FADD基因遗传多态与子宫颈癌易感性
摘要
背景和目的:子宫颈癌是发病率最高的女性肿瘤之一。高危型人乳头瘤病毒(humanpapillomavirus,HPV)通过产生的癌蛋白E6和E7结合并抑制P53及RB1等蛋白,干扰细胞凋亡和细胞周期调控机制的正常运行,从而导致子宫颈癌的发生。FADD是参与细胞凋亡和细胞周期调控的重要蛋白,其表达水平的改变与多种肿瘤相关。鉴定FADD基因及其调控序列中与子宫颈癌发病相关的遗传多态并初步探讨其生物学机制。
方法:对30个随机挑选的中国人的FADD基因编码区、5′UTR、3′UTR及5′侧翼序列进行重新测序,筛查遗传多态。用463例子宫颈癌患者和876例正常对照对遗传多态进行病例-对照研究。用PCR-RFLP方法进行基因分型。用非条件logistic回归模型计算各基因型的比值比(odds ratio,OR)及95%可信区间(confidence interval,CI)。用电泳迁移率变动实验(EMSA)和双荧光素酶报告基因实验分析遗传多态的生物学功能。
结果:检测到3个在中国人群中频率超过3%的遗传多态,包括2个单核苷酸多态和1个插入/缺失多态,分别位于5′侧翼区(-16C/T),5′非翻译区(255A/G)和3′非翻译区(3859 TGT/-,简称3N ins/del或3N I/D)。3个多态处于一定程度的连锁不平衡。病例-对照分析结果表明,-16CT,255AG和3859 3N ins/del基因型增加患子宫颈癌的风险,相应OR值(95%可信区间)分别是1.41(1.06-1.88),1.48(1.16-1.89)和1.34(1.05-1.72),而纯合子变异基因型并不增加子宫颈癌风险。分层分析显示,3个多态的杂合基因型主要增加子宫颈鳞癌的发病风险,且在35岁以上人群中尤为显著。-16CC基因型在中晚期子宫颈癌患者(Ⅱ期至Ⅳ期)中的频率显著高于早期患者(0期和Ⅰ期),而255A/G和3859 3N ins/del多态和子宫颈癌进展无关。以分布频率最高的-16CC/255GG/3859II基因型为参照,-16TT/255AG/3859ID基因型(OR=2.52,95%CI=1.49-4.27)和-16CT/255AG/3859ID基因型(OR=1.71,95%CI=1.23-2.38)增加患病风险。双荧光素酶报告基因实验结果表明,从-304位至297位核苷酸的启动子转录活性最强,-780位至-304位有负调控作用,-1070位至-780位有正调控作用,5′非翻译区有一定的转录活性。C_(-16)G_(255),T_(-16)G_(255)和C_(-16)A_(255)单体型的转录活性均显著高于T_(-16)A_(255)单体型,提示-16C和255G的转录活性分别大于其相应等位基因,且2个位点之间可能存在交互作用。报告基因实验还显示,3′非翻译区3859 3N del基因型的报告基因表达水平显著低于3859 3N ins基因型。电泳迁移率变动实验显示,-16C和-16T等位基因均与核蛋白结合,但前者结合能力比后者强,该核蛋白可能是转录刺激蛋白SP1;255G和255A均可与某一转录因子结合,但结合强度无明显区别。
结论:FADD-16C/T,255A/G和3859 3N ins/del多态增加患子宫颈癌尤其是子宫颈鳞癌的风险,其机制可能与这些多态影响FADD的转录或翻译有关。
Background and Objective:Cervical cancer is the second most common malignancy in women worldwide.Human papillomavirus are the major cause of cervical cancer and one of the underlying mechanisms is the interaction of their oncoproteins E6 and E7 with cellular proteins P53 and RB1,which interferes with normal cellular apoptosis and cell cycle.FAS-associated via death domain(FADD) is an important player both in apoptosis pathways and in cell cycle regulation,and implicated in the development of various human cancers.The present study examined the association between polymorphisms in FADD and susceptibility to cervical cancer and its underlying mechanism.
Methods:Coding regions,5'UTR,3'UTR and 5' flanking region of FADD were resequenced in 30 Chinese adults to detect polymorphisms.The associations between the identified polymorphisms in FADD and risk of cervical cancer were examined in a case-control analysis consisted of 463 patients with cervical cancer and 876 controls. PCR-RFLP was used to identify genotypes and unconditional logistic regression model was used to compute the odds ratio(OR) and its corresponding 95%confidence interval (CI).Electrophoretic mobility shift assays and dual-luciferase reporter gene assays were used to examine the functions of the polymorphisms.
Results:Three genetic variations,i.e.-16C/T,255A/G and 3859TGT/-(3N ins/del,3N I/D) were detected,which are in some extent of linkage disequilibrium.We showed that heterozygotes of these polymorphisms were all associated with significantly increased risk for cervical cancer,with the ORs(95%CI) being 1.41(1.06-1.88),1.48(1.16-1.89) and 1.34(1.05-1.72),for-16CT,255AG and 3859 3N ins/del genotypes,respectively, while homozygotes of each variant seemed not to be associated with risk of the cancer. Stratification analyses revealed that the risk seemed to restrain in squamous cell carcinoma developed in women more than 35-year old.The frequency of the -16CC genotype was significantly higher in advanced cervical cancer(stageⅡto stageⅣ) than in early stage cancer(stage 0 andⅠ),while 255A/G and 3859 3N ins/del polymorphisms appeared not to be associated with cancer progression.Subjects carrying the -16TT/255AG/3859ID(OR=2.52,95%CI=1.49-4.27) or-16CT/255AG/3859ID(OR =1.71,95%CI=1.23-2.38) genotype had significantly increased risk compared with the -16CC/255GG/3859Ⅱgenotype.Reporter gene assay showed that the region between -304 bp and 297 bp had the highest promoter activity,while the regions between -1070 bp and -780 bp and -780 bp and -304 bp displayed activities of positive and negative regulation,respectively.The 5' untranslated region also exhibited transcriptional activity. For the 3859 3N ins/del polymorphism in the 3' untranslated region,the deletion allele had decreased reporter gene expression compared with the insertion allele.Haplotypes C_(-16)G_(255),T_(-16)G_(255) and C_(-16)A_(255) had higher promoter activity than haplotype T_(-16)A_(255), suggesting that both -16C and 255G alleles have higher transcription activity than their counterparts,but it seemed that epistasis existed between these two polymorphisms. Electrophoretic mobility shift assay showed that the -16C allele binds to potential Sp1 transcription factor more robustly than the -16T allele does.However,the 255G and 255A alleles did not show any difference in binding to nuclear proteins.
Conclusion:FADD polymorphisms are associated with increased risk for developing cervical cancer,especially cervical squamous cell carcinoma,which might be caused by their impact on transcription and/or translation regulation of the gene.
方法:对30个随机挑选的中国人的FADD基因编码区、5′UTR、3′UTR及5′侧翼序列进行重新测序,筛查遗传多态。用463例子宫颈癌患者和876例正常对照对遗传多态进行病例-对照研究。用PCR-RFLP方法进行基因分型。用非条件logistic回归模型计算各基因型的比值比(odds ratio,OR)及95%可信区间(confidence interval,CI)。用电泳迁移率变动实验(EMSA)和双荧光素酶报告基因实验分析遗传多态的生物学功能。
结果:检测到3个在中国人群中频率超过3%的遗传多态,包括2个单核苷酸多态和1个插入/缺失多态,分别位于5′侧翼区(-16C/T),5′非翻译区(255A/G)和3′非翻译区(3859 TGT/-,简称3N ins/del或3N I/D)。3个多态处于一定程度的连锁不平衡。病例-对照分析结果表明,-16CT,255AG和3859 3N ins/del基因型增加患子宫颈癌的风险,相应OR值(95%可信区间)分别是1.41(1.06-1.88),1.48(1.16-1.89)和1.34(1.05-1.72),而纯合子变异基因型并不增加子宫颈癌风险。分层分析显示,3个多态的杂合基因型主要增加子宫颈鳞癌的发病风险,且在35岁以上人群中尤为显著。-16CC基因型在中晚期子宫颈癌患者(Ⅱ期至Ⅳ期)中的频率显著高于早期患者(0期和Ⅰ期),而255A/G和3859 3N ins/del多态和子宫颈癌进展无关。以分布频率最高的-16CC/255GG/3859II基因型为参照,-16TT/255AG/3859ID基因型(OR=2.52,95%CI=1.49-4.27)和-16CT/255AG/3859ID基因型(OR=1.71,95%CI=1.23-2.38)增加患病风险。双荧光素酶报告基因实验结果表明,从-304位至297位核苷酸的启动子转录活性最强,-780位至-304位有负调控作用,-1070位至-780位有正调控作用,5′非翻译区有一定的转录活性。C_(-16)G_(255),T_(-16)G_(255)和C_(-16)A_(255)单体型的转录活性均显著高于T_(-16)A_(255)单体型,提示-16C和255G的转录活性分别大于其相应等位基因,且2个位点之间可能存在交互作用。报告基因实验还显示,3′非翻译区3859 3N del基因型的报告基因表达水平显著低于3859 3N ins基因型。电泳迁移率变动实验显示,-16C和-16T等位基因均与核蛋白结合,但前者结合能力比后者强,该核蛋白可能是转录刺激蛋白SP1;255G和255A均可与某一转录因子结合,但结合强度无明显区别。
结论:FADD-16C/T,255A/G和3859 3N ins/del多态增加患子宫颈癌尤其是子宫颈鳞癌的风险,其机制可能与这些多态影响FADD的转录或翻译有关。
Background and Objective:Cervical cancer is the second most common malignancy in women worldwide.Human papillomavirus are the major cause of cervical cancer and one of the underlying mechanisms is the interaction of their oncoproteins E6 and E7 with cellular proteins P53 and RB1,which interferes with normal cellular apoptosis and cell cycle.FAS-associated via death domain(FADD) is an important player both in apoptosis pathways and in cell cycle regulation,and implicated in the development of various human cancers.The present study examined the association between polymorphisms in FADD and susceptibility to cervical cancer and its underlying mechanism.
Methods:Coding regions,5'UTR,3'UTR and 5' flanking region of FADD were resequenced in 30 Chinese adults to detect polymorphisms.The associations between the identified polymorphisms in FADD and risk of cervical cancer were examined in a case-control analysis consisted of 463 patients with cervical cancer and 876 controls. PCR-RFLP was used to identify genotypes and unconditional logistic regression model was used to compute the odds ratio(OR) and its corresponding 95%confidence interval (CI).Electrophoretic mobility shift assays and dual-luciferase reporter gene assays were used to examine the functions of the polymorphisms.
Results:Three genetic variations,i.e.-16C/T,255A/G and 3859TGT/-(3N ins/del,3N I/D) were detected,which are in some extent of linkage disequilibrium.We showed that heterozygotes of these polymorphisms were all associated with significantly increased risk for cervical cancer,with the ORs(95%CI) being 1.41(1.06-1.88),1.48(1.16-1.89) and 1.34(1.05-1.72),for-16CT,255AG and 3859 3N ins/del genotypes,respectively, while homozygotes of each variant seemed not to be associated with risk of the cancer. Stratification analyses revealed that the risk seemed to restrain in squamous cell carcinoma developed in women more than 35-year old.The frequency of the -16CC genotype was significantly higher in advanced cervical cancer(stageⅡto stageⅣ) than in early stage cancer(stage 0 andⅠ),while 255A/G and 3859 3N ins/del polymorphisms appeared not to be associated with cancer progression.Subjects carrying the -16TT/255AG/3859ID(OR=2.52,95%CI=1.49-4.27) or-16CT/255AG/3859ID(OR =1.71,95%CI=1.23-2.38) genotype had significantly increased risk compared with the -16CC/255GG/3859Ⅱgenotype.Reporter gene assay showed that the region between -304 bp and 297 bp had the highest promoter activity,while the regions between -1070 bp and -780 bp and -780 bp and -304 bp displayed activities of positive and negative regulation,respectively.The 5' untranslated region also exhibited transcriptional activity. For the 3859 3N ins/del polymorphism in the 3' untranslated region,the deletion allele had decreased reporter gene expression compared with the insertion allele.Haplotypes C_(-16)G_(255),T_(-16)G_(255) and C_(-16)A_(255) had higher promoter activity than haplotype T_(-16)A_(255), suggesting that both -16C and 255G alleles have higher transcription activity than their counterparts,but it seemed that epistasis existed between these two polymorphisms. Electrophoretic mobility shift assay showed that the -16C allele binds to potential Sp1 transcription factor more robustly than the -16T allele does.However,the 255G and 255A alleles did not show any difference in binding to nuclear proteins.
Conclusion:FADD polymorphisms are associated with increased risk for developing cervical cancer,especially cervical squamous cell carcinoma,which might be caused by their impact on transcription and/or translation regulation of the gene.
引文
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