高氡暴露地区人群中hOGG1基因启动子区多态性单体型功能研究

Radon and its daughters could induce DNA damage injury and closely correlate with development of lung cancer.8-oxoguanine DNA glycosylase plays an important role in the cellular DNA oxidative damage repair process induced by radon and its daughters. The difference of capacity of repairing DNA damage among individuals is in the polymorphisms of DNA damage repair genes. To clarify the role of polymorphism haplotypes in the regulatory region of hOGG1 gene in carcinogenesis induced by radon and its daughters, we will search and identify the polymorphisms in the 5'-flanking region of hOGG1 and construct its haplotypes. We are to perform EMSA, reporter gene assay, ChIP and transcriptional factor expression plasmids co-transfection in HBE cells to obtain the direct evidence for effects of polymorphisms on the transcription of hOGG1 gene that regulated by transcriptional factor. To confirm the effects of hOGG1 expression with variant polymorphism haplotypes on cellular function activity after radon exposure, we will establish cell modes with different hOGG1 regulatory expression and silenced expression. This study aims to clarify the molecular mechanism for cellular outcomes ragulated by polymorphisms haplotypes via the interaction with transcriptional factor and further to provide the foundation for biomarker of lung cancer susceptibility in high radon-exposured area and protections for radon hazards.

氡及其子体可以引起DNA氧化损伤，与肺癌的发生密切相关。8-羟基鸟嘌呤DNA糖苷酶(hOGG1)在DNA氧化损伤修复过程中起着重要的作用。而个体间DNA氧化损伤修复能力的差异主要取决于DNA损伤修复基因的多态性。为了明确hOGG1基因调控区多态性单体型在氡及其子体致癌过程中的作用，本课题拟筛查并确认高氡暴露人群中hOGG1基因5'侧翼区的多态性,并构建该人群单体型；利用永生化人支气管上皮细胞株，通过EMSA、报告基因检测、体内ChIP分析、转录因子表达质粒共转染等实验，获得hOGG1启动子区多态性影响转录因子调控基因转录的直接证据；构建hOGG1调控表达和缺陷表达细胞模型证实不同多态性单体型对氡暴露细胞功能活性影响。由此，本研究将揭示hOGG1基因启动子区多态性单体型经由与转录因子相互作用影响细胞转归的分子机制，为确立hOGG1基因作为高氡暴露地区人群肺癌易感性标志和氡危害防护提供依据。

本研究选择广东高本底地区汉族人群60例，获取其基因组DNA，PCR扩增获得hOGG1基因5′侧翼区目的片段产物后纯化测序，发现该人群中8个SNP位点，其中3个已知位点－1493 G>A、－834 G>C 和－18 G>T和5个新位点－1455 G>A、－1293 A>T、－1187 C>A、－337 C>A和－323G>A。其中－1493 G>A的等位基因频率在该人群的分布与HapMap数据库中HapMap-CEU人种/人群的分布差异有统计学意义(P<0.05)。该目标人群hOGG1基因目的片段存在4种主要单体型，合计频率为96.7%；其中最主要的野生单体型H1频率为87.4%，其余4种为变异单体型(H2~H4，频率为9.3%)。同时本研究通过PCR扩增和成功测序60例目标人群MGMT基因5’侧翼区目的片段，序列比对发现该人群中6个已知多态性位点：－797G>A、-245T>G、－177T>G、－19C>T、+73C>T和+336G>A。其中+336G>A的等位基因频率在该人群的分布与HapMap-JPT和HapMap-CEU人种/人群分布差异具有统计学意义(P < 0.05)。采用HaploView软件分析表明各遗传多态性位点在该人群的杂合度不同，连锁不平衡分析结果表明－797G>A与－19C>T、－797G>A与+73C>T均呈较强的连锁不平衡关系，-19C>T与+73C>T之间呈完全连锁不平衡关系。该目标人群中存在5种多态性单体型，其中最主要的野生单体型(H1)频率为51.4%，其余4种为变异单体型(H2~H5，频率为46.3%)。采用分子克隆的方法构建不同长度多态性单体型报告基因重组质粒，进行双荧光素酶报告基因检测结果表明MGMT基因目的片段区域中的3种单体型都具有较高启动子转录活性且转录活性不同，提示目的基因启动子活性的主要调控位点位于中端启动子区。本研究结果为低剂量电离辐射致机体适应性反应机制及对人群健康影响研究提供一定科学依据和理论基础。