【作者】 李莹；

【导师】 程黎明；

【作者基本信息】 华中科技大学 ， 临床检验诊断学， 2019， 硕士

【摘要】 目的:筛选染色体5p15.33区域与中国人群肺癌发病风险相关的遗传变异,评估其与外周血白细胞端粒长度的关系,探索功能性遗传变异在端粒调节和肺癌发生中的生物学效应。方法:将400份非小细胞肺癌患者5p15.33区域的二代测序结果,与公共数据库“千人基因组计划”东亚人群的基因型数据比对,筛选出肺癌易感候选位点。对985份非小细胞肺癌病例和970份健康对照基因分型,评估774份健康人群的外周血白细胞端粒长度与肺癌易感位点的相关性。结合生物信息学预测,完成荧光素酶报告基因实验和电泳迁移率变动分析及超迁实验,进一步探索肺癌易感位点的生物学功能。结果:5p15.33区域二代测序共识别到1478个变异,基因型比对筛选出17个常见变异候选位点。病例-对照的关联分析得到4个显著性肺癌易感位点,分别是位于TERT基因的rs7726159(OR=1.34,95%CI:1.18-1.52,P=7.78×10^-6)、rs10054203(OR=1.29,95%CI:1.13-1.46,P=1.37×10^-4)、rs2853677（OR=1.23,95%CI:1.08-1.39,P=0.002）和TERT基因上游的rs2736107(OR=1.28,95%CI:1.11-1.47,P=5.14×10^-4)。在健康人群中,rs7726159和rs10054203与外周血白细胞相当端粒长度显著相关（加性模型下,P值分别为0.008和0.036）。在肺癌细胞系A549和SK-MES-1中,双荧光素酶报告基因实验显示,与插入rs7726159-C片段的质粒相比,含rs7726159-A等位基因的片段具有更高的荧光素酶活性(P_A549=2.17×10^-6,P_SK-MES-1=2.69×10^-4)。电泳迁移率变动分析及超迁实验表明,rs7726159野生型位点C与转录因子MYC特异性结合能力更强。结论:位于TERT基因3号内含子区的功能性遗传变异rs7726159 C>A可能干扰MYC转录因子结合以提高TERT基因转录,并影响端粒长度,增加肺癌的发病风险。更多还原

【Abstract】 Objective:We intended to identify polymorphisms on chromosome 5p15.33associated with lung cancer susceptibility in Chinese population,evaluate the relevance between genetic variations and leukocyte telomere length,and explore the functional effects of causal variants in telomere biology and tumorigenesis.Methods:Firstly,we compared the genotypic frequency in 400 non-small cell lung cancer（NSCLC）cases by targeted sequencing with the data in East Asian population from 1000 Genomes Project.Candidate variants were selected,and their associations with lung cancer were subsequently validated in 985 cases and 970 controls.We evaluated the relationships between risk variants and telomere length in 774 healthy subjects.Based on bioinformatic analyses,luciferase assays and electrophoretic mobility shift assays（EMSA）were conducted to explore potential functions and reveal carcinogenic mechanism.Results:We identified 1478 variants through targeted sequencing on chr5p15.33region and selected 17 candidates by genotypic comparison.Four polymorphisms exhibited prominent associations with lung cancer risk,including rs7726159(OR=1.34,95%CI:1.18-1.52,P=7.78×10^-6),rs10054203(OR=1.29,95%CI:1.13-1.46,P=1.37×10^-4),rs2736107(OR=1.28,95%CI:1.11-1.47,P=5.14×10^-4)and rs2853677（OR=1.23,95%CI:1.08-1.39,P=0.002）.The minor allele of rs7726159and rs10053203 were associated with long telomeres（under additive model,P=0.008 and 0.036,respectively）.In luciferase assays,the fragment containing rs7726159-A increased luciferase activity comparing with the allele C in both A549and SK-MES-1 cell lines(P_A549=2.17×10^-6;P_SK-MES-1=2.69×10^-4).EMSA and supershift assays demonstrated the specific affinity of MYC to the rs7726159 wild type allele C.Conclusion:The functional variant rs7726159,located in 3th intron of TERT,confers lung cancer susceptibility might by affecting MYC binding and inducing telomere lengthening.更多还原