EIF5A2通过c-myc/miRNA-29b通路诱导非小细胞肺癌干性抵抗放疗的作用与机制研究

EIF5A2 is an oncogene which promotes cancer cell proliferation, invasion, metastasis and chemotherapeutic drug resistance. However, the role of EIF5A2 in cancer resistance to radiation is totally unknown. In our previous study, we found EIF5A2 is a poor prognostic factor of NSCLC patients. In our preliminary experiment, we found: ① overexpression of EIF5A2 increased the stemness while decreased the radiosensitivity of cancer cells in NSCLC; ② c-Myc/miRNA-29b is responsible for the EIF5A2 maintained NSCLC cell stemness. Based on the results of our previous work, we will carry out the followings experiments in the present study: ① via gain-function, loss function and rescue assay, we will explore the influence of the expression levels of EIF5A2 and miRNA-29b on the stemness and radiobiological effect of NSCLC; we will predict the downstream potential genes using target prediction programs, screen and verify the targeted genes by the methods of qRT-PCR, western blot, up/down regulation and rescue assay, and explore the regulation mechanism with the methods of CHIP and luciferase reporter system; ③ IHC will be utilized to detect the expression status of EIF5A2 and its downstream targeted genes in a NSCLC tissue microarray, and the clinical prognostic value of which will be analyzed. We aim to explore the function and mechanisms of EIF5A2 promoting NSCLC radioresistance by inducing cancer cell stemness; and serve as evidence that EIF5A2 may be a new target in the treatment of NSCLC.

EIF5A2是一个与肿瘤增殖、侵袭、转移、化疗抵抗相关的癌基因，但EIF5A2在放疗抵抗中的作用，目前尚属空白。我们前期研究证实EIFA52是NSCLC的不良预后指标；进一步预实验发现：①上调EIF5A2增强NSCLC干性同时降低NSCLC放疗敏感性；②EIF5A2可通过c-Myc/miRNA-29b通路影响NSCLC干性。本研究将承前启后，①通过功能获得、缺失及回复实验探讨EIF5A2及miRNA-29b对NSCLC干性及放射生物效应的影响；②通过miRNA 靶基因预测软件预测、qRT-PCR、Western blot、功能回复等实验验证下游靶基因，经CHIP和荧光素酶实验明确具体调控机制；③IHC检测EIF5A2及其下游基因在大病例NSCLC组织芯片中的蛋白表达，分析其临床预测价值。本研究旨在深入解析EIF5A2诱导NSCLC干性抵抗放疗的作用机制，为NSCLC治疗寻找新靶点提供依据。

肺癌的高发病率和高死亡率，严重影响全球生命健康。而非小细胞肺癌（non-small-cell lung cancer, NSCLC）是肺癌中最主要的病理类型，放疗是NSCLC的主要治疗手段。EIF5A2与肿瘤增殖、侵袭、转移、化疗抵抗相关，但其在放疗抵抗中的作用仍不明确。本课题通过体外细胞实验发现EIF5A2的表达增加非小细胞肺癌（NSCLC）细胞放疗抵抗，降低NSCLC细胞放疗敏感性，进一步通过WB，qRT-PCR，和体外球体形成实验发现EIF5A2通过增加CD133的表达，增强NSCLC细胞干性来减低放疗敏感性。生物信息学分析肿瘤干细胞自我更新和分化相关的qPCR miRNA array发现，miRNA-29b是CD133的潜在调控因子。体外和体内实验证实了miR-29b参与了EIF5A2对CD133的调控进而影响NSCLC干细胞特性和放疗敏感性。既往发现miRNA-29b-1/miR-29a的启动子区域包含了c-Myc的结合位点染色质，我们通过免疫沉淀（CHIP）和双荧光素酶报告基因检测证明EIF5A2介导c-Myc结合miRNA-29b启动子以抑制miRNA-29b的表达。我们研究也在分子生物学水平证明EIF5A2通过c-Myc调节miR-29b来调控CD133进而影响NSCLC干细胞特性和放疗敏感性。该项目基本阐明EIF5A2/c-Myc/miRNA-29b/CD133通路在NSCLC放疗抵抗中的作用和机制，为诠释NSCLC放疗抵抗的分子机制和寻找靶向治疗提供科学依据。