细胞凋亡相关基因单核苷酸多态与铂类治疗晚期非小细胞肺癌

【作者】 顾少华；

【导师】 金力；

【作者基本信息】 复旦大学 ， 遗传学， 2010， 博士

【摘要】 本研究从8个细胞凋亡相关基因(BCL2、BAX、CASP3、CASP8、CASP9、 CASP10、TNF-α和MIF)中,选定10个推断的功能SNP位点,通过这些SNP位点与连续收集的445例晚期非小细胞肺癌患者铂类联合化疗疗效、血液毒性和胃肠道毒性的关联分析,以期发现能预测疗效或毒性的分子标记。关联研究表明,CASP3rs6948变异等位基因C显著降低血液毒性风险：AC相对于AA,OR0.588,95%CI0.369-0.940; CC相对于AA, OR0.174,95%CI0.038-0.784; p for trend0.010,呈现基因剂量效应。TNF-αrs1800629变异等位基因A显著增加胃肠道毒性风险：GA相对于GG, OR3.354,95%CI1.302-8.640,p值0.012。携带BAX rs4645878变异等位基因A的52例个体(有效病例389)不发生高度血小板毒性。CASP8rs3834129、CASP10rs13006529和CASP10rs3900115三个位点杂合子相对于野生纯合子显著降低白细胞毒性,OR(95%CI)分别为0.557(0.334-0.930)、0.587(0.325-0.980)和0.575(0.344-0.959),p值分别为0.025、0.041和0.034。在顺铂-长春瑞滨治疗组中,BCL2rs2279115变异等位基因A在显性模型中显著降低疗效,OR0.454,95%CI0.207-0.998, p值0.049。在BCL2rs1564483与疗效的年龄分层关联分析中,出现了截然不同的结果：在≤57岁年龄组中,BCL2rs1564483变异纯合子加杂合子相对于野生纯合子显著降低疗效,OR为0.327,95%CI为0.116-0.924,p值为0.035；在>57岁年龄组中,则相反,该位点变异纯合子加杂合子相对于野生纯合子显著提高疗效,OR为3.088,95%CI为1.528-6.239,p值为0.002。上述关联分析结果还需进一步扩大样本量和利用其它独立样本加以验证。为证实CASP3rs6948与血液毒性的关联,我们另外招募52例经铂类治疗的肺癌患者,对其外周血白细胞中的1mRNA量进行荧光实时定量PCR分析。携带rs6948变异等位基因C的个体,其CASP3mRNA相对表达量的平均值相对于野生纯合子AA个体,下降约27.5%,与变异等位基因C降低CASP3mRNA表达量、从而降低血液毒性风险的预期相一致。我们在52例样本中取20例对CASP33’UTR进行测序,发现除了rs6948多态外,仅存在rs1049216多态。根据PHASE2.0.2估算,两位点紧密连锁,存在两种单倍型：A/C(A为rs6948等位基因,C为rs1049216等位基因)和C/T(C为rs6948等位基因,T为rs1049216等位基因)。据此,我们构建了包含CASP3整个3’UTR的pGL3-PromoterA/C和pGL3-PromoterC/T重组子。双荧光分析表明,变异等位基因重组子pGL3-PromoterC/T相对于野生等位基因重组子pGL3-PromoterA/C,萤火虫荧光酶相对表达量显著降低。经TargetScan预测,rs6948可能位于miR-24的靶序列中,rs1049216可能位于miR-664的靶序列中。进一步研究首先应证实引起翻译抑制的原因是rs6948多态还是rs1049216多态或者两者皆是；其次应证实rs6948是否位于miR-24的靶序列中或者rs1049216是否位于miR-664的靶序列中。更多还原

【Abstract】 The development of predictive markers to identify patients who will derive significant benefit with minimal toxicity from chemotherapy is a continuing focus in lung cancer research. In this study,10putative functional single nucleotide polymorphisms in8apoptosis-related genes (BCL2, BAX, CASP3, CASP8, CASP9, CASP10, TNF-a and MIF) were selected. We then investigated whether these polymorphisms were associated with severe hematological toxicity, gastrointestinal toxicity or objective response rate among445platinum-based chemotherapy treated advanced NSCLC patients.The incidence of severe hematological toxicity was significantly lower in variant C allele carriers of CASP3rs6948polymorphism (adjusted OR of0.588and95%CI of0.369-0.940for carriers of one C allele, adjusted OR of0.174and95%CI of0.038-0.784for carriers of two C alleles), compared with their wild-type homozygotes AA and the significance levels of the increasing trend were apparent for an indication of a dose-dependent relationship between the polymorphism and severe hematological toxicity risk (P for trend=0.010). The GA genotype of TNF-α rs1800629was significantly associated with increased gastrointestinal toxicity risk compared with the GG genotype (adjusted OR=3.354;95%CI=1.302-8.640,P=0.012). No severe throbocytopenia observed in52variant A allele carriers of BAX rs4645878polymorphism among389samples suggested that this SNP was associated with lower risk for thrombocytopenia. The heterozygotes of CASP8rs3834129(adjusted OR=0.557;95%CI=0.334-0.930, P=0.025), CASP10rs13006529(adjusted OR=0.587;95%CI=0.325-0.980, P=0.041) and CASP10rs3900115(adjusted OR=0.575;95%CI=0.344-0.959, P=0.034) were significantly associated with lower risk for leukocytopenia compared with their wild type homozygotes, respectively. The three polymorphisms are in high linkage disequilibrium in Chinese population. In the subgroup analysis for patients receiving Navelbine in combination with cisplatin treatment, poor response was significantly associated with the minor A allele of BCL2rs2279115in a dominant genetic model (adjusted OR=0.454;95%CI=0.207-0.998, P=0.049). BCL2rs1564483polymorphism was related to response in both elder patients (>57years old) C allele carriers of CASP3rs6948and younger (≤57years old) patients. However, the effects on response were distinct in age-stratified patients. The variant C allele increased the possibility to have objective response in elder patients (adjusted OR=3.088;95%CI=1.528-6.239, P=0.002), but decreased the possibility in younger patients (adjusted OR=0.327;95%CI=0.116-0.924, P=0.035) in a dominant genetic model.Further studies with larger group of study subjects or other samples are warranted to validate the associations as indicated above.To validate the association between rs6948and hematological toxicity, we recruited other52patients with newly diagnosed lung cancer treated with platinum combination chemotherapy and examined the relative CASP3mRNA levels in the peripheral blood leukocytes of these patients by real-time PCR, whose CASP3rs6948SNP genotypes were also determined. We found that the CASP3rs6948C variant in the3’UTR was associated with a relatively lower mRNA level. The average mRNA level of CASP3C variant genotypes was about72.5%compared to that of wild genotype AA. The result is consistent with the assumption that the minor C allele suppresses CASP3expression and lowers the risk for hematological toxicity.Besides rs6948we detected another SNP, rs1049216in the CASP33’UTR from20samples. Using PHASE2.0.2program we found only two haplotypes, A/C (A for rs6948, C for rs1049216) and C/T (C for rs6948, T for rs1049216) in this region, which suggested that the two SNPs were highly linked. By cloning the CASP33’UTR into pGL3-Promoter vector, we constructed two recombinants, pGL3-PromoterA/C and pGL3-PromoterC/T. Luciferase assays revealed that reporter expression levels of pGL3-PromoterA/C were significantly lower than those of pGL3-PromoterC/T in ECV304, HEK293, H1299and HeLa cell line, separately.TargetScan predicts that rs6948is located in miR-24seed region and rs1049216is located in miR-664seed region. Further studies are needed to verify whether rs6948or rs1049216causes the translation suppression. If does, whether miR-24or miR-664contributes to the translation suppression.更多还原