多梳基因家族蛋白EZH2对鼻咽癌细胞的放射敏感性及其相关机制的研究

Nasopharyngeal carcinoma (NPC) is a remarkably distinctive type of head and neck cancer, which is prevalent in the coast of China. Local residual and recurrent is still the major cause of failure in NPC treatment. Previously, we have found that the amplification and overexpression of EZH2 is closely assciated with invasiveness/metastasis of NPC cells,and that the EZH2 target histone marker, H3K27me3, is correlated with chemoradioresistance in NPC. However, the relationship between EZH2 and chemoradioresistance of NPC is unclear up to now..In this study, we first investigate the alteration of NPC cells Radiosensitivity and side population when EZH2 was knocked down by lentivirus or was induced by gene transfection. Next, we utilize the cDNA array to screen target genes of EZH2, which is closely related to NPC stem cell pathways. Then, we will explore the exact mechnism .of EZH2 regulating target genes by luciferase reporter assays and chromatin immunoprecipitation(ChIP) assays. Finally, we identify the relationship of EZH2 and its target genes with NPC radiatherapy sensetivity in clinical tissue samples using immunohistochemistry (IHC) staining, and summerize the high risk of biomarker closely related to the NPC radiotherapy resistance, providing some references for clinical treatment in the future.

鼻咽癌是中国沿海地区常见的一种特殊类型的头颈部肿瘤.局部或区域治疗失败而导致的肿瘤残余或者复发仍然是鼻咽癌治疗失败的很重要的原因。我们在前期的研究已经发现EZH2扩增和过度表达与鼻咽癌的浸润转移密切相关，并且发现其主要靶点组蛋白H3K27位的三甲基化与鼻咽癌的放化疗敏感性相关。目前，关于EZH2与鼻咽癌的放疗敏感性的关系仍不清楚。.本课题拟首先采用慢病毒介导RNA干扰技术和基因转染技术，观察EZH2基因沉默和导入后对鼻咽癌细胞株的放射敏感性的影响和侧群细胞群的变化；进一步通过cDNA芯片技术，筛选出EZH2与鼻咽癌干细胞通路密切相关潜在下游基因；之后通过荧光素酶报告基因系统和染色质免疫共沉淀技术探讨EZH2对靶基因调控的具体机制；最后用免疫组化在临床组织标本中验证EZH2及其下游靶基因与鼻咽癌放射敏感性的关系，分析总结与鼻咽癌患者放射抵抗有关的高危的分子指标，为今后临床治疗提供参考。

我们在前期的研究已经发现EZH2扩增和过度表达与鼻咽癌的浸润转移密切相关，并且发现其主要靶点组蛋白H3K27位的三甲基化与鼻咽癌的放化疗敏感性相关。本项目应用慢病毒介导RNA干扰技术，PI-annexinⅤ，克隆形成实验观察及线性二次模型分析EZH2基因沉默后对鼻咽癌细胞株的放射敏感性的影响；结果发现：沉默EZH2基因可以显著提高鼻咽癌细胞株对射线的敏感性。进一步通过western blot，亚细胞器蛋白分离技术及染色质免疫共沉淀技术等技术，分析和证明：EZH2基因对通过在组蛋白的修饰调控P14ARF的表达，进而通过P14ARF/MDM2/P53/Bax通路诱导的线粒体凋亡途径调控鼻咽癌细胞株的放疗敏感性；我们还发现在非射线处理情况下EZH2基因可以通过P14ARF/MDM2/P53/P21通路调控鼻咽癌细胞的增殖活性。提示EZH2对下游通路的调控可能是环境依赖性的。