LncRNA调控P2X7R/NLRP3信号通路介导CD4+T淋巴细胞亚群分化：幼年类风湿性关节炎发病新机制

RP11-340F14.6 is a long non-coding RNA (lncRNA) with unknown function that suggested to be associated with juvenile idiopathic arthritis (JIA) identified by Digital Gene Expression Tag Profiling. Our previous work and bioinformatic analysis demonstrated that RP11-340F14.6 was significantly over-expressed in peripheral blood mononuclear cells and Th17 cells of JIA and positively correlated with the Th17 differentiation levels. Silencing RP11-340F14.6 by siRNA significantly suppressed the Th17 differentiation. Moreover, bioinformatic analysis and preliminary experiment revealed that P2X7R and NLRP3 expression levels were highly correlated with the RP11-340F14.6 .Silencing RP11-340F14.6 could significantly down-regulate the expression of P2X7R,NLRP3,IL-1 and IL-17 through cis regulatory mechanisms. Based on the findings above, we hypothesized that RP11-340F14.6 modulate the differentiation of CD4+T lymphocyte through P2X7/NLRP3 inflammasome pathway. In this proposal, firstly, we plan to clarify the functional role of RP11-340F14.6 in the differentiation of CD4+T cells. To achieve this goal, we design to over-express and knock-down RP11-340F14.6 both in vivo and in vitro. Secondly, we aim to explore the underlying mechanism of RP11-340F14.6 modulating CD4+Tcells differentiation through P2X7/NLRP3 inflammasome pathway via CHIP, ChIRP, MSP and rescue experiments. Finally, we design to evaluate the expression level of RP,P2X7R and NLRP3 in a large sample of JIA patients .Our study is the original source of innovation and can provide new potential biomarkers for the prevention and treatment of JIA.

RP11-340F14.6（简称RP）是应用高通量测序技术筛选的幼年类风湿性关节炎相关、功能未知的lncRNA。前期发现：RP特异高表达于关节炎患儿Th17细胞，沉默RP显著抑制CD4+T细胞向Th17细胞分化，生物信息学分析和实验提示RP可与P2X7结合，沉默RP显著抑制P2X7R及其下游炎症因子表达，临床标本中RP与P2X7R、NLRP3高度相关，因此我们推测RP通过调控P2X7R/NLRP3信号通路介导关节炎CD4+T细胞亚群分化。本研究拟采用过表达/沉默策略，结合体内体外实验，明确RP在CD4+T细胞亚群分化中的作用，论证RP通过调控P2X7R/NLRP3信号通路介导CD4+T细胞亚群分化的分子机制，最后通过大样本病例评价RP、P2X7R及NLRP3在关节炎诊疗中的临床价值。本研究有望为幼年类风湿性关节炎的防治提供新靶标。

幼年特发性关节炎(Juvenile idiopathic arthritis,JIA)是儿童时期最常见的高度致残性风湿性疾病，常持续到成人期，引起关节变形、生长发育异常、骨质酥松症、疼痛、心理异常、生活自理困难等明显的功能残疾。JIA发生与CD4+T 细胞功能紊乱有关，Th17细胞的自身优势应答反应强化，交叉调节抑制Treg细胞分化。LncRNA可在表观遗传学、转录、转录后及翻译等多个水平调控CD4+T淋巴细胞的发育和分化过程，进而参与JIA的发病过程。本研究通过高通量测序技术、流式细胞仪、实时定量PCR、酶联免疫吸附测定、构建过表达/敲低慢病毒、双荧光素酶报告基因等方法在临床、细胞、动物水平进一步验证其机制。首先，JIA 与正常儿童PBMC开展高通量测序，并进行PCR验证芯片验证后发现lncRNA RP11-340F14.6（简称RP）在JIA（RF阳性多关节型）与健康儿童CD4+T细胞中表达存在显著差异，其表达水平与疾病活动度JADAS27呈正相关，生物信息学分析和实验提示RP可与P2X7R结合，通过逆转实验进一步阐明RP11-340F14.6通过激活P2X7R来调控CD4+T细胞分化。其次再用DBA小鼠构建CIA模型，取小鼠脾脏细胞通过高通量测序及PCR验证筛选出一条差异表达显著的与Th17/Treg细胞有相关性的lncRNA ENSMUST00000197208（简称EN），并可调控下游P2X7R/NLRP3通路。接着，通过构建过表达/敲低EN慢病毒载体，分别转染初始CD4+T细胞和动物模型，发现敲低EN可抑制小鼠CD4+T细胞向Th17细胞分化，并且P2X7R/NLRP3表达显著降低，关节病理显示敲低EN后关节炎症较CIA模型组明显缓解，且在NLRP3基因敲除组HE染色亦可见炎症较CIA模型组减轻。综上，lncRNA可通过P2X7R/NLRP3途径在Th17/Treg分化中发挥重要作用，为JIA干预靶点提供了一个新的视角。