辐射诱导PD-L1异常表达调控肿瘤干细胞介导鼻咽癌辐射抗拒

Radioresistance seriously restricts the radiotherapeutic efficacy of nasopharyngeal carcinoma (NPC). In the past, people often focused on DNA damage and repairment to study the mechanism of radioresistance of NPC. Recently, aberrant upregulation of PD-L1 which caused by the mutation in cancer cell genomes or transcripts and its role in radioresistance are of concern. Also excessive reparation can be caused by radiation as a result of over-activated negative feedback, transforming the residual cells into cancer seeds with radioresistance. It is believed that PTEN /Stat3 regulates PDL1’s expression and PDL1 can regulates the function of cancer stem cells (CSC) in published literature. However, the key nodes and regulation network that abnormal expression of PDL1 regulates CSC and mediates NPC radioresistance still remain elusive. Our previous study suggested that 1)the expression of PD-L1 in NPC radioresistant cells CNE2R6 was specifically up-regulate, and radiation could up-regulate the percentage of CSC, the expression of PDL1 and CSC related genes (SOX9, Nanog) in CNE2R6 cells; 2)radiation could affect the cell cycle and apoptosis of NPC cells by regulating the miR205-PTEN-AKT and miR24-Stat3-Jab1 pathway, which resulted in NPC radioresistance; and 3)overexpression of miR24 downregulated the expression of eight CSC-related genes. Therefore, the hypothesis that "abnormal expression of PD-L1 specifically induced by radiation regulates cancer stem cell that further mediates radioresistace in nasopharyngeal carcinoma" is proposed. Here we attempt to explore the assumption with experimental and clinical studies to elucidate the mechanism of NPC radioresistance and to provide a new target for predicting and reversing radioresistance.

辐射抗拒严重制约鼻咽癌(NPC)放疗疗效，过去常从DNA损伤抗损伤角度研究，近期癌细胞基因组或转录组异常上调PD-L1或辐射过度激活负反馈增强修复使细胞残存形成癌种子（CSC）与辐射抗拒备受关注。文献示PTEN/Stat3调控PDL1表达，PDL1可调控CSC功能。但PDL1异常调控CSC介导NPC辐射抗拒关键节点及网络调控仍未阐明。我们前期研究提示：NPC辐射抗拒细胞CNE2R6高表达PD-L1，辐射可上调CNE2R6细胞PD-L1、CSC百分率及相关基因（SOX9、Nanog）；辐射干扰miR205-PTEN-Akt和miR24-Stat3-Jab1通路影响细胞周期和凋亡介导NPC辐射抗拒；过表达miR24下调稳转株8个CSC相关基因。故提出“辐射诱导PD-L1异常表达调控CSC介导NPC辐射抗拒”假设，拟经实验和临床验证假设,以阐明NPC辐射抗拒机制，为预测和逆转辐射抗拒提供新靶点。

严重制约鼻咽癌（NPC）放疗疗效的辐射抗拒机制至今尚未阐明，此外NPC的转移和化疗耐受也限制着患者的长期生存。本项目在成功构建多株同源不同辐射抗拒NPC细胞的基础上深入探讨介导NPC辐射抗拒、化疗耐受和转移的相关分子机制。经4年努力按计划完成原约定研究内容，并拓展部分研究内容。通过分析比较多组细胞间miRNA、circRNA和转录组的差异表达，分析筛选出诱导NPC辐射抗拒的调控网络及潜在关键基因SOX9和PD-L1，并验证二者在辐射抗拒细胞的高表达；随后确证SOX9在介导辐射抗拒和化疗耐受中发挥的效应，发现其可能与c-Fos协同调控细胞周期及DNA损伤修复介导辐射抗拒和放化疗耐受；同时发现circRNA0013181在NPC辐射抗拒细胞中相对高表达，可能通过下调miR-195-5p表达上调PD-L1表达而诱导NPC辐射抗拒。此外，转录组测序分析发现与辐射敏感细胞CNE1相比，辐射抗拒细胞CNE1R中促癌相关基因和通路显著激活，尤其转移相关基因；体内外实验确证CNE1R细胞具有较强迁移能力和肺转移形成能力，且在射线诱导下其转移能力增强，提示辐射抗拒NPC细胞转移能力增强，且射线可诱导辐射抗拒细胞转移。新增探讨姜黄素及其衍生物逆转NPC放化疗耐受的作用及其机制，发现NPC常见p53突变与NPC细胞5-FU耐受相关，下调突变型p53表达或联用姜黄素可显著增强5-FU对NPC的抑癌效应；进一步研发酸性敏感性姜黄素纳米粒F68-姜黄素，体外实验证实F68-姜黄素可通过诱导细胞凋亡增强姜黄素对NPC辐射抗拒细胞的抑癌效应，提示F68-姜黄素可作为逆转NPC辐射抗拒的新剂型。本研究已发表SCI论文2篇，已投稿SCI论文1篇，正在整理待发表SCI论文1篇；新增申请发明专利1项；培养硕士与博士研究生各1名。本研究成果为临床预测和逆转NPC放化疗耐受和转移提供新靶点及靶向抗癌先导物。