长链非编码RNACUST52998调控T细胞的功能及其对SLE的作用和机制研究

The relationship between epigenetic regulation on gene expression of immune cells and autoimmune disease have been highly concerned by researchers. Systemic lupus erythermatosus (SLE) is a fatal autoimmune disease with complex etiology. Although the pathogenesis of SLE is still unclear, abnormal activity of immune cells caused by disregulation of gene expression play a key role in the development of SLE. Long non-coding RNAs(lncRNAs), an important fraction of the untranslated RNA molecules, participate in diverse important biological processes through distinct mechanisms. The roles of lncRNA on immune cells regulation in the pathogenesis of SLE have attracted much attentions.In this study, we applied lncRNA microarray to elucidate the lncRNA expression profile of T cells in SLE, and found that several lncRNA expressions such as CUST52998 were markedly elevated in SLE patients.We intend to further explore the role of lncRNA on T cell regulation.We use bioinformatic，luciferase reporter gene and RNA pull down techniques to conform the target genes of lncRNA CUST52998, and further construct lentiviral vector overexpressing or inhibiting lncRNA CUST52998 to analyze the detailed mechanisms of CUST52998 on T cell function. This reasearch will be helpful for us to further clarify the epigenetic regulation mechanisms of T cell abnormality in SLE development and provide experimental clues for the disease prevention and treatment.

免疫细胞基因表达的表观调控机制与自身免疫性疾病的关系受到高度关注。SLE是严重危害人类健康的自身免疫病，发病机制复杂，但基因表达调控导致的免疫细胞功能异常是关键。长链非编码RNA(lncRNA)作为细胞内大量存在的重要非编码RNA，其调控免疫细胞的作用和SLE的关系受到人们重视成为新的研究热点。我们采用lncRNA芯片筛选出SLE患者外周血T细胞中差异表达的CUST52998等多个，在此基础上本课题拟进一步研究lncRNA 调控T细胞的功能和相关机制。采用生物信息学分析、荧光素酶共转染及RNA pulldown技术确定与CUST52998直接作用的分子。构建慢病毒高表达或干扰表达T细胞中CUST52998，探讨lncRNA对T细胞表型、性状和功能发挥的调节作用和机制，为进一步认识lncRNA对T细胞功能的表观调控机制以及lncRNA在SLE发生发展中的作用、更有效地防治SLE提供实验依据。

lncRNA可作为疾病诊断和预后的重要标志物和潜在的治疗靶点，具有重要的临床价值。lncRNA可能在SLE 发病过程中也起着重要的作用。基于目前lncRNA 与SLE 发病关系的研究尚未深入，本课题拟深入探讨lncRNA对T细胞功能的调控及其机制。本课题主要研究内容为：1）筛选SLE 患者CD4+T 细胞中差异表达的lncRNA；2）体外过表达lncRNA CUST52998，研究其对JAK/STAT1信号通路、CD4+T细胞表型、性状及功能的影响；3）体外干扰lncRNA CUST52998 的表达，研究其对SLE患者活化CD4+T细胞功能的抑制作用；4）解明lncRNA CUST52998对靶基因STAT1的调控方式。我们采用lncRNA 芯片筛选出SLE患者外周血CD4+T细胞中差异表达的lncRNA 表达谱，通过生物信息学分析预测出lncRNA CUST52998作用于JAK/STAT1通路中的STAT1基因；SLE 患者外周血CD4+T细胞中lncRNACUST52998过表达。我们成功构建lncRNA CUST52998过表达和干扰表达慢病毒载体，将其转染CD4+T细胞，结果显示过表达lncRNA CUST52998可上调STAT1及IFN诱导基因的表达，并可导致CD4+T细胞异常活化，诱导Th1和Th17分化，抑制Treg，与B细胞共培养发现可激活B细胞分泌IgG水平。进一步发现干扰表达lncRNA CUST52998可下调STAT1及IFN诱导基因的表达，并抑制CD4+T细胞的异常活化，与B细胞共培养后可抑制B细胞分泌IgG水平。lncRNA CUST52998可能通过调控STAT1及IFN诱导基因参与CD4+T细胞异常活化的调控。通过RNA-pull down实验发现lncRNA CUST52998跟STAT1蛋白并没有直接的相互作用，可能通过其他间接途径起作用。lncRNA CUST52998与miR-145存在13bp相配对，我们将lncRNA CUST52998慢病毒转染miR-145过表达细胞系，STAT1基因无显著差异变化。lncRNA52998并不一定通过miR-145调控STAT1基因的表达。该课题的研究为进一步认识lncRNA对T细胞功能的表观调控机制以及lncRNA在SLE 发生发展中的作用具有重要意义。

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