LncRNA CUST21719调控树突状细胞分泌趋化因子CCL5在SLE发病中的作用及机制研究

Systemic lupus erythermatosus (SLE) is a fatal autoimmune disease with complex etiology. The abnormal expression of CCL5 recruited inflammatory cells into inflammation site, which is related to the imbalance of immune system and organ damage of SLE. Long non-coding RNAs (lncRNAs), an important and new molecule of epigenetic regulation, have a genome-wide transcription in mammalians and participate in diverse important biological processes. The relationship between the regulation of lncRNA on immune cells and the pathogenesis of SLE has attracted much attention and gradually became a new hotspot. In this study, we used lncRNA and mRNA co-expression array to investigate the lncRNA and mRNA expression profile of dendritic cells in SLE, and found that several lncRNA expressions such as CUST21719 and several mRNA including CCL5 were markedly increased in SLE patients. On the basis of our results, We intend to further explore the regulation and mechanisms of lncRNA CUST21719 on the secretion of CCL5 by dendritic cells in systemic lupus erythematosus. We firstly constructed lentiviral vector overexpressing or inhibiting lncRNA CUST21719 respectively to analyze the specific effect on the expression of CCL5 and the function of attraction CD4+ T cells by DCs. We then use RAP techniques and RNA stability experiment to confirm the mechanism between lncRNA CUST124090 and CCL5. The reasearch will help us to further clarify the epigenetic regulation mechanisms of immunological abnormalities in SLE development and provide experimental basis for the disease prevention and treatment.

SLE是严重危害人类健康的自身免疫病，发病机制复杂，其中CCL5的异常表达募集炎症细胞进入炎症部位与SLE的免疫紊乱及脏器损伤密切相关。lncRNA是新发现的细胞内广泛转录的表观遗传调控分子，其调控免疫细胞的作用和SLE的关系成为新的研究热点。我们采用lncRNA和mRNA共表达芯片筛选出SLE患者树突状细胞中差异表达的CUST21719和CCL5等多个lncRNA和mRNA，在此基础上我们拟深入研究CUST21719调控树突状细胞分泌趋化因子CCL5在SLE发病中的作用及机制研究。首先通过构建慢病毒过表达或干扰表达DCs中CUST21719，探讨其对CCL5表达水平和DCs趋化CD4+T细胞功能的调节作用,进一步用RNA酶保护实验及RNA稳定性实验探讨CUST21719调控CCL5基因表达的作用方式。本课题有助于我们从表观遗传学的角度进一步明确SLE免疫学异常的发病机制。

树突状细胞(Dendritic cells, DCs)功能异常是介导SLE患者免疫异常的关键因素。课题组前期体外实验发现SLE患者单核细胞来源的树突状细胞（moDCs）异常表达炎症因子，其lncRNA表达谱与正常对照组存在差异。本研究选取其中一个上调的lncCUST21719 (lncTAF15:1-1)作为研究对象，探索了lncTAF15:1-1通过调控树突状细胞功能从而促进SLE发病，并对其分子机制进行了初步的探讨。. 首先我们发现SLE患者来源的moDCs的lncTAF15:1-1的表达较健康对照组显著上调，将SLE患者moDCs中lncTAF15:1-1的表达水平与患者的临床严重水平进行了相关性分析，发现lncTAF15:1-1在重度活动组（SLEIDAI≥15）中表达高于非重度活动组（SLEDAI<15）。接着我们构建了lncTAF15:1-1的过表达腺病毒载体，发现过表达lncTAF15:1-1能显著上调成熟moDCs中IL-6、CCL5的表达。通过qRT-PCR还测定了与moDCs功能密切相关的重要细胞因子及转录因子的表达，未发现显著改变。我们利用transwell小室评估了lncTAF15:1-1对成熟moDCs迁移功能的影响，发现过表达lncTAF15:1-1能显著上调成熟moDCs的基础迁移能力。这些结果进一步表明lncTAF15:1-1确实可以调控moDCs的促炎状态，且主要表现在促进炎症因子分泌上。最后我们初步探究lncTAF15:1-1调节moDCs功能的分子机制。我们发现lncTAF15:1-1在胞浆和胞核均有分布，胞核分布为主。运用RNA测序技术筛选出差异化表达的mRNA，其中348个上调，140个下调。基因本体（gene ontogeny）聚类分析表示主要集中在白细胞、淋巴细胞的激活和增殖及细胞因子产生、细胞因子受体活性等等；而pathway聚类分析集中在感染、C型凝集素受体通路及细胞因子-细胞因子受体互作等。通过生信分析我们进一步证实了炎症因子是lncTAF15:1-1调控moDCs促炎状态的主要靶基因。. 综上所述，我们发现lncTAF15:1-1可通过调控树突状细胞分泌CCL5、IL-6促进SLE的发病。这一研究补充了lncRNA对树突状细胞功能的调控作用，为SLE的机制研究提供了新的思路。