DNA聚合酶 β 突变体影响食管癌化疗敏感性的机制研究

Resistance of esophageal cancer to chemotheraptic agents is the main reason of chemotherapy failure. DNA polymerase β (pol β) is a key enzyme in DNA base excision repair (BER), and overexpression or mutation of pol β is closely related to drug resistance. In our previous study, nine types of mutation in hot spots of pol β gene were identified in human esophageal squamous cell carcinoma (ESCC). Compared with the wild-type pol β group, the median survival of patients with Δ117-335 mutation was significantly shorter, while improved survival was found among patients with mutation of G179R, which is corresponding to their effect on sensitivity of ESCC cells to chemotherapeutic agents. However, the mechanisms of pol β variants in regulating chemotherapy sensitivity of ESCC cells have not been investigated. Bioinformatics predicts that these two pol β mutations might have some influences on their BER function, and change the repair ability of tumor cells to chemotherapy injury. In this study, The BER function of these two variants will be fully checked. In addition the interaction between pol β variants and BER related proteins will be checked to analyze the molecular mechanism of abnormal BER function of pol β mutations. On this basis, construction of ESCC cell stablely transfected with mutant pol β to investigate the mechanism of abnormal BER function of pol β variants in regulating cisplatin sensitivity of human esophageal carcinoma cells. Finally the patient-derived esophageal squamous cell carcinoma xenograft mouse models with pol β variants will be established to further evaluate the effects of these pol β variants on the sensitivity to the chemotheraptic agents The polβ gene alteration may serve as a biomarker for individualized treatment and evaluation of the prognosis in human esophageal carcinoma.

肿瘤耐药是影响食管癌化疗效果的主要因素。DNA 聚合酶 β（pol β）是碱基切除修复（BER）系统的关键酶，其蛋白异常与肿瘤耐药密切相关。课题组前期检测到9 种食管癌组织相关的 pol β突变体，其中携带Δ117-335等突变体的患者预后差，而携带G179R突变体预后较好，与这些突变体对食管癌细胞株化疗敏感性的影响基本一致，但作用机制尚未明确。生物信息学预测这两种突变可能影响pol β的BER功能，进而改变肿瘤细胞对化疗损伤的修复能力。本项目拟通过检测这两种突变对BER功能及pol β与BER系统中其它分子之间相互作用的改变，分析其BER功能异常的分子机制。在此基础上，从细胞水平探讨pol β 突变体BER功能异常影响食管癌细胞化疗敏感性的机制；并建立人食管癌组织来源移植瘤模型，进一步评估pol β 突变对食管癌化疗敏感性的影响，为临床食管癌的个体化治疗及预后评估提供有效的标记物。

DNA聚合酶β（polβ）是碱基切除修复（BER）系统的关键酶，其蛋白结构功能异常与肿瘤耐药密切相关。课题组前期检测到9种食管癌组织相关的polβ突变体，其中部分突变体影响患者预后，但作用机制尚未明确。本项目首先成功构建了polβ突变体稳定表达的KYSE150和EC109食管癌细胞株。细胞毒性实验和软琼脂糖克隆实验显示G179R和R183G两种polβ突变体减弱细胞化疗药物的敏感性。免疫荧光及彗星实验结果显示G179R 和R183G两种pol β突变能够增加细胞的γ-H2AX表达水平及DNA链断裂程度，减弱细胞的DNA损伤修复能力。选取polβ-R183G突变体做蛋白质组学分析，结合免疫沉淀验证，结果显示染色质解旋酶DNA 结合蛋白1样蛋白（CHD1L）在polβ-R183G突变体耐药中可能发挥关键作用。相关实验结果显示CHD1L敲低能够进一步降低细胞对化疗药物的敏感性和DNA损伤修复能力。与此同时本项目也初步研究polβ敲低对食管癌化疗敏感性的影响。该研究为后期研究CHD1L敲低与polβ-R183G突变之间合成致死提供依据，也为临床食管癌化疗提供理论基础。