Annexin A1表达下调/缺失在鼻咽癌放疗抵抗中的作用及其分子机制研究

Annexin A1 is a potential tumor suppressor gene, is closely related to tumor cell proliferation, differentiation and apoptosis. In our previous study, we found that Annexin A1 was down-regulated or deletion in the nasopharyngeal carcinoma(NPC) tissue and cell line with radioresistance, and was one of the proteins that were involved in p53-mediated radioresponse in NPC. And it might be related to radioresistance of NPC. In this study, firstly, we will increase the expression of Annexin A1 in CNE2-IR cell line using gene transfection and knock down the expression of Annexin A1 in CNE2 cell line using siRNA techlique, analyze the radiosensitivity of these cell lines in vitro and in vivo, then evaluate its effect of the biological activity of p53 and its correlation with NPC radioresistance. Secondly, we will identify the proteins interacting with Annexin A1 using tandem affinity purification and 2D-LC-MS/MS technology, build the protein regulatory networks of Annexin A1 in NPC radioresistance, screen the important targetprotein regulated by Annexin A1, and find the key signaling pathway related to NPC radioresistance by bioinformatics analysis. This project helps not only to reveal the role and mechanism of Annexin A1 downregulation / deletion in the radioresistance for NPC, but also to provide a scientific basis for radiation sensitivity prediction, targeted therapy and individualized radiotherapy of NPC.

Annexin A1是一个候选抑瘤基因，与肿瘤细胞增殖、分化和凋亡密切相关。申请者前期研究发现：Annexin A1在放疗抵抗鼻咽癌组织和细胞中均表达下调/缺失，是p53参与鼻咽癌放射应答的蛋白质之一，可能与鼻咽癌放疗抵抗有关。本项目拟建立Annexin A1稳定上调的CNE2-IR细胞系，Annexin A1稳定下调的CNE2细胞系，通过体内和体外实验研究Annexin A1在鼻咽癌放疗抵抗中的作用。以建立的细胞系为样本，研究Annexin A1与p53的放射生物学活性及鼻咽癌放疗抵抗的关系；采用串联亲和纯化耦联质谱技术，鉴定Annexin A1的相互作用蛋白，构建Annexin A1在鼻咽癌放疗抵抗中的蛋白质调控网络，明确Annexin A1调控的关键靶蛋白及信号通路，阐明Annexin A1在鼻咽癌放疗抵抗中的作用机制，为鼻咽癌的放疗敏感性预测、靶向治疗和个体化放疗提供科学依据。

我们前期的研究发现，Annexin A1蛋白在放射抵抗的鼻咽癌组织细胞和鼻咽癌细胞系中表达均下调，提示Annexin A1蛋白表达下调增强鼻咽癌的放疗抵抗性。为揭示Annexin A1蛋白表达下调增强鼻咽癌放射抵抗的作用及其分子机制，项目首先建立Annexin A1表达改变的鼻咽癌细胞系，分析Annexin A1表达改变对体外和体内鼻咽癌放射敏感性的影响；再以建立的细胞系为样本，分析发现Annexin A1表达下调通过降低p53的生物学活性而导致鼻咽癌放疗抵抗；采用串联亲和纯化（TAP）和二维液相色谱串联质谱（2D-LC-MS/MS）技术，分离、鉴定NPC细胞中Annexin A1的相互作用蛋白。通过生物信息学分析构建Annexin A1在NPC放疗抵抗中的蛋白质调控网络，筛选在NPC放疗抵抗中Annexin A1所调控的关键靶蛋白分子。通过研究，我们证实Annexin A1表达下调增强鼻咽癌细胞和鼻咽癌裸鼠移植瘤的放疗抵抗性；发现Annexin A1表达下调可降低p53的生物学活性及抑制P53-ATM/ BRCA1信号通路而增强鼻咽癌放疗抵抗；项目研究不仅证实了Annexin A1表达下调在鼻咽癌放疗抵抗中的作用，而且提示Annexin A1能作为鼻咽癌放疗敏感性预测以及放疗增敏的靶标。至今，已发表标注项目资助的SCI论文6篇，CSCD论文3篇，接收论文2篇。