铁离子调控自身免疫疾病中TNFα表达的机制研究

It has been proved that proinflammatory cytokines produced from CD4+ T cells are essential to autoimmune diseases development. Our previous study revealed that intracellular iron drives autoimmune disease by increasing proinflammatory cytokine production, such as GM-CSF and TNFα. Mechanistically, intracellular iron promoted GM-CSF mRNA stability via a specific RNA binding protein-PCBP1. However, the molecular mechanism of how iron regulates TNFα expression in T cells is still undefined. Through a short hairpin (sh) RNA screen, we have identified that Poly(rC) binding protein 2 (PCBP2), another RNA binding protein with iron chaperon function, was an specific regulator of TNFα, and the protein level of PCBP2 was dynamically adjusted by intracellular iron. These results suggest a possible model that iron control TNFα production by PCBP2 mediated post-transcriptional regulation..In the proposed study, we will explore the function of PCBP2 in TNFα regulation by iron and in the pathogenesis of autoimmune disease, by analyzing PCBP2 knockout mice. Then we will construct RNA sequencing and CLIP sequencing, to identify transcriptome-wide targets of PCBP2 and its gene post-transcriptional regulation pattern. Further more, we will analyze the correlation among iron level, PCBP1 expression and TNFα expression in patients with autoimmune disease.Our proposed studies aim to construct the network model that iron regulates different proinflammatory cytokine expression through specific RNA binding protein, and provide novel theoretical basis for autoimmune disease treatment by targeting particular cytokine.

促炎性细胞因子的异常表达与自身免疫疾病密切相关。我们之前的研究发现T细胞中铁离子在转录后水平调控GM-CSF、TNFα等促炎性细胞因子表达，促进自身免疫疾病发生；并证明铁离子通过PCBP1特异性调控GM-CSF的mRNA稳定性。但是，铁离子调控TNFα表达的分子机制并不明确。前期高通量筛选鉴定了RNA结合蛋白PCBP2是TNFα的特异性调控因子，且其表达受铁离子动态调控。本项目将聚焦“铁-PCBP2-TNFα”调控模型，利用基因敲除小鼠，分析PCBP2在铁离子调控TNFα表达和自身免疫疾病中的作用；通过CLIP等高通量方法，鉴定PCBP2的靶点RNA及调控模式；分析人类自身免疫疾病中铁离子，PCBP2及TNFα的表达相关性。本项目旨在揭示铁离子通过不同RNA结合蛋白转录后调控不同促炎性细胞因子的网络，为靶向特定促炎性细胞因子的自身免疫疾病治疗提供理论依据。

自身免疫疾病包括类风湿性关节炎、成年型斯蒂尔综合症（AOSD）等100多种疾病,常伴随促炎性细胞因子的异常高表达。靶向促炎性细胞因子是自身免疫疾病治疗的重要方向，研究其表达调控机制对开发治疗该疾病的新型药物具有重要意义。我们前期研究发现，辅助性T细胞中铁离子能通过RNA结合蛋白PCBP1调控GM-CSF、TNFα等促炎性细胞因子表达参与自身免疫疾病。本项目旨在继续探索PCBP2蛋白在CD4+T细胞中功能和机制，并研究其与人类自身免疫疾病（如AOSD）的相关性。我们调整了部分研究内容和方向，并进行了拓展和延伸，包括：（1）发现了Pcbp2在CD4+T细胞中的功能与Pcbp1相反，敲除Pcbp2能促进Th1细胞中多种促炎性细胞因子表达，但对Th17细胞中细胞因子表达没有影响，明确了PCBP2在Th1细胞中抑炎功能；（2）鉴定了PCBP2能够与RNA乙酰转移酶NAT10互作，并调控T细胞中RNA ac4C水平，延伸了PCBP2通过调控RNA乙酰化修饰参与基因表达的转录后调控分子机制；（3）完成了AOSD病人的单细胞测序和验证工作，不仅首次解析了这一自身免疫疾病外周血免疫全景图谱，还发现了另一个RNA结合蛋白ZFP36L2在AOSD中细胞因子风暴中的重要作用，拓展了AOSD病人中细胞因子表达调控的新机制。综上，我们的研究不仅为靶向促炎性细胞因子治疗自身免疫疾病提供理论基础，并且丰富了T细胞中RNA结合蛋白参与基因转录后调控的网络模型。