泛素蛋白连接酶RNF2、BMI1与食管癌细胞DNA损伤修复和放射敏感性关系的研究

Esophageal carcinoma is one of the most common malignant tumor in our country. Radiotherapy(RT) is one of the most important methods to therapied esophageal carcinoma, however, the curative effect was not satisfied. It is still a challenged question how to enhance radiosensitivity of esophageal carcinoma cells, to decrease local recurrent, and to increase local control and long-time survival. DNA is believed the main target of irradiation, cells have evolved a complex protein network to detect, signal and repair DSBs. Our previous research indicated one of the initial events occurring at DNA damage loci is phosphorylation of H2AX, at Ser 139 of its carboxy-terminal tail (γ-H2AX) by one or more members of the PI3K-like kinase group, including ATM, ATR .γ-H2AX decorates the chromatin flanking DSBs and recruits many early DDR proteins, such as MDC1 and 53BP1, to generate IR-induced foci. MDC1 and 53BP1 both appear to play a central role in transducing DNA damage signals to downstream effectors by serving perhaps as adaptor proteins connecting the upstream signal from γ-H2AX to the downstream effector Chk2. Chk2 is a major target of ATM, which phosphorylates threonine 68 to activate its kinase activity in response to DNA damage. Activated Chk2, in turn, phosphorylates p53 at Ser20, CDC25A at Ser123, and CDC25C at Ser216, contributing to the G1/S, S, and G2/M checkpoint, arrest of cell cycle were induced to enhance repair for DNA lesions. Monoubiquitination of H2AX is induced by E3 ligase enzymes RNF2 and BMI1 upon DNA double-strand breaks (DSBs) and plays a critical role in H2AX S139 phosphorylation (γ-H2AX). Furthermore, BMI1 functions as an enhancer of RNF2 E3 ligase activity to promote the H2A ubiquitination.Howecer,the molecular mechanism of ubiquitination is still unclear.Basing on the above observations, we presume that E3 ligase enzymes RNF2 and BMI1 play an importmant roles in transmitting DNA damage signal and regulating radiosensitivity of esophageal carcinoma cells， RNF2-BMI1-mediated H2AX monoubiquitination is a very upstream event.We plan to carry out our research in following aspects to demonstrate our assumption:1. seeking the site of ubiquitination of H2AX by using site-directed mutagenesis technique.2.using gene transfection to enunciate the molecular mechanism of ubiquitination of H2AX by gene RNF2 and BMI1.Detect the works factor of esophageal carcinoma at the molecular level, offer the theoretical foundation to improve the effect of radiotheraphy in the clinical practice.

DNA 损伤信号感应、传导在DNA损伤修复中至关重要，亦是调控细胞放射敏感性的重要途径之一，但其机制复杂。申请人前期研究工作发现H2AX、MDC1/53BP1、CHK1/CHK2在调控辐射诱导的食管癌细胞DNA损伤修复、进而影响放射敏感性中起着重要的作用，且呈自上而下的调控关系。令人感兴趣的是DNA损伤位点的H2AX泛素化在调控其磷酸化过程中发挥作用，并且发现泛素连接酶RNF2和BMI1对H2AX泛素化的发生及程度具有调控作用。根据这些初步结果，申请人认为RNF2和BMI1在调控食管癌细胞DNA损伤信号传导及放射敏感性中发挥关键作用，拟从以下进行研究：1.利用定点突变技术寻找RNF2和BMI1对H2AX泛素化的具体位点；2.利用基因转染研究RNF2、BMI1表达水平对H2AX泛素化程度及食管癌放射敏感性的影响。

本课题的预期目标是通过采用BMI1和RNF2的shRNA病毒表达载体转染体外及体内食管癌细胞，抑制BMI1和RNF2的蛋白表达，阻断DNA损伤信号传导，消除照射后细胞周期阻滞，减少DNA损伤修复，从而增加放射敏感性。研究发现BMI1和RNF2的mRNA水平及其蛋白表达均呈现时间剂量依赖性，促进了食管癌细胞株ECA109的增殖，并将细胞周期阻滞在G2/M期，利于细胞凋亡的发生。针对BMI1和RNF2设计构建RNA干扰质粒，与慢病毒包装后感染食管癌细胞，筛选BMI1和RNF2蛋白低表达稳定转染细胞株，降低了食管癌细胞的增殖能力，在一定程度上消除照射后细胞周期阻滞，增加体外食管癌细胞放射敏感性。并且研究发现，抑制BMI1和RNF2的蛋白表达，会使H2AX的泛素化和磷酸化受到抑制，且对MDC1、53BP1、CHK1、CHK2表达有不同程度的影响。在此基础上，课题组利用BMI1和RNF2蛋白低表达稳转细胞株建立了荷瘤裸鼠模型，发现实验组的肿瘤生长速度慢于对照组，证明在体内抑制BMI1和RNF2蛋白表达确实发挥了抑制肿瘤增长的作用。本课题基本达到预期研究目标。