Wang Maoxin, Chen Xianming, Chen Hui, Zhang Xian, Li Jianzhong, Gong Hongxun, Shiyan Chen, Yang Fan
Fuzong Clinical College of Fujian Medical University, Fuzhou General Hospital of Nanjing Command Zone of PLA, Fuzhou, Fujian, P.R. China.
Oncol Rep. 2015 Jul;34(1):341-9. doi: 10.3892/or.2015.3958. Epub 2015 May 7.
Tumor residue or recurrence is common after radiation therapy for nasopharyngeal cancer (NPC) since the tumor cells can repair irradiation-induced DNA damage. The ubiquitination cascade mediates the assembly of repair and signaling proteins at sites of DNA double-strand breaks (DSBs). Ring finger protein 8 (RNF8) is an E3 ubiquitin ligase that triggers ubiquitination at the site of DSBs. The present study aimed to identify whether and how RNF8 small interfering RNA (siRNA) treatment enhances the radiosensitivity of irradiated human NPC cell lines. The CNE1, CNE2, and SUNE human NPC cell lines were stably transfected with a constructed RNF8-targeting siRNA expression vector. Western blotting was used to detect the effectiveness of RNF8 downregulation by RNF8 siRNA. The siRNA-transfected (RNF8-) and non-transfected (RNF8+) cells were irradiated at different doses by a linear accelerator. The growth inhibition ratio and apoptosis rate were detected by the methyl thiazolyl tetrazolium (MTT) assay and flow cytometry, respectively. The ataxia-telangiectasia mutated (ATM), DNA-PKcs, Chk1, Chk2, Nbs1 and Ku80 protein levels in each group were determined. The growth inhibition ratio and apoptotic percentage of RNF8- cells were higher than those of the RNF8+ cells in each of the three cell lines. Lower protein expression levels of Chk1, Chk2, ATM, and Nbs1 were observed in the irradiated RNF8- cells compared to the irradiated RNF8+ cells in each of the three cell lines (P<0.01). As a result, a conclusion could be drawn that RNF8 recruits and ubiquitinates many factors to repair DNA damage, including DSBs, thereby conferring radioresistance to NPC cells.
鼻咽癌（NPC）放疗后肿瘤残留或复发很常见，因为肿瘤细胞能够修复辐射诱导的DNA损伤。泛素化级联反应介导修复和信号蛋白在DNA双链断裂（DSB）位点的组装。环指蛋白8（RNF8）是一种E3泛素连接酶，可在DSB位点触发泛素化。本研究旨在确定RNF8小干扰RNA（siRNA）处理是否以及如何增强经辐射的人NPC细胞系的放射敏感性。用构建的靶向RNF8的siRNA表达载体稳定转染CNE1、CNE2和SUNE人NPC细胞系。采用蛋白质免疫印迹法检测RNF8 siRNA下调RNF8的有效性。用直线加速器对转染siRNA（RNF8-）和未转染（RNF8+）的细胞进行不同剂量照射。分别通过噻唑蓝（MTT）法和流式细胞术检测生长抑制率和凋亡率。测定每组中共济失调毛细血管扩张突变（ATM）、DNA依赖蛋白激酶催化亚基（DNA-PKcs）、细胞周期检查点激酶1（Chk1）、细胞周期检查点激酶2（Chk2）、Nbs1和Ku80蛋白水平。在三种细胞系中，RNF8-细胞的生长抑制率和凋亡率均高于RNF8+细胞。与三种细胞系中经照射的RNF8+细胞相比，经照射的RNF8-细胞中Chk1、Chk2、ATM和Nbs1的蛋白表达水平较低（P<0.01）。因此，可以得出结论，RNF8招募并泛素化许多修复DNA损伤的因子，包括DSB，从而赋予NPC细胞放射抗性。
