miR-34a通过Foxp3调控Treg功能及其在SLE疾病中的作用

Tregs play a critical role in maintaining immune homeostasis, so they have the potential to be used in autoimmune diseases and transplantation rejection targeting therapy. However, recent studies have shown that Treg in patients with autoimmune disease (such as SLE) in despite retaining the immunosuppression features, but they own some characteristics of Th17 and secret IL-17. In addition, adoptive transfer Treg cells to treat the autoimmune disease animal model, it was found that the Tregs had been shifted to Th17 cells in sites of inflammation. Foxp3 is the key transcription factor controlling the development, differentiation and function of natural T regulatory cells. Recently studies have found that miRNA play the critical role in the development, differentiation and function of T cells. Our previous studies found that miR-34a and miR-31 can be expressed in mouse T cells and much high in SLE mice. Bioinformatics prediction found miR-34a and miR-31 (who has been confirmed in human) can target on Fxop3 the 3'-UTR directly.Therefore, we set up a hypothesis: miR-34a regulates the development, differentiation and function of Tregs via direct targeting on the expression Foxp3. It may also promote the development of SLE. In this project, we will study on the effect of miR-34a on Foxp3 expression of Tregs in mice, declare the mechanism of Tregs shift toward Th17 and the role of miR-34a in the development of SLE mice models.

Treg在维持机体免疫自稳中具有重要作用，有可能被应用于自身免疫病治疗。然而近年来研究表明Treg在自身免疫病患者中表现出分泌IL-17等Th17特征，过继治疗的Treg在炎症部位变成Th17细胞，说明Treg可以向Th17转化。Foxp3是nTreg的关键调控因子，目前已发现Foxp3表达调控受表观遗传调控，长期研究发现T细胞发育分化和功能受miRNA的调控。我们采用生物信息学预测及预实验验证，发现miR-34a、31负向调控Foxp3作用最为显著。因此我们设想：miR-34a可直接负向调控Foxp3表达，从而调控Treg发育分化及功能，有可能促进SLE等自身免疫性病的发生、发展。本项目通过研究miR-34a对小鼠Treg中Foxp3表达调控机制，阐明miR-34a在Treg与Th17之间转化和SLE等自身免疫病发病过程中的作用机制，为治疗自身免疫病寻找新靶标奠定基础。

调节性T细胞（regulatory T cells，Treg）在预防自身免疫病及维持免疫稳态中发挥重要作用。Foxp3是Treg细胞的关键性转录调控因子。近年来发现miRNA对T、B细胞的发育起关键的作用，但目前有关miRNA对Foxp3表达调控的相关报道较少。我们采用生物信息学预测调控人、鼠Foxp3的miRNAs，发现人和鼠的miR-34a和鼠的miR-31对Foxp3具有调控作用，因此我们假设miR-34a和miR-31可以通过负向调控Foxp3的表达，从而抑制Treg细胞的分化和功能，促进Treg细胞向Th17转化，引起或加剧自身免疫性疾病的发生、发展。本课题从①生物信息学预测负向调控Foxp3的miRNAs；②不同T细胞miR-34a、miR-31表达水平与Foxp3表达的相关关系；③miRNAs表达载体、抑制性载体的构建及验证对Foxp3的调控作用；④miR-34a、miR-31对Foxp3的表达调控机制; ⑤建立miR-34a基因敲除的DBA1和DBA2。⑥在miR-34a基因敲除的C57B6小鼠和DBA2以及正常小鼠中建立CIA模型，采用miR-34a及其inhibitor对上述小鼠进行治疗；⑦部分临床SLE患者的标本验证等七个方面进行了研究。研究初步结果表明①预测到作用于小鼠和人Foxp3基因的miRNAs中miR-34a与miR-31的效果最为明显；②而在体外诱导的Th17细胞中miR-34a和miR-31的表达升高。miR-34a和miR-31在iTreg细胞和TGF-β诱导的EL4细胞中的表达水平下调；③成功构建miR-34a和miR-31的逆转录载体和慢病毒载体及初步验证对Foxp3的调控作用；④IL-6和/或TNF-α信号通路通过NF-κB介导了miR-34a及miR-31对Foxp3的表达调控；⑤成功建立miR-34a基因敲除的DBA1和DBA2小鼠；⑥在miR-34a转基因C57BL/6小鼠和正常小鼠中建立CIA模型，miR-34a转基因C57BL/6小鼠关节炎更为严重。⑦SLE患者PBMCs中Treg细胞群体变化与miR-34a的表达变化呈负相关。本研究初步结果表明miR-34a和miR-31可以负向调控Foxp3的表达，进而影响Treg/Th17细胞的平衡，为自身免疫性疾病的诊断、治疗以及疾病预后的判断提供了一个新的靶点。