c-Jun通过PI3K/AKT-mTOR信号轴介导人鼻咽癌放射抗拒细胞自噬的发生及其机制的研究

The local recurrence and distant metastasis rate of Nasopharyngeal carcinoma（NPC)following the radiotherapy and chemotherapy is about 20 percent. It is mainly dueto the radioresistance to NPC. However, radioresistance is attributed to the autophagy related to NPC. In our previous study, the radiation caused autophagy in CNE-2 cells (radiosensitive cell line of NPC) and such autophagy served as a cell survival mechanism against irradiation-mediated cell death;C - Jun in radiation resistant cells CNE - 2 R has high expression.In addition, c-Jun was involved in the radioresistance of NPC and knockdown of the c-Jun gene enhanced the radiation sensitivity of CNE-2R cells (radioresistant cell line of NPC).Nowadays, few research adances focus on c-Jun, autophagy and radioresistance related to NPC. Research shows that PI3K/AKT-mTOR pathway is activated in many tumors related to autophagy. So, wein CNE-2R cells and such autophagy serves as a cell survival mechanism against irradiation-mediated cell death. To verify this hypothesis, RNA interference technology, RT-PCR, Western blot analysis, Transmission electron microscopy analysis, MTT assay, Clonogenic survival assay, Flow cytometry, and Xenograft mice.radiation causes autophagy and the pro-survival function of autophagy in CNE-2R cells. Secondly, c-Jun regulates the PI3K/AKT-mTOR pathway leads to autophagy in CNE-2R cells and such autophagy serves as a cell survival mechanism against irradiation-mediated cell death. Finally, down-regulation of c-Jun andphagy will enhanced radiosensitivity in CNE-2R cells and the nude mice xenograft model of CNE-2R cells. The completion of the study will offer some innovative ideas to enhance the radiosensitivity of NPC therapy.

临床上约有20%的鼻咽癌患者经治疗后仍出现局部复发和远处转移，主要归因于鼻咽癌放射抗拒，而自噬作为鼻咽癌放射抗拒的重要原因。我们前期研究显示:放射致鼻咽癌细胞发生自噬，且自噬促进鼻咽癌细胞存活；c-Jun在人放射抗拒细胞CNE-2R中呈高表达，沉默CNE-2R细胞c-Jun基因，能增加其放射敏感性。迄今为止，国内外尚未有人研究c-Jun、自噬及鼻咽癌放射抗拒的关系。基于PI3K/AKT-mTOR是自噬发生的经典通路。我们提出假说：c-Jun可能通过PI3K/AKT-mTOR信号轴介导自噬的发生而参与鼻咽癌放射抗拒。本课题拟通过鼻咽癌放射抗拒细胞和裸鼠放射抗拒移植瘤模型，采用多种实验技术手段，研究c-Jun在鼻咽癌放射抗拒中的作用，探讨c-Jun通过PI3K/AKT-mTOR信号轴介导自噬参与放射抗拒的机制，从c-Jun介导自噬的新视点揭示鼻咽癌放射抗拒的机制，为鼻咽癌的增敏治疗提供新的思路

临床上约有20%的鼻咽癌患者经治疗后仍出现局部复发和远处转移，主要原因之一是鼻咽癌放射抗拒，而自噬是鼻咽癌放射抗拒发生的重要原因。我们前期研究显示：放射可导致鼻咽癌放射抗拒细胞株CNE-2R 发生自噬，且自噬促进CNE-2R细胞存活。本研究利用基因芯片及蛋白互作网络图筛选出放射抗拒CNE-2R细胞和放射敏感CNE-2细胞的差异基因c-Jun；通过电子显微镜技术、慢病毒沉默，q-PCR、Western blot、双荧光素酶报告基因检测系统、CCK-8、克隆形成、裸鼠模型及免疫组化等实验手段，发现：放射致鼻咽癌放射抗拒细胞CNE-2R 发生自噬；c-Jun在CNE-2R细胞中呈明显高表达；沉默c-Jun基因表达可增加CNE-2R细胞放射敏感性；进而对c-Jun进行体内、外功能试验。免疫组化实验验证c-Jun在鼻咽癌组织中的表达明显高于正常鼻咽粘膜组织，且c-Jun高表达是影响鼻咽癌预后的独立危险因素；沉默c-Jun可通过抑制自噬水平进而增强CNE-2R细胞对放射线的敏感性，提示沉默c-Jun可能是提高鼻咽癌患者放疗敏感性的有效靶点。通路网站预测c-Jun与 PI3K/AKT存在结合位点，并通过双荧光素酶报告基因检测系统及Western blot验证c-Jun靶向PI3K/AKT，进一步在CNE-2R中验证c-Jun敲低后激活PI3K/AKT-mTOR 通路抑制自噬减少放射抗拒；此外，c-Jun敲除可以通过自噬抑制异种移植瘤生长。本研究为通过抑制c-Jun表达来治疗放射抗拒鼻咽癌提供了理论依据。