NFBD1-RPA1轴调控同源重组修复通路介导鼻咽癌放射抵抗的机制研究

Nasopharyngeal carcinoma (NPC) is one of the most common eight malignant tumors in China, and radiotherapy is the primary treatment for NPC. But the main reasons of treatment failure for NPC are radioresistance which often results to tumor local control failure, tumor recurrence and distant metastasis. Recent studies reveal that the excessive activation of DNA damage repair is responsible for the development of radioresistance. NFBD1 is identified as an important mediator in DNA damage repair, and RPA1 is a key protein to maintain to the high-fidelity of homologous recombination repair. Our recent study shows an increased NFBD1, RPA1 and HR related proteins in radioresistant NPC tumors, and both of them have significant correlation. Silencing NFBD1 or RPA1 enhanced the radiosensitivity of radioresistant CNE2-RR cells, furthermore, our preliminary data also revealed a novel role of NFBD1 in the regulation of ionizing radiation inducing RPA1 foci formation. Our study will focus on the role of NFBD1-RPA1 axis in the development of NPC radioresistance. Firstly, we study the expression of NFBD1, RPA1 and HR genes in the NPC patients with different radiosensitivity. Secondly we will investigate the effects of NFBD1-RPA1 axis on the homologous recombination repair in radioresistant NPC cells. Thirdly we will examine the role of NFBD1-RPA1 axis on the development of radioresistance in nude mice and its effects on homologous recombination repair. Collectively this project will provide a new insight into the development of NPC radioresistance and be hopefully expected to offer a new strategy in the study of the prevention and treatment of patients with NPC radioresistance.

鼻咽癌是我国常见肿瘤之一，放射治疗是其主要治疗方式。但是，放射抵抗常常导致肿瘤局部控制失败、肿瘤复发和远处转移。研究发现DNA损伤修复系统过度激活是肿瘤放射抵抗发生的重要机制。NFBD1作为DNA损伤修复重要的调节分子，而RPA1是维持同源重组（HR）高保真特点的关键蛋白，我们课题组前期基因芯片结果显示，NFBD1、RPA1及HR通路基因在鼻咽癌放射抵抗细胞株和放射抵抗组织中异常高表达，且NFBD1和RPA1蛋白表达有明显相关性，进一步发现沉默NFBD1、RPA1均一定程度的逆转了鼻咽癌细胞放射抵抗性，且二者存在直接相互作用，本项目以此为切入点，进一步研究NFBD1-RPA1轴调控鼻咽癌放射抵抗的分子机制，从临床大样本分析NFBD1-RPA1与鼻咽癌放射抵抗的相关性和预后判断价值，同时在细胞和动物模型中进一步深入探讨其在鼻咽癌放射抵抗调控中的作用方式和调控分子机制，阐明下游效应途径及靶点。

鼻咽癌是我国常见肿瘤之一，放射治疗是其主要治疗方式。但是，放射抵抗常常导致肿瘤局部控制失败、肿瘤复发和远处转移。研究发现DNA损伤修复系统过度激活是肿瘤放射抵抗发生的重要机制。NFBD1作为DNA损伤修复重要的调节分子，而RPA1是维持同源重组（HR）高保真的关键蛋白，我们课题组前期研究显示，沉默NFBD1、RPA1均一定程度的逆转了鼻咽癌细胞放射抵抗性，且二者存在直接相互作用，本项目通过研究NFBD1-RPA1轴与同源重组修复通路的关系，阐明鼻咽癌放射抵抗的分子机制。首先我们通过梯度增加放射剂量的方法（总剂量60Gy）建立鼻咽癌放射抵抗细胞株，并通过细胞增殖实验、细胞周期、G2/M checkpoint实验、凋亡、彗星实验、细胞克隆实验验证鼻咽癌细胞的放射抵抗性；然后采用基因芯片技术筛选鼻咽癌放射抵抗细胞中的差异基因表达，发现NFBD1、RPA1、HR相关蛋白均高表达，进一步我们在鼻咽癌临床样本中发现，NFBD1、RPA1、HR相关蛋白在鼻咽癌放射抵抗组织中明显高表达，并建立了基于HR相关蛋白表达预测鼻咽癌放射抵抗的预测模型，且NFBD1与RPA1的表达存在明显相关性；随后我们采用慢病毒技术沉默NFBD1的表达，明显提高了鼻咽癌细胞的放射敏感性，进一步发现沉默NFBD1明显抑制了电离辐射诱导的RPA1蛋白及HR通路的激活，从而增加电离辐射诱导的DNA损伤，并通过裸鼠移植瘤实验证实了沉默NFBD1抑制了肿瘤细胞的生长，并且HR修复通路也处于抑制状态；为了阐明NFBD1调控HR通路的分子机制，我们进一步沉默了RPA1蛋白，发现其一定程度上逆转了鼻咽癌细胞的放射抵抗性，通过激光共聚焦实验发现，电离辐射诱导NFBD1蛋白与RPA1发生共定位，说明二者存在直接的相互作用，沉默NFBD1抑制了RPA1及HR相关蛋白DNA损伤位点聚集点形成，从而干扰了HR修复通路的激活，进而增加鼻咽癌细胞的放射敏感性，所以，NFBD1通过调控RPA1蛋白而介导同源重组修复通路的激活，进而导致鼻咽癌放射抵抗的形成，本研究可能为鼻咽癌放射抵抗患者的预防和治疗寻找到重要的靶分子和突破口，也可能为其他放射抵抗肿瘤的预防和治疗提供重要的借鉴资料。