雌激素受体途径关键基因FOXA1的顺式和反式功能变异在中国人群受自然选择并与肝细胞癌易感性关联

FOXA1 is the core component in ERɑ pathway and they co-regulate a panel of target genes in a functional interaction mode and are implicated in sexual dimorphism in Hepatocellular carcinoma (HCC) susceptibility. We have previously found that regulatory and coding variants of FOXA1 gene are associated with HCC risk. The trans-acting −173VNTR enhances the transactivation of FOXA1 by EGR1, a HCC related transcription factor, correlates with allelic expression imbalance of the coding variant Thr83Ala, which is in strong linkage disequilibrium with −173VNTR. FOXA1 Thr83Ala variant impairs its interaction with ERα and attenuates the transactivation toward their HCC protective target genes. Furthermore, we observed that the HCC protective derived allele FOXA1 Thr83 has been under positive selection in Chinese population. This proposal is aimed to validate the genetic association between FOXA1 and HCC, investigate the in vitro and in vivo phenotype of FOXA1 Thr83Ala using CRISPR/Cas9 gene editing technique, analyze its functional effects on the cistromes and target genes of Foxa1 and ERɑin DEN induced HCC mice model using ChIP-Seq strategy, and to reveal the underlying specific functional target genes and network for FOXA1 Thr83Ala using integrate genomics and bioinformatics tools. This study will construct the genetic model for the association between FOXA1 and HCC, uncover the phenotypic relevance and biological mechanism, broaden our knowledge on molecular epidemiology and evolutionary genetic of HCC.

肝细胞癌易感性呈现性别差异，作为雌激素受体途径关键因子，FOXA1与ERɑ结合并共同调控HCC抗性基因的表达。我们前期研究发现，FOXA1基因变异和HCC易感性关联，其中顺式变异−173VNTR增强转录因子EGR1对FOXA1的转录，并通过连锁不平衡导致编码区Thr83Ala的等位表达不平衡；而反式变异Thr83Ala则减弱了FOXA1与ERɑ的相互作用，降低了对若干HCC抗性靶基因的表达；并且抗性衍生型Thr83在中国人群受正选择。本项目拟在独立人群验证FOXA1与HCC的关联，基于CRISPR/Cas9技术建立Thr83Ala突变体的细胞系和小鼠模型，分析FOXA1反式作用变异的体外和体内表型，并以ChIP-Seq技术鉴定突变体相关的Foxa1/ERɑ的功能性差异靶基因及网络。本项目有望建立FOXA1和HCC关联的遗传模型并阐明其体内表型和功能的生物学机制，丰富HCC进化遗传学新观点。

肝癌在我国的发病率高，肝癌易感性有着显著的性别差异，性激素及其受体相关通路参与了肝癌的发生。FOXA1作为先导转录因子，它与雌激素受体ERα及雄激素受体AR结合，并介导它们对下游靶基因的转录表达，FOXA1和ERα及AR在基因组有重叠的结合区或叫顺式元件组。基于两个独立的肝癌病例对照人群的研究（合并样本后病例组为2085人，对照组为2272人），我们发现FOXA1基因该功能性错义变异和肝癌易感性关联（OR 1.28; 95% CI 1.11-1.48），并且与此位点连锁的顺式元件区的VNTR多态性，也和肝癌易感性关联。通过基于细胞和组织的研究，我们发现FOXA1启动子区VNTR高重复等位增强与转录因子EGR1的结合并增加FOXA1基因本身的表达；FOXA1基因的错义变异rs7144658(Thr83Ala)，能减弱FOXA1与ERα的蛋白相互作用但不影响对AR的结合，降低对FOXA1对DIO1, UGT2B17, NTCP等ERα共同的靶基因的顺式元件的结合，并降低靶基因的表达，但增强甲胎蛋白基因AFP的表达并与肝癌病人血清AFP浓度增高关联（n=1096）。通过基于基因组多态性公共数据库的进化群体遗传学分析，我们进一步发现对肝癌有保护作用的FOXA1基因的衍生型等位基因Thr83，其等位基因频率存在显著的人群差异，单倍型结构的EHH检验结果显示，FOXA1的Thr83在中国人群可能经历了达尔文正向自然选择。通过本项目的工作，我们建立了FOXA1基因变异和肝癌易感性的关联关系，并初步阐明了FOXA1基因顺式和反式作用变异对FOXA1-ERα通路调控网络的生物学机制，丰富肝癌进化遗传流行病学新观点，深入肝癌病因学认识，从而助益于肝癌的人群预防预警和个体干预。

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