MiR205/24与DNA甲基化相互作用调控NPC辐射抗拒

It's essential to clarify the mechanism of NPC radioresistance which affects the radiotherapetic efficacy severely.More and more attention has been paid to microRNA(miR)or/and DNA methylation for its extensive targets and reversibility.However,there is no report on their interaction and combined function in regulating NPC radioresistance.Our work has demonstrated that miR-205 overexpressed was in company with low level of miR-24-1 in NPC radioresistance cells or tissues compared to ralatively radiosensitive cells and tissues.And also miR-205 can activate PI3K-Akt pathway by targeting PTEN and then suppress cell apoptosis contributed to NPC radioresistance.In addition ,Jab1 overexpression induces p27 dislocation from nucleus to cytoplasm ,which leads to NPC radio-chemo resistance .Genome wide methylation levels ,6 main gene elements and many windows in 24 chromosomes between CNE2R and CNE2(with the same isogeny but different radiosensitivity) cells vary obviously.Furthermore,data from NCBI indicates that CpG islands are included in both miR-205 and miR-24-1 promoters.Consequently,we put forward the hypothesis that "The Interaction Between miR205/24 and DNA methylation Regulates NPC Radiation Resistance".Methods including MSP combined with BSP,in situ hybridization and luciferase reporters and etc. will be used to validate our hypothesis experimentally and clinically.We are going to clarify the mechanism of NPC radioresistance from the perspective of epigenetics, which will provide the new biomarkers and targets for predicting and reversing NPC radioresistance.

辐射抗拒严重影响鼻咽癌（NPC）放疗疗效，阐明其机制至关重要。微小RNA（miR）和DNA甲基化因其靶点广且可逆备受关注，尚未见两者相互作用调控NPC辐射抗拒的报道。我们已证实辐射抗拒NPC细胞或组织miR205过表达且伴miR-24-1下调；miR205靶向PTEN激活PI3K-Akt通路，抑制细胞凋亡介导NPC辐射抗拒；过表达Jab1促p27核浆转位致NPC放化疗抵抗。预实验显示同源不同辐射抗拒NPC细胞CNE2R/CNE2间全基因组、六个主要基因元件和24条染色体多窗口甲基化水平存在差异。NCBI数据库显示miR205/24-1基因启动子区均含CpG岛，故提出"miR205/24与DNA甲基化相互作用调控NPC辐射抗拒"假设。拟采用MSP结合BSP、原位杂交和报告基因分析等实验和临床验证假设，旨在从表观遗传调控角度揭示NPC辐射抗拒机制，确立预测和逆转NPC辐射抗拒新标记物和靶点。

严重制约鼻咽癌（NPC）放疗疗效的辐射抗拒的机制至今尚未阐明。本项目在成功构建同源不同辐射抗拒NPC细胞（辐射抗拒CNE2R/亲本CNE2）的基础上，提出和验证miR205/24与DNA甲基化相互作用调控NPC辐射抗拒的假设。经4年努力高质量按计划完成原约定研究内容，且某些方面超额完成。通过筛查和鉴定CNE2R/CNE2细胞间差异表达miRNA、全基因组甲基化水平和异常DNA甲基化差异区域及位点，发现与CNE2相比，CNE2R细胞 miR24低表达最显著，且全基因组甲基化水平降低，CDS、intron、downstream 2k、upstream2k、3’UTR及5’UTR等六个功能元件区域甲基化差异显著，提示miR24低表达与甲基化水平异常相关；同时阐明miR24低表达与DNA甲基化相互调控介导NPC辐射抗拒的机制，发现辐射通过升高miR-24前体mir-24-1启动子区DNA甲基化而抑制miR-24转录，减弱miR-24靶向Jab1的3’UTR和5’UTR抑制其表达的功能，导致Jab1过表达，进而干预细胞周期和减少凋亡而介导NPC辐射抗拒；新增探讨miR-24异常表达与NPC转移的作用及其机制，确证miR-24可靶向c-Myc的3’UTR下调其蛋白表达，干预高转移NPC细胞发生上皮间质转化，而抑制NPC转移。由此提示升高的mir-24-1启动子区DNA甲基化为NPC辐射抗拒细胞miR24低表达的原因，作为抑癌微小RNA，miR24的低表达与NPC辐射抗拒及转移等相关。其次，新增探索并确立c-Myc和XBP1等多个与HBV/EBV感染相关的预测NPC病人转移和预后的分子标记物；研发和探索多种NPC靶向抗癌先导物的作用及其机制。本研究已发表论文8篇，其中SCI收录论文5篇；培养研究生7名，其中博士4名；本研究成果为临床预测和逆转NPC辐射抗拒提供新靶点及靶向抗癌先导物。