DNA聚合酶β突变体在食管癌发生中的作用和机制研究

DNA polymerase β (polβ)，a key enzyme responsible for DNA base excision repair (BER)，is relatively low and constant expression in normal somatic cells. The major function of DNA polβ is keeping genetic stability of mammalian cells. Mutation of DNA polβ is found in various human cancer tissues. In our previous study, three types of mutation in hot spots of DNA polβ gene were found in human esophageal carcinoma．However, the role of polβ mutants in tumorigenesis have not been fully investigated. In the present study, the enzyme activity of three polβ mutants(58 bp deletion(177-234nt), 466G→A(Gly118Glu) and 648G→C(Gly179Arg)) will be checked. Normal human esophageal epithelial cells (HEECs) stablely transfected with mutant DNA polβ will be established. Tumor related phenotypes, including cell growth, cell transformation, cell cycles and formation of tumors of immunodeficient nude mice will be checked after treatment of the low dose of N-nitrosomethylbenzylamine（NMBA). The BER function and genetic stability will be investigated. Genomic DNA sequences will be detected in transformed cells. A conditional knockin modle of polβ mutant with 58 bp deletion in adult rat esophagus will be generated . The rat modle will be induced to suffer from esophageal carcinoma by NMBA and the role of polβ mutants in tumorigenesis will be explored. The purpose of this study is to elucidate the role and mechanism of polβ mutants in esophageal tumorigenesis by cell and animal modles and the possibility of DNA polβ used as a target for esophageal carcinoma prevention.

DNA聚合酶β（DNA polβ）是碱基切除修复中的关键酶，在维持细胞基因组稳定性方面有重要意义。DNA polβ基因的突变在多种肿瘤组织中得到证实。课题组前期研究发现人食管鳞癌组织中热点突变形式有3种。但是突变的DNA polβ与食管癌发生的关系和机制有待深入研究。本课题拟在体外检测polβ突变体酶活性和功能的变化；并将构建的polβ3种突变型的载体转染人正常食管上皮细胞系，以低剂量甲基苄基亚硝胺（NMBA)为诱变剂，通过对细胞转化表型观察和转化细胞中polβ功能检测，研究polβ突变体对细胞恶性转化能力的影响和机制；建立食管组织条件性敲入58bp缺失突变型polβ的大鼠模型， 经NMBA诱变致食管肿瘤，研究polβ突变体在食管鳞状上皮癌变过程中的作用和机制。本课题旨在通过polβ突变体的细胞模型和动物模型阐明其在食管癌发生中的作用机制。

DNA 聚合酶β（DNA polβ）是细胞中重要的修复酶，其基因的突变和功能的丧失，与肿瘤的发生密切相关。我们利用前期研究发现的人食管鳞癌组织中DNA polβ 3种热点突变，探讨其在食管癌发生发展中的作用。研究表明，在体外实验中polβ突变体的聚合酶功能降低，而稳定表达polβ突变体G179R的细胞，表现出克隆形成能力增加。polβ的敲低表现为对DNA 损伤剂的敏感性增加。在食管上皮polβ特异性敲除的小鼠模型上，经甲基苄基亚硝胺的诱导，建立了小鼠食管癌前病变模型，发现polβ的功能缺失，是促进小鼠食管癌前病变的重要原因。进一步的探索表明，PLK1可能在小鼠食管黏膜上皮polβ缺失的情况下，促进了食管上皮的癌前病变的发生。