lncRNA CUST_7079调控树突状细胞活化在SLE中的作用及其分子机制研究

SLE is a serious harm to human health autoimmune diseases, the pathogenesis is complex, immune cell dysfunction is the key.The research of lncRNA regulation of immune cells in SLE become a new hot spots. We had found Mo-DCs abnormally activated in SLE, The lncRNACUST_7079 high expressing which was detected by high-throughput microarray and qPC, it was certified to directly interact with Ago2 by RNA - binding protein immunoprecipitation(RIP). In this study, we proposed to construct lentiviral vector overexpressing or inhibiting lncRNA CUST_7079 to analyze the detailed mechanisms of on Mo-DCs‘ function in vitro; And the subcellular localization was determined by RNA FISH and confocal microscopy. The bioinformatics was used to predict the miRNA and to be verified by the method of luciferase reporter gene system. Further to verify its interaction with micrornas in SLE Mo - DCs cell model which had been "knocked down" lncRNA CUST_7079. This reasearch will be helpful for us to further clarify the molecular mechanism of lncRNA CUST_7079 as a competing endogonous RNA to regulate the dendritic cells' abnormality in SLE , to understand the role of lncRNA in the pathogenesis of SLE; and to provide experimental clues for the disease prevention and treatment.

SLE是严重危害人类健康的自身免疫性疾病，发病机制复杂，免疫细胞功能异常是关键。lncRNA调控免疫细胞在狼疮发病中的作用成为新的研究热点。前期我们发现SLE中Mo-DCs细胞异常活化，采用高通量芯片技术结合qPCR发现lncRNACUST_7079在该细胞中高表达，RIP试验证其可与Ago2直接作用。在此基础上，我们拟采用体外构建其过表达/抑制表达的慢病毒载体，转染Mo-DCs细胞，观察其细胞功能的改变；应用RNA FISH和共聚焦显微镜确定其亚细胞定位；利用生物信息学预测与其发生作用miRNA并通过和荧光素酶报告基因系统验证；进一步在“敲除”lncRNA CUST_7079的SLE Mo-DCs细胞模型中验证其与靶miRNA的交互作用；从而阐明其可作为竞争内源性RNA参与调控SLEMo-DCs活化这一分子机制，揭示lncRNA在SLE发病中的作用，为更有效地防治SLE提供实验依据。

系统性红斑狼疮（systemiclupuserythematosus，SLE）是一种可以造成多个器官脏器损伤的自身免疫性疾病，其发病机制与免疫系统的异常激活密切相关，树突状细胞（dendriticcells，DCs）是最强的抗原提呈细胞，DCs的异常活化参与其发病。lncRNA在机体各种生理病理过程中发挥了强大的调控作用，成为自身免疫相关性疾病中的一个研究热点。本项研究运用高通量芯片技术比较了SLE患者与正常对照之间单核细胞来源的树突状细胞（monocyte-deriveddendriticcells，Mo-DCs）lncRNA、mRNA的表达谱，筛选出并用QRT-PCR验证lncRNA NEAT1在SLE患者中的高表达。利用生信分析，构建了lncRNA、microRNA、mRNA三者的共表达网络，阐明SLE发病的潜在机制。进而调控lncRNA NEAT1的表达水平，发现其会影响Mo-DCs IL-6的分泌。为了进一步探究其机制，我们通过生物信息学技术筛选了可能在lncRNA NEAT1与IL-6之间起到调控作用的分子has-miR-365a-3p，并通过双荧光素酶报告基因试验验证了这三者之间的互作关系。当我们在Mo-DCs中同时调控lncRNA NEAT1和has-miR-365a-3p水平时，发现IL-6的表达水平可以随着lncRNA NEAT1和has-miR-365a-3p的改变而改变，证实了lncRNA NEAT1可以作为“分子海绵”改变has-miR-365a-3p的表达量从而影响IL-6的表达水平，发挥ceRNA的作用。项目的研究结果表明lncRNA NEAT1可作为SLE发病的一个重要分子靶点，为今后的进一步诊断治疗研究提供科学依据。