CAST、ERCC1基因重叠区域多态及microRNA 调控在肺癌遗传易感中的生物学意义

The Polymorphism of DNA repair enzyme in Neucleotide Exision Repair(NER) can determine the DNA repair capability to the damage caused by environmental carcinogen such as PAHs, which can mainly explain the indivadual cancer susceptibility.Our previous study supported by NSFC found that A allele mutation of ERCC1 C8092A (rs3212986) were significantly associated with high levels of BPDE-DNA adducts. Meanwhile RNA PolⅠsubunit CAST mRNA levels in participants carrying A allele of ERCC1C8092A has been found decreased compared with the homozygous CC.Therefore it suggests that the polymorphism located in overlaping region of CAST and ERCC1 can impact DNA repair capablity by altering CAST expression and function. Our present project manages to clarify that CAST can involve in NER pathway, and ERCC1 C8092A polymorphism can impact indivadual repair capability by altering CAST transfection and translation and ERCC1 microRNA modulation. ERCC1 C8092A polymorphism as a significant biomaker in cancer susceptibility can be confirmed by the smoking case-control study, DNA repair test related to RNAi silence and different expression of CAST in vitro, and molecular characteristic analysis of lung cancer such as genomic stablity and gene microarray test, and the study on microRNA modulation of CAST and ERCC1. The purpose of our present project is to provide much more scientific clues to study the polymorphism of DNA repair gene and cancer susceptibility.

核苷酸切除修复NER通路的DNA修复酶多态决定多环芳烃类环境致癌因子所致DNA损伤的修复效率，故成为肿瘤遗传易感的主要原因。本项目在前一个国家自然基金发现ERCC1C8092A(rs3212986)位点A等位基因突变可降低BPDE-DNA加合物修复能力，同时降低RNA聚合酶Ⅰ亚单位CAST 基因mRNA水平的基础上，提出这个位于CAST和ERCC1 基因重叠区域的SNP可能通过影响CAST蛋白而改变DNA修复能力。本项目拟通过吸烟暴露的肺癌病例对照研究、体外细胞的CAST基因沉默及不同表达与DNA修复能力关联研究、不同基因型肺癌分子特征分析及micorRNA调控机制研究从遗传及表遗传层次阐明CAST参与核苷酸切除修复的关键环节，ERCC1C8092A多态可通过影响CAST转录和翻译及ERCC1转录后microRNA调控而影响个体DNA修复能力，进而成为意义明确的肺癌遗传易感的生物学标志。

本项目从人群研究、体外试验验证及micorRNA功能挖掘等角度探讨共同位于ERCC1和 CAST(CD3EAP)重叠区域的C8092A(rs3212986)多态与肺癌发生风险的因果关联。首先300例肺癌病例对照研究显示无论整体或分层分析均发现rs3212986多态与肺癌，尤其吸烟型肺癌发生风险的关联。CAST沉默的体外试验提示其可能通过影响ERCC1在DNA损伤修复中发挥重要作用。另外通过设计分别以ERCC1和CAST(CD3EAP)为靶的rs3212986多态不同基因型的系列质粒，转染16HBE及293T细胞，获得分别过表达ERCC1和CD3EAP的两系列转染细胞，比较明确施加致癌剂BPDE所致两种细胞系中该多态不同基因型之间的细胞存活率及DNA损伤修复能力的差别，结果发现ERCC1过表达转染细胞的rs3212986 CC基因型更优于AA，而在CD3EAP过表达细胞中两者尚无显著性差异，提示rs3212986可能通过影响ERCC1表达而发挥作用。随后我们检测到的肺癌及癌旁组织中ERCC1mRNA水平与蛋白表达的不一致提示ERCC1转录后miRNA调控的作用。我们通过生物信息学软件预测rs3212986不同基因型的ERCC1 mRNA序列二级结构和最小自由能差异，及可能结合在rs3212986位点的9种miRNA，并分别检测肺癌病例及对照的血浆、肺癌组织及癌旁组织中9种miRNA表达水平。综合分析后认为miR-15a和miR-16可结合在ERCC13’UTR上，并具有生物学意义。由于其同源性，我们以miR-15a为例进行双荧光素酶实验：发现rs3212986基因型差异可影响miR-15a与ERCC13’UTR区的绑定。而miR-15a转染A549细胞也明显降低了ERCC1的蛋白表达。综上，本项目首次系统研究rs321298多态可以作为一个有效的肿瘤易感性生物标志预测吸烟型肺癌发病风险的价值，提出该多态通过影响miR-15a对ERCC1转录后的调控而影响环境致癌因子所致DNA损伤修复。为肺癌易感性生物标志的深入研究提供了重要的研究思路和科学依据。本项目已发表SCI源期刊论文4篇，中文核心期刊论文3篇，项目组邀请荷兰专家来华进行学术交流，参加学术会议交流7次，发表8篇会议论文，共培养博、硕士研究生5名。已达到预期目标。