞，但免疫系统细胞始终处于激活增殖与凋亡的动态平衡中。这种激活-凋亡平衡一旦失常，就可能影响对恶性细胞的免疫监视，导致肿瘤发生。免疫状态与肿瘤的关系一直是肿瘤研究领域的焦点之一。人们早就注意到在免疫缺陷病患者和使用免疫抑制剂的人群中，恶性肿瘤的发生率异常增高。随着认识的深入，对免疫与肿瘤关系的

2、观念也进一步更新。目前认为，体内随时可能发生细胞恶变，但只有当恶变细胞逃逸免疫系统监视，肿瘤才能发生，而肿瘤进一步发展也必须成功逃脱免疫监视。在肿瘤免疫中，T 淋巴细胞发挥关键作用。但是，T 淋巴细胞被肿瘤相关抗原或特异抗原激活后，会自行凋亡或自相残杀称激活诱导的细胞死亡(activation-induced cell death, AICD)。AICD 是肿瘤浸润淋巴细胞凋亡的主要机制，也是恶性转化细胞逃逸抗肿瘤免疫反应，得以发生发展的关键途径。T 淋巴细胞 AICD 过程由死亡通路(death pathway)介导，死亡配体(FasL)和死亡受体(Fas)结合产生“死亡信号” ，激活下游的

3、半胱氨酸蛋白酶家族(caspases)的级联反应引起细胞凋亡。死亡受体通路及其调节基因存在遗传变异，这些遗传变异可能影响个体的免疫状态从而影响对肿瘤的易感性。我们曾对 FASL/FAS 基因启动子区单核苷酸多态与肿瘤发病风险以及在 T 淋巴细胞 AICD 过程中的功能进行了研究，发现 FASL 和 FAS 遗传变异与食管癌(Sun et al, J Natl Cancer Inst 2004)、肺癌(Zhang et al, J Med Genet 2005)、子宫颈癌(Sun et al, J Exp Med 2005)、乳腺癌(Zhang et al, Carcinogenesis 200

4、7)等易感性相关，FASL启动子区遗传变异显著影响 T 淋巴细胞被肿瘤抗原激活后的凋亡率(Sun et al, J Exp Med 2005)。然而，FasL/Fas 信号通路下游一些重要效应分子如 caspase-8、caspase-10 等编码基因的遗传变异是否与肿瘤易感性也有关系尚不清楚。研究表明，caspases 对免疫细胞的生死存亡起决定性作用，其表达或激活异常与包括肿瘤在内的多种疾病有关。caspase-8 由 CASP8 基因编码，它是死亡配体-受体相互作用介导的细胞凋亡过程的起始型 caspase。来自死亡配体与受体结合的凋亡信号，首先激活caspase-8，后者将凋亡信号传递

5、给 caspase-10(由 CASP10 基因编码)引起 caspase 级联反应导致细胞凋亡。在该过程中，CFLAR(由 CFLAR 基因编码)对 caspase-8 起负调节作用。这三个凋亡相关分子在凋亡信号传导过程共同作用，是细胞凋亡事件中的关键分子。鉴于死亡通路在 T 淋巴细胞凋亡过程中的关键作用，我们设想该通路关键基因遗传变异可能影响肿瘤遗传易感性。为验证这个假设，我们研究了位于染色体 2q33 区域的 CASP8-CASP10-CFLAR 基因簇遗传变异与多种肿瘤易感性的关系。方法方法：( (1) )遗传变异与肿瘤易感性研究遗传变异与肿瘤易感性研究：本研究采用候选基因关联研究策略

6、，研究分两个阶段，第一阶段以小样本量筛选，第二阶段以大样本量验证。我们利用国际 HapMap 计划公布的北京地区汉族人群 CASP8-CASP10-CFLAR 基因簇区域 SNP数据，用 Haploview3.2 软件构建出单体型域并据此挑选出 19 个单体型标签 SNP 进行基因分型。我们先在较小样本量(730 个病人和 700 个对照)的肺癌病例-对照中进行关联研究，以检查该区域是否存在与肿瘤易感性相关的遗传变异位点。获得阳性结果后，又设计了独立的大样本量的多种肿瘤病例-对照队列，对所发现的肿瘤易感性位点进行第二阶段的验证，研究队列包括 1150 个肺癌病人和 1120 个对照、1050个

7、食管癌病人和 960 个对照、420 个胃癌病人和 410 个对照、930 例个结直肠癌病人和 892 个对照、1125 个乳腺癌病人和 1005 个对照、320 个子宫颈癌病人和 585 个对照。( (2) )遗传变异的功能和作用机制研究遗传变异的功能和作用机制研究：我们对与肿瘤易感性相关的遗传变异进行了从 DNA、RNA、蛋白质、细胞到人群的生物学功能和作用机制的系统研究。采用的主要研究方法为 DNA 测序、双荧光素酶报告基因实验、电泳迁移率变动实验、定量 PCR 检测 CASP8 RNA、染色质免疫共沉淀实验、caspase-8 酶活性检测、淋巴细胞肿瘤抗原激活实验、流式细胞术检测 T

8、淋巴细胞凋亡等。( (3) )统计学分析统计学分析：以非条件性 logistic 回归模型计算的比值比(odds ratio, OR)及其 95%可信区间(confidence interval, CI)表示基因型与各种肿瘤发生的相对风险度。所有的 OR 值均经混杂因素如年龄、性别和吸烟状况校正。多重比较的显著性检验用排列变换检验(permutation test)，经 104次检验后 P 值仍然 0.01 才被认为有统计学显著性。结果：结果：( (1) )CASP8-CASP10-CFLAR 基因簇区域中存在肿瘤易感位点：基因簇区域中存在肿瘤易感位点：我们在730 个肺癌病人和 700 个对

9、照的筛查中发现，位于 CASP10 基因 3端和 CASP8 基因5端之间的 SNP 的基因型频率在病人和对照中的分布有显著差异，说明可能存在易感位点。为了明确易感位点的位置，对 CASP8 基因启动子区及其相邻区域进行重新测序。结果发现 CASP8 基因启动子区在652 核苷酸起始处有一 6 个碱基插入/缺失(652 6N ins/del)变异，该变异与肺癌风险降低相关性最强，提示可能是易感位点。( (2) )CASP8 652 6N ins/del 变异的生物学功能：变异的生物学功能：我们对 CASP8 652 6N ins/del 变异进行了一系列生物化学和功能研究。荧光素酶报告基因实验

10、结果显示，6N del 等位基因驱动的报告基因表达水平显著低于 6N ins 等位基因。电泳迁移率变动实验证明，该 6 个核苷酸缺失破坏了转录因子激活蛋白 1(Sp1)的结合位点，使 Sp1 转录因子不能与 CASP8 652 6N del 等位基因启动子结合。染色质免疫共沉淀实验结果显示，CASP8 652 6N ins/ins 基因型的染色质经 Sp1 抗体沉淀后可扩增出含652 6N ins 片段的 DNA，但 CASP8 652 6N del/del 基因型的染色质则无相应产物，表明在活细胞内确实存在 Sp1 与 6N ins 等位基因的结合。我们进一步检测不同基因型个体淋巴细胞经或未

11、经植物凝集素(PHA)激活后 CASP8 RNA 表达量，结果显示在未经 PHA 处理的淋巴细胞中，652 6N ins/ins 基因型表达的 CASP8 RNA 中位拷贝数为1033.01(675.631868.70; n=11)，而652 6N ins/del 和 del/del 基因型表达的拷贝数则分别为 493.43 (198.821058.99; n=12)和 109.33(38.43293.93; n=4)，显著低于ins/ins 基因型（P=0.008 和 0.001） 。但652 6N ins/del 基因型与652 6N del/del 基因型之间的表达量无显著差异（P=0.

12、131） 。经 PHA 处理的淋巴细胞中，652 6N del/del 基因型的 CASP8 RNA 拷贝数为 183.62（128.43382.78; P=0.001） ，652 6N ins/del 基因型的拷贝数为 1445.60（324.563719.79; P=0.002)，两者均显著低于652 6N ins/ins 基因型的 3868.70（1659.005967.06） 。酶活性检测结果显示，经 Fas 抗体或 PHA 激活后，携带 CASP8 652 6N del/del 基因型 T 淋巴细胞的 caspase-8 活性增加倍数显著低于携带 CASP8 652 6N ins/i

13、nsl 基因型的 T 淋巴细胞1.64 倍(1.363.85; n=8)比 3.27 倍(2.049.99; n=30); P=0.01，PHA 激活后的差异更大2.62 倍(1.555.79; n=8)比 13.54 倍(6.2533.14; n=30); P = 0.0008。我们进一步用流式细胞术检测了携带不同 CASP8 基因型的 T 淋巴细胞在 PHA、Fas 抗体或肿瘤细胞抗原激活后的凋亡率。结果表明，经 Fas 抗体激活后，携带 652 6N del/del 基因型的 T 淋巴细胞凋亡率显著低于携带652 6N ins/ins 基因型的淋巴细胞3.97%(2.986.95, n=

14、8)比 7.35%(1.5722.45, n=30); P=0.029; PHA 激活诱导的 T 淋巴细胞凋亡率也呈相似趋势17.81%(7.1723.10, n=8)比 51.10%(24.3472.78，n = 30); P=0.0003。T淋巴细胞接触肿瘤细胞 A549 或 KYSE150 抗原后也发生凋亡，不同基因型凋亡率有显著差异。经 A549 抗原激活后，携带 6N del/del、ins/del 和 ins/ins 基因型 T 淋巴细胞的凋亡率分别为 3.99% (2.395.60; n=2)、7.81%(2.3911.29; n=11)和11.57%(4.7420.61; n=

15、24)，差异有极显著性(P=0.002)。经 KYSE150 细胞抗原激活后，上述三种基因型 T 淋巴细胞的凋亡率分别为 7.15%(5.488.81; n=2)、9.14%(5.1116.82; n=11)和 16.52%(7.6627.81; n=24)，差异有极显著性(P=0.001)。这些差异主要是由于 6N ins/ins 与 6N ins/del 和 6Ndel/del 基因型之间的差异造成的，而 6N ins/del 和 6N del/del 基因型之间的差异无统计学显著性。这些结果清楚地表明，CASP8 652 6N ins/del 遗传变异对激活诱导的 T 淋巴细胞凋亡有显著

16、的影响。( (3) ) CASP8 652 6N ins/del 与多种肿瘤易感性相关：与多种肿瘤易感性相关：鉴于第一阶段关联研究和功能实验的阳性结果，我们进一步进行了第二阶段大样本量的验证。我们总共分析了包括肺癌、食管癌、胃癌、结直肠癌、子宫颈癌和乳腺癌在内的 4,995 个肿瘤病人和 4,972个对照。结果表明，与携带652 6N ins/ins 基因型比较，携带652 6N ins/del 基因型个体不同肿瘤的患病风险均降低，OR 值分别为 0.650.83，而携带652 6N del/del基因型的个体罹患各种肿瘤的风险更低，OR 值分别为 0.490.60，呈现显著的等位基因剂量效应

17、关系，趋势检验除胃癌以外的各种肿瘤均 P0.8, and htSNPs were selected with Haploview 3.2 software on a block-by-block basis using the method described by Carlson et al. with the sample size inflation factor Rh2 0.8. We performed two-stage association analysis; stage I was carried out with 730 lung cancer patients and 7

18、00 cancer-free controls and stage II was carried out with 1150 lung cancer patients, 1050 esophageal squamous cell carcinoma patients, 420 gastric cancer patients, 930 colorectal cancer patients, 1125 breast cancer patients, 320 cervical cancer patients, and 4972 frequency-matched controls. SNPs and

19、 other variants in the CASP8 promoter region were identified by direct sequencing of PCR product of genomic DNA from 40 individuals. SNPs in stage I association analysis were genotyped by the MassARRAY system. (2) Functional and genotype-phenotype analysis: Functional significances of variants in th

20、e CASP8 promoter region were analyzed by a set of biochemical assays including dual-luciferase reporter assays, electrophoretic mobility-shift and -supershift assays (EMSA), chromatin immunoprecipitation (ChIP) assays. CASP8 RNA levels were determined by real-time quantitative PCR, caspase-8 activit

21、y was measured using Caspase-Glo 8 Assay kit. T cell apoptosis was analyzed by flow cytometry using Annexin V-FITC Apoptosis Detection Kit. (3) Statistical analysis: Unconditional logistic regression was used to analyze the association between a single locus and cancer risk, adjusted for sex, age, a

22、nd smoking status, where it was appropriate. Kruskal-Wallis one-way analysis of variance tests were performed to analyze the results of CASPASE-8 expression, activity, and apoptosis rate of PBMCs treated with anti-FAS antibody. Non-parametric covariance analysis, using CD25+/CD4+ and CD69+/CD4+ cell

23、s as covariants, was performed to analyze the results in PBMCs treated with PHA and cancer cells.Results: (1) Identification of candidate cancer susceptibility locus: In the pilot analysis of 730 lung cancer patients and 700 controls, a significant association with lung cancer was observed in the 3

24、region of CASP10 or 5part of CASP8, with the P values being lower for SNPs located within the 25-kb region containing CASP8 promoter and 5untranslation region. We then resequenced most possible causative regions, i.e., the CASP8 promoter and its adjacent area, and three variants, CASP8 856GC, CASP8

25、652 AGTAAG insdel, and CASP8 614GA were identified in 40 DNA samples. Among these 3 variation, the 652 6N ins/del variation was most strongly associated with risk of lung cancer, suggesting that this variation may be the susceptibility locus. (2) Functional significance of CASP8 652 6N ins/del varia

26、tion: To evaluate the CASP8 652 6N ins/del variation on CASP8 transcriptional activity, two luciferase reporter vectors (pGL3 basic) were generated, and they were used to transiently transfect HEK293, Jurkat E6-1, and HeLa cells. It was shown that the 6N del-containing promoter drove a significantly

27、 lower luciferase expression than the 6N ins-containing promoter. We then examined whether the CASP8 652 6N del allele has an effect on binding of transcriptional factor Sp1 as suggested by in silico analysis. EMSA and ChIP assays demonstrated that the 652 6N deletion destroys an Sp1 binding site. W

28、e next examined levels of CASP8 RNA in non-activated or PHA-activated lymphocytes of 27 individuals TaqMan real-time quantitative PCR analysis and the results showed that in non-activated lymphocytes, the median levels of CASP8 RNA (range) for the CASP8 652 6N del/del and ins/del genotypes were 109.

29、33 (38.43293.93; n=4) and 493.43 (198.821058.99; n=12) copies compared with 1033.01 (675.631868.70; n=11) copies for the 6526N ins/ins genotype (P=0.001 and 0.008, respectively). However, the levels between the 652 6N ins/del and 652 6N del/del genotypes were not significantly different (P=0.131). S

30、imilar results were obtained from PHA-stimulated lymphocytes, with the value being 183.62 (128.43382.78) and 1445.60 (324.563719.79) copies for the 6526N del/del and 652 6N ins/del genotype compared with 3868.70 (1659.005967.06) copies for the 652 6N ins/ins genotype (P=0.001 and 0.002, respectively

31、). We further examined the impact of genetic CASP8 variants on caspase-8 activity and AICD of T cells ex vivo in PBMCs from subjects with different CASP8 652 6N ins/del genotypes stimulated with anti-FAS, PHA, or cancer cells. It was found that when the apoptosis pathway was triggered by anti-FAS an

32、tibody or PHA, lymphocytes with the 652 6N del/del genotype had significantly less increased caspase-8 activity (median fold, range) compared with those with the 652 6N ins/ins genotype (1.64 fold 1.363.85; n=8 versus 3.27 fold 2.049.99; n=30; P=0.01 and 2.62 fold 1.555.79; n=8 versus 13.54 fold 6.2

33、533.14; n=30; P=0.0008). Flow cytometry analysis showed that the median (range) apoptosis rate of T lymphocytes was significantly lower for the 652 6N del/del genotype than that for the 652 6N ins/ins genotype when stimulated by anti-FAS antibody (3.97% 2.986.95; n=8 versus 7.35% 1.5722.45; n=30; P=

34、0.029) or PHA (17.81% 7.1723.10; n=8 versus 51.10% 24.3472.78; n=30; P=0.0003). Furthermore, the caspase-8 activity and T cell apoptosis rate for the 6N ins/del genotype (n=20) were also significantly lower than those for the 6N ins/ins genotype, whereas they were not significantly different between

35、 the 6N ins/del and 6N del/del genotypes. In assays that mimic tumor microenvironment where tumor-infiltrating lymphocytes encounter tumor cells, the apoptosis rate of T lymphocytes with the 652 6N del allele was significantly lower than that of T lymphocytes with the 652 6N ins allele, with the val

36、ues being 3.99% (2.39, 5.60; n=2), 7.81% (2.3911.29; n=11) and 11.57% (4.7420.61; n=24) for the 6N del/del, ins/del and ins/ins genotype, respectively (P=0.002) upon co-cultured with A549 lung adenocarcinoma cells. Similar results were also obtained from assays with KYSE-150 esophageal squamous cell

37、 carcinoma cells, with the apoptosis rate of T lymphocytes being 7.15% (5.48, 8.81; n=2), 9.14% (5.1116.82; n=11) and 16.52% (7.6627.81; n=24) for the 6N del/del, ins/del and ins/ins genotype, respectively (P=0.001). These results clearly demonstrate that the CASP8 652 6N ins/del variation may have significant impact on activation-induced cell death of tumor-related T lymphocytes. (3) Associations of CASP8 genotype with cancer susceptibility: In total, we genotyped 1150 lung cancer patients, 1050 esophageal squamous cell carcinoma