乏氧条件下外泌体lncRNA机制介导的人支气管上皮辐射旁效应研究

Ionizing radiation can lead the unirradiated tissues to a biologic reaction similar to the irradiated tissue, namely radiation-induced bystander effect, which leads to radiation injury and carcinogensis in unirradiated tissues. The lung is one of the sensitive organs for the radiation-induced bystander effect, and the damage caused by hypoxia is more severe than that of oxygen. We previously found the methylation status of DNA was obviously changed in the radiation induced bystander effective bronchial epithelium under hypoxic, but the mechanism was unclear. LncRNA can regulate DNA methylation in a variety of ways, and intercellular communication can be carried out in the form of exosomes. Therefore, this research focuses on human bronchial epithelial cells as the research object. The lncRNA gene chip screening is employed to screen out the exosomal lncRNA which have a significant influence on the radiation-induced bystander effect. Through exosomes intake observation, DNA methylation, DNA methylation enzymes and gene expression detection, radiation biology detection, oxidative stress related detection and the application of expression vector and siRNA, we try to explore the possible role of exosomes and lncRNA on radiation induced bystander cells, especially on the DNA methylation. Also the mice unilateral lung irradiation model will be used to study in vivo influence. This research will be the breakthrough point to provide experimental basis for lung radiation protection in the future.

辐射可导致未受照组织产生辐射诱导旁效应,造成辐射损伤和癌变。肺是辐射旁效应敏感器官，乏氧下引起的损伤较有氧更重。课题组发现乏氧下辐射诱导人支气管上皮旁效应细胞中DNA甲基化明显改变，但机制不明。lncRNA可通过多途径调节DNA甲基化并以外泌体形式进行细胞间通讯。因此本研究拟开展乏氧条件下外泌体lncRNA介导的人支气管上皮辐射旁效应机制研究，特别是对旁效应细胞DNA甲基化的作用：以lncRNA基因芯片筛选显著影响辐射旁效应的外泌体lncRNA；通过荧光标记外泌体观察摄入、通过lncRNA表达检测、lncRNA过表达载体和siRNA转染、以及DNA甲基化、甲基化相关酶及调控基因表达检测和放射生物学、氧化应激相关检测，观察乏氧辐射后外泌体及外泌体中lncRNA对旁效应细胞的影响。同时以小鼠单侧肺照射模型研究体内影响。以此为切入点，为今后开展肺辐射防护提供实验基础。

辐射可导致未受照组织产生辐射旁效应，造成辐射损伤。肺是辐射旁效应敏感器官，乏氧下引起的损伤较有氧更重。外泌体可能是辐射诱导旁效应的重要途径， lncRNA在DNA 损伤、氧化应激中起重要作用。本研究探讨乏氧条件下外泌体lncRNA途径介导的辐射诱导的支气管上皮细胞旁效应作用。.研究建立人支气管上皮细胞电离辐射诱导旁效应细胞模型，ELISA检测显示辐射后共培的旁细胞中TNF-α、IL-6、IL-8的浓度上升，TGF-β表达下降，且乏氧处理细胞组变化更大，辐射对旁细胞炎症程度增加，且乏氧处理加重辐射带来的炎症损伤，旁细胞凋亡率上升，G0/G1期以及S期的细胞比例上升，微核形成率和DNA迁移距离上升，DNA损伤水平更高，细胞的ROS损伤程度加剧， SOD及CAT活性下降，MDA含量上升。GEO数据库（GSE151120）数据进行生信分析，并通过qPCR对备选LncRNAs筛选，选定LncRNA KLF3-AS1 作为备选基因进行后续验证。乏氧辐射处理后，提取外泌体，将外泌体和旁细胞共同培养后，qPCR显示外泌体对旁细胞中KLF3-AS1表达明显上升，且在乏氧条件下表达更高。建立sh-KLF3-AS1稳转细胞株，降低旁细胞中KLF3-AS1表达，旁细胞微核形成率下降、DNA迁移距离下降、CCK-8检测细胞增殖活力回升、细胞凋亡率下降、G0/G1期以及S期的细胞比例下降、G2/M期细胞比例回升，SOD以及CAT活性上升，MDA含量下降。双荧光素酶实验、Ago-RIP实验、qPCR检测及细胞转染表明KLF3-AS1可通过靶向关系调节hsa-miR-138-5p与HIF1α的表达，乏氧且辐射处理的肺癌细胞外泌体中KLF3-AS1通过与hsa-miR-138-5p结合并调节HIF1α加重旁细胞效应对旁细胞的ROS以及DNA损伤。对鼠肺原位移植转染sh-KLF3-AS1的A549细胞，成瘤后进行X线照射，HE染色发现sh-KLF3-AS1组小鼠未辐射肺组织病理状况明显缓解、纤维化程度降低，SOD以及CAT活性较对照组上升，MDA含量下降。表明外泌体LncRNA KLF3-AS1参与肺癌小鼠放射性肺损伤的调控。本研究结果为今后开展肺的辐射防护提供靶点和实验基础。