eEF2K在食管癌放疗抵抗中的作用及机制研究

Eukaryotic elongation factor 2 kinase (eEF2K) is a structurally and functionally unique protein kinase in the calmodulin-mediated signaling pathway. It inhibits the elongation stage of protein synthesis by phosphorylating eEF2 under stressful conditions. Some studies demonstrate that eEF2K is over expressed in many tumors and also participates in the regulation of tumor progress. However, people have little knowledge about the association with eEF2K to chemoradio-resistence.Our preliminary studies revealed that patients with overexpression of eEF2K had a poor prognosis in esophageal cancer. We also found that, as an inhibitor of eEF2K, NH125 could increase the apoptosis of irradiated esophageal cancer cell but decrease its autophagic activity in vitro. We propose a hypothesis that mTOR/p70S6K signal pathway may regulate this procedure. For this hypothesis，human esophageal cancer cell line, nude-mouse transplanted tumor model and clinical specimens will be used to investigate the relationship between eEF2K and raidoresistance of esophageal cancer by real-time PCR, Western-blot, Lentiviral Transfection, RNA interference in vivo, vitro and molecular level. In addition, autophagy related proteins, such as LC3, Atg5 and Beclin 1, will be detected by Western-bolt and Laser Scanning Confocal Microscopy to demonstrate the correlation between autophagy and eEF2K.The present study will investigate the mechanism of radioresistance of esophageal cancer via the regulation of apoptosis and autophagy, and provide the potential application in improving the radiosensitivity of esophageal cancer.

真核延伸因子2激酶（eEF2K）是细胞在应激状况下激活的蛋白激酶。已有研究表明eEF2K在多种肿瘤中高表达并参与肿瘤进程的调控，但其对肿瘤放疗敏感性的影响迄今仍知之甚少。我们的前期研究提示：高表达eEF2K的食管癌患者放疗疗效较差，细胞实验证实eEF2K抑制剂在促进食管癌细胞放疗后凋亡的同时抑制了自噬；故推测eEF2K可能通过推动细胞由凋亡向自噬转化介导食管癌放射抵抗；这一过程可能受mTOR/p70S6K通路调节。为验证此假说，我们将应用人食管癌细胞株、裸鼠移植瘤模型和临床标本，采用定量PCR、Western blot、慢病毒转染、RNA干扰等技术，从分子、细胞及动物整体水平等方面探讨eEF2K调节肿瘤细胞放射敏感性的分子机制。本研究将从eEF2K调控肿瘤细胞凋亡自噬转化这一新视点探究食管癌放疗抵抗的机制，为提高食管癌放射治疗敏感性提供新思路。

目的：研究eEF2K在食管鳞状细胞癌（ESCC）中的生物学功能。.方法：组织芯片共完成100对ESCC肿瘤及邻近正常组织检测。在ECA-109和TE-13食管癌细胞中构建eEF2K的过表达和敲除株。分别用免疫荧光法、CCK-8法、transwell法、集落形成法、流式细胞仪和westernblot法检测DNA损伤、细胞活力、迁移和侵袭、放射抗性、凋亡和自噬。肿瘤生长和放射抗性采用裸鼠移植瘤模型进行评估。.结果：食管鳞状细胞癌组织中eEF2K的表达高于相应的非肿瘤组织（P<0.05）。与对照组相比，eEF2K高表达细胞的增殖速率增加（P<0.05），而沉默eEF2K则减弱了肿瘤细胞增殖（P<0.05）。此外，低水平的eEF2K表达与迁移和侵袭能力降低相关（P<0.05），而较高水平的eEF2K表达与更快的迁移和侵袭率相关（P<0.05）。与对照组相比，eEF2K过表达可导致辐射抵抗和辐射诱导的自噬，并减少辐射诱导的细胞凋亡，但沉默eEF2K可促进辐射敏感性和细胞凋亡，减少自噬。另外，eEF2K的高表达促进了肿瘤的生长（P<0.01）。NH125（eEF2K的小分子抑制剂）联合放疗延缓食管癌移植瘤生长的效果优于NH125或单纯放疗（P<0.05）.结论：eEF2K诱导食管鳞状细胞癌的进展和抗辐射性，这可能是提高食管鳞癌放射敏感性的一个新的治疗靶点。