长链非编码RNA在介导白藤梨多糖佐剂效应中的作用及其机制研究

Long noncoding RNAs (lincRNAs) are important regulators of gene expression. Although lincRNAs are expressed in immune cells, their functions in immunity are largely unexplored. We previously found and identified a novel immunoregulatory lincRNA, lincRNA-AR, during the study of the mechanisms of adjuvant action of Actinidia eriantha polysaccharides (AEPS). lincRNA-AR was precisely regulated in RAW264.7 macrophages to control the expression of immune response genes. Here, using lincRNA-AR-deficient mice, the transcriptome analysis of bone-marrow derived macrophages (BMDMs), gain-of-function and rescue experiments will first be carried out to reveal an in vivo specific role for this lincRNA in regulating the expression of immune response genes. Consistently, the role of lincRNA-AR in mediating adjuvant activity of AEPS was investigated by determining the effect of lincRNA-AR deletion on the innate immune and adaptive immune responses in ovalbumin-immunized mice induced by AEPS. Furthermore, the underlying molecular mechanism of lincRNA-AR in regulating the expression of immune response genes in BMDMs was also investigated to mine the functional association between lncRNA and the experimentally defined key regulator. The project was performed to excavate new lncRNA involved in immune response, to discover the brand-new pattern of mechanism of adjuvant action, as well as provide a reference for the study on new molecular targets of immunological adjuvant, the high throughput screening of novel adjuvant and the development of safe, effective and novel immunological adjuvant.

前期发现和鉴定一个新的免疫调节活性长链非编码RNA (lncRNA)--lincRNA-AR，其在白藤梨多糖激活RAW264.7细胞及调控免疫应答基因表达中发挥特异性功能。本项目针对多糖佐剂作用机制研究陷入瓶颈、lncRNA体内免疫学功能及其作用机制亟待阐明的现状，拟采用已构建成功的lincRNA-AR缺失小鼠，通过对骨髓来源巨噬细胞基因表达谱分析、功能获得性和功能挽救性实验，研究lincRNA-AR调控免疫应答基因表达的功能；检测lincRNA-AR缺失对白藤梨多糖诱导卵清蛋白免疫小鼠先天性免疫和适应性免疫应答的影响，阐明在介导白藤梨多糖佐剂效应中的作用。研究调控免疫应答基因表达的作用机制，确定lincRNA-AR的伴侣蛋白，揭示其与伴侣蛋白的相互作用及结构基础。旨在挖掘新的lncRNA及其免疫学功能，发现佐剂作用机制新的模式，为高通量筛选佐剂活性天然产物及研制新型免疫佐剂提供新的靶标。

linc-AAM是本项目组发现和鉴定的一个新的lncRNA，在AEPS激活RAW264.7细胞及调控免疫应答基因（DEGs）表达中发挥特异性功能。然而，体内免疫学功能及其作用机制尚未阐明。通过检测AEPS处理及对照linc-AAM缺失和WT型小鼠BMDMs IRGs mRNA表达水平、培养上清中细胞因子含量、吞噬活性、以及表面分子表达水平，以及挽救实验证实linc-AAM介导AEPS促进小鼠BMDMs活化和功能。测序分析AEPS处理及对照linc-AAM缺失和WT型小鼠BMDMs，通过GSEA分析、Metascape和Cytoscape软件预测AEPS和linc-AAM特异性Hub基因和转录因子，从转录组水平阐明linc-AAM和AEPS调控小鼠BMDMs的作用机制。采用FCM检测腹腔及腹股沟引流淋巴结七类先天性免疫的数量，研究linc-AAM缺失对AEPS诱导先天性免疫应答的影响。测定血清中OVA特异性IgG、IgG1和IgG2b等抗体效价，淋巴细胞增殖反应，NK细胞杀伤活性，以及OVA刺激脾细胞细胞因子和转录因子mRNA表达水平，研究linc-AAM缺失对AEPS促进免疫小鼠适应性免疫应答的影响。linc AAM缺失显著抑制AEPS诱导小鼠对OVA的先天性免疫应答及抗原特异性细胞和体液免疫反应。采用特异性抑制剂、多位点ChIP-qPCR及报告基因检测研究linc-AAM转录的调控机制，发现p65调控巨噬细胞linc AAM转录。采用RNA pull-down、液相色谱‒质谱联用技术、Western blot和RIP方法确定linc-AAM互作分子hnRNPL。通过点突变、siRNA、挽救实验、RIP、Co-IP和ChIRP等方法研究linc-AAM调控免疫应答基因表达的机制。linc AAM通过两个CACACA基序与hnRNPL相互作用，导致其与组蛋白H3分离，激活染色质并促进IRG的转录。此外，采用ChIRP-MS方法鉴定linc-AAM微量互作蛋白ANXA2，并评价了linc-AAM缺失对小鼠抗肿瘤效应及对伪狂犬病毒感染小鼠存活率的影响。研究结果不仅证实linc-AAM作为转录增强子激活巨噬细胞并促进适应性免疫，而且揭示lncRNA调节hnRNPL功能的机制。不仅为多糖机制研究提供新的思路，而且为筛选佐剂活性多糖以及研制安全、高效、新型免疫佐剂提供新的靶标。