辐射诱导NRP1-HOXs通路在肺癌辐射抵抗中的组蛋白甲基化机制研究

NRP1 gene was a recently confirmed oncogene. It was involved in tumorigenesis and development by influence on the process of tumor microenvironment, angiogenesis and deterioration. Our previous studies have demonstrated that the expression of NRP1 was increased in radiation response of immune system, while it has an enhanced expression in radioresistant tumor cells and played an important role in radioresistance of lung cancer cells. But the mechanism of NRP1-regulated target genes (HOXs and MLL) in lung cancer radioresistance and the regulatiom of NRP1-mediated histone methylation modification rebuild have not been fully clarified. This research intends to confirm the key biological function of radiation-induced NRP1-HOXs pathway in lung cancer radioresistance with multiple biological models including MLLf/fCre ER gene knockout animal models, NRP1-regulated target gene interference stable cell lines and clinical tissue samples of lung cancer; to determine the accurate binding site of NRP1 in targeted gene transcriptional regulatory regions under radiation stress condition with key technologies such as ChIP; to reveal the regulation of HOXs transcription histone methylation rebuild responses to radiation stress and its targeted gene networks; to expound the epigenetic mechanism of HOXs gene transcription mediated lung cancer radioresistance regulated by NRP1 and its biological implications. The purpose of this research is to provide theoretical foundation for finding key target molecules in lung cancer radioresistance, which may be useful to improve clinical effectiveness of radiotherapy for the treatment of lung cancer.

NRP1基因是新近证实的癌基因，通过肿瘤微环境、血管生成以及细胞的恶化等过程参与肿瘤的发生与发展中。我们发现，NRP1在免疫系统的辐射反应中表达增高的同时，在辐射抗性肿瘤细胞中表达增强，并在肺癌细胞辐射抵抗中起到很重要的作用，但NRP1调控的靶基因在肺癌辐射抵抗中的作用机制和其介导的组蛋白甲基化修饰重建规律及机制尚不清楚。本项目拟利用MLLf/fCre ER基因敲除动物模型、NRP1调控的靶基因干扰稳定细胞系以及临床肺癌标本等多种生物学模型，证实辐射诱导NRP1-HOXs通路在肺癌细胞辐射抵抗中的生物学功能，利用ChIP等关键技术鉴定NRP1在靶基因转录调控区域的精确结合位点，深入揭示辐射应激调控HOXs转录的组蛋白甲基化重建规律及靶基因网络，阐明NRP1通过调控HOXs基因转录而介导肺癌细胞辐射抵抗的表观遗传学机制及其生物学意义，为临床合理制定肺癌放疗方案提供新思路。

在肺腺癌患者中，因肺癌细胞的辐射反应不同其放疗疗效不同，因此寻找肺癌辐射抵抗中的关键分子，探讨其生物学功能及表观遗传学机制，有利于肺癌放疗方案的细化和完善，为肺癌的精准放疗提供崭新的思路。.1. 证实了特异性阻断剂 miR-9与 NRP1的靶向调控关系，探讨其在肺癌细胞辐射抗性中的作用机制。发现miR-9 可以抑制 A549细胞的增殖、迁移、侵袭能力，并通过多条信号通路的激活提高 A549 细胞的放射敏感性。.2. 从全基因组表达谱芯片结果中筛选出MLL5及其下游的HOXA6/9基因，利用已建立的NRP1表达差异稳定肺癌细胞系，证实了在具有辐射抗性的A549 细胞中NRP1对HOXA6、HOXA9和MLL5具有正向调控作用，当干扰HOXA6/9和MLL5基因后导致受照后的A549细胞出现G2/M期阻滞，诱导细胞凋亡。.3. 进一步对45套肺癌患者的正常组织、癌旁组织和肿瘤组织中，NRP1、HOXA6、HOXA9及MLL5四种基因的表达量进行了检测，并进行了回顾性分析与年龄、性别、临床分期、分化程度、病理分型等因素的关系。结果显示，NRP1和MLL5促进肺癌的淋巴结转移，并证实了NRP1与HOXA6、HOXA9、MLL5均具有相关性。同时，利用裸鼠荷瘤模型证实了A549细胞产生辐射抗性中，NRP1- MLL5-HOXA6/ 9通路起重要作用。.4. 进一步探讨NRP1下游的MLL5和EZH2分别对H3K4me3和H3K27me3的组蛋白甲基化修饰影响及其分子机制、下游靶分子调控作用和细胞周期进程的影响。结果提示，产生辐射抗性的A549-RR细胞MLL5蛋白表达上调，增强OGT的招募形成复合物，进而显著增强H3K4me3修饰，在此过程中MLL5对下游靶基因HOXA9 起到正向调控作用，进而改变细胞周期进程，导致S期缩短、G1期和G2/M期阻滞的方式对辐射抗性细胞产生影响。另一方面，A549 细胞产生辐射抗性后EZH2表达显著升高，同时H3K27me3表达水平显著高于对照组；当干扰EZH2基因后，H3K27me3显著下降，说明在肺癌细胞产生辐射抗性时EZH2增强H3K27me3的甲基化修饰，进而导致细胞周期中的S期延长、G1期缩短。.本项目的研究内容已在学术期刊上发表8篇论文，在全国学术会议上发表5篇摘要，其中6篇SCI收录论文，2篇核心期刊，并均已标注本项目的编号。