靶向抑制CPEB4对鼻咽癌细胞的影响及放射增敏的机制研究

Effect of Radiation Sensitization and Targeted Inhibition of CPEB4 on Nasopharyngeal Carcinoma Cell Line

目的本研究于2016年3~6月期间探讨靶向抑制CPEB4对鼻咽癌细胞生物学行为以及放射敏感度的影响。方法利用RNAi技术构建慢病毒载体靶向抑制CPEB4基因的干扰序列;采用CCK-8法绘制CNE-2-sh CPEB4、5-8Fsh CPEB4、CNE-2及5-8F的生长曲线(增殖能力);采用Transwell法检测CNE-2-sh CPEB4、5-8F-sh CPEB4迁移能力的改变;采用流式细胞术法检测CNE-2-sh CPEB4、5-8F-sh CPEB4凋亡率的改变;采用克隆形成实验检测CNE-2-sh CPEB4、5-8F-sh CPEB4、CNE-2及5-8F细胞的放射敏感度差异。采用Western blot法检测CNE-2-sh CPEB4,5-8F-sh CPEB4、CNE-2及5-8F细胞EMT相关蛋白表达改变。结果 CNE-2-sh CPEB4、5-8F-sh CPEB4分别与CNE-2及5-8F对照,增殖速度以及迁移能力减弱;凋亡率无明显改变,放射敏感度增加。Western blot法检测了CPEB4靶向抑制后EMT表型相关蛋白表达改变,结果提示E-cadherin表达上调,slug、snail以及vimentin的表达下调相关,推测靶向抑制CPEB4后鼻咽癌放射敏感度增强可能与EMT相关蛋白及因子的表达改变相关。结论靶向抑制CPEB4蛋白表达后,鼻咽癌细胞系的增殖能力、体外迁移能力显著减弱,放射敏感度明显增强,其机制可能与CPEB4抑制EMT表型相关,E-cadherin表达上调,vimentin表达下调,E-cadherin表达上调可能与抑制E-cadherin表达的转录因子slug、snail表达下调相关。

Objective To explore the emerging evidence that indicates a key role for the CPEB4 in the regulation of radiosensitivity and biological behavior of nasopharyngeal carcinoma cells.Methods The inhibition of CPEB4 gene was worked with the RNAi technigue and lentiviral vector. Routine culture of nasopharyngeal carcinoma cells CNE-2 and 5-8F, logarithmic phase cells were selected for the experiment. CCK-8 assay was used for the detection the proliferative capacity of CNE-2-shCPEB4, 5-8F-shCPEB4, CNE-2 and 5-8F. Transwell assay was used for the detection the migration capacity of CNE-2-shCPEB4, 5-8F-shCPEB4, CNE-2 and 5-8F. The flow cytometry assay was used for the detection the apoptosis rate of CNE-2-shCPEB4, 5-8F-shCPEB4, CNE-2 and 5-8F. The expression EMT related proteins of CNE-2-shCPEB4, 5-8F-shCPEB4, CNE-2 and 5-8F were detected by Western blot.Results The proliferation capacity and the migration capacity diminished in CNE-2-shCPEB4, 5-8F-shCPEB4. No obvious changes were detected in the apoptosis rate.The radiosensitivity of CNE-2-shCPEB4, 5-8F-shCPEB4 were increased. The possible mechanism about radiosensitization seemed to be related with the up regulated expression of EMT phenotype relative E-cadherin and the down regulated expression of slug, snail, vimentin in the situation of CPEB4 inhibition.Conclusion The proliferation capacity and the migration capacity diminished in CNE-2-shCPEB4, 5-8F-shCPEB4. No obvious changes were detected in the apoptosis rate.The radiosensitivity of CNE-2-shCPEB4, 5-8F-shCPEB4 were increased,which may be related to the up regulation of EMT phenotype relative E-cadherin and the down regulation of EMT phenotype relative vimentin, slug and snail.