miR-let-7i/E2F1/NEAT1前馈环调控tol-DC诱导心脏移植免疫耐受的机制

It is an essential scientific challenge that the state of immune tolerance could be induced after heart transplantation. Recently, tolerogenic dendritic cell (tol-DC) has developed from bench to bedside in treating immune diseases. Long non-coding RNA(lncRNA) is crucial in immune regulation, whether it involves in inducing tol-DC is still unknown. We demonstrate that lncRNA NEAT1 plays a critical role in immune tolerance of heart transplantation by lncRNA microarray, and it is the first time that we have obtained tol-DC by regulating NETA1. We find that miR-let-7i/E2F1/NEAT1 feedforward loop regulates costimulatory molecules of DC and cytokines through the method of bioinformation, which feedforward loop affects the quantity and function of Treg. And NEAT1 regulates downstream target gene CXCL8 by paraspeckle protein SFPQ, affecting migration, proliferation and differentiation of T cells, and then induces immune tolerance. We will reveal the important mechanism of tol-DC in immune tolerance from cell to mice,solving the problem of immune tolerance of heart transplantation.Our group illuminate the complicated network of NEAT1 in tol-DC and find the key point for the first time. It may provide new therapeutic target for immune tolerance of heart transplantation.

心脏移植患者如何诱导术后免疫耐受状态是本领域重要科学问题。近年应用耐受性树突状细胞（tol-DC）调控机体免疫相关疾病研究逐渐从基础走向临床。长链非编码RNA（lncRNA）在免疫调控中起重要作用，但在tol-DC中作用不明。我们前期应用lncRNA芯片分析，发现NEAT1在心脏移植免疫耐受中起重要作用，并首次应用NEAT1诱导tol-DC形成。通过生物信息方法预测并验证miR-let-7i/E2F1/NEAT1以前馈环方式调控DC表面共刺激分子和相关细胞因子，影响Treg数量和功能，而NEAT1下游通过SFPQ介导的旁斑作用调控CXCL8，影响T细胞迁移、增殖和分化，从而诱导免疫耐受。我们分别从细胞到动物水平揭示NEAT1诱导免疫耐受的重要作用，解决心脏移植免疫耐受临床应用的具体问题。首次阐明NEAT1在tol-DC中的复杂调控网络，找出关键节点，为诱导心脏移植免疫耐受提供新的治疗靶点。

移植排斥和难以诱导特异性免疫耐受是心脏移植治疗的主要瓶颈，既往研究表明耐受性树突状细胞（tol-DC）在免疫相关疾病中发挥重要作用，并通过塑造T细胞反应诱导免疫耐受。越来越多的证据证明，长链非编码RNA（LncRNAs）在免疫系统中起着重要调控作用。然而，lncRNAs在tol-DC中的潜在作用和潜在机制仍不清楚。本课题发现lncRNA NEAT1在tol-DC中发挥重要作用。本研究通过RNA原位杂交（FISH）和qRT-PCR方法检测NEAT1在DC中的分布和表达，流式细胞仪分析NEAT1对DC表面共刺激分子影响，对NEAT1基因进行小RNA测序，通过荧光素酶报告实验、染色质免疫共沉淀（CHIP）和免疫荧光等方法探讨了NEAT1的作用机制，最后通过体内实验验证敲除NEAT1获得tol-DC对免疫相关疾病免疫耐受影响。结果表明，NEAT1主要位于DC胞核中，DC中敲减NEAT1能够降低DC表面共刺激分子CD80、CD86、MHCII，与T细胞共培养，增加调节性T细胞（Treg）数量，降低TH17数量，从而诱导tol-DC形成。机制方面，NEAT1上游受到let-7i/E2F1调控，tol-DC中let-7i表达降低，let-7i使转录因子E2F1升高，转录因子E2F1进入细胞核通过组蛋白H3K27ac修饰使NEAT1表达降低；NET1通过ceRNA机制调控miR-3076-3p/NLRP3表达，tol-DC中NEAT1表达降低，吸附miR-3076-3p减少，使miR-3076-3p与NLRP3结合增多，促进NLRP3的mRNA降解，使NLRP3表达降低。我们构建自身免疫心肌炎及心脏移植模型，分别回输NEAT1所诱导的tol-DC，发现自身免疫心肌炎症降低，移植心生存时间延长，进一步证实敲减NEAT1诱导的tol-DC能够诱导免疫耐受。综上，我们的研究阐明了NEAT1诱导tol-DC的具体分子机制，并强调了以NEAT1为靶点治疗免疫相关疾病的潜力，为临床治疗免疫相关疾病提供了靶点。