BCL6调控CD4+T细胞miR-142-3p/5p表达的分子机制及其在系统性红斑狼疮发病中的作用

Our previous study has proved that decreased miR-142-3p/5p expression plays an important role in aberrant CD4+ T cell responses in systemic lupus erythematosus. However, the molecular mechanism of down-regulation of miR-142-3p/5p remains poorly understood. In our preliminary study, we found that expression of BCL6 (mRNA/protein) were up-regulated in CD4+ cells of SLE patients, which is negative correlation with expression of miR-142-3p/5p. Over-expression of BCL6 in vitro T cell can lead to decreased expression of miR-142-3p/5p. These results suggest that BCL6 may regulate the expression of miR-142-3p/5p. Recently studies have shown that BCL6 and EZH2 cooperate to regulate gene expression. Meanwhile, our previous study showed H3K27 trimethylation in MIR-142 promotor is enriched in SLE CD4+ T cells, which is also inverse correlation with expression of miR-142-3p/5p. Considering our previous study and latest researches abroad, we propose a new hypothesis of SLE pathogenesis that a transcriptional repressor complex of BCL6/EZH2 may elevate H3K27me3 level in MIR-142 promotor, and leading to down-regulation of miR-142-3p/5p, resulting in over-activation of T cells and overproduction of auto-antibodies, and finally, SLE. In this project, this hypothesis should be confirmed through investigating molecular mechanism of BCL6 regulating miR-142-3p/5p expression in CD4+ T cells, and its role in the pathogenesis of systemic lupus erythematosus.

我们的研究已证实SLE T细胞异常降低的miR-142-3p/5p对T细胞异常活化的关键调控作用,但其表达下调的分子机制尚不清楚。我们预实验发现SLE T细胞中BCL6表达明显上调，与miR-142-3p/5p表达呈负相关；同时体外实验发现T细胞过表达BCL6可导致其表达下降，提示BCL6调控miR-142-3p/5p表达。最新研究发现BCL6可协同组蛋白甲基化酶EZH2调控基因表达，而我们前期研究发现SLE T细胞MIR-142基因启动子区H3K27me3水平异常升高。因此我们提出BCL6可能通过与EZH2形成转录复合体，调控MIR-142基因启动子区H3K27me3水平，抑制miR-142-3p/5p表达，从而诱导T细胞异常活化和自身抗体过度产生，导致SLE发病这一全新假说。本项目将通过研究BCL6调控miR-142-3p/5p表达的分子机制及其异常在SLE发病中的作用来证实这一假说。

我们的前期研究证实SLE 患者CD4+ T 细胞中miR-142-3p/5p表达降低在系统性红斑狼疮发病中扮演重要角色。但SLE患者CD4+ T细胞miR-142-3p/5p 低表达的分子机制目前尚不清楚。我们通过realtime PCR和WB检测发现SLE患者CD4+ T 细胞中BCL6表达水平明显高于正常对照， ChIP-PCR及荧光素报告载体实验证实BCL6与MIR-142基因启动子存在结合作用。随后通过IP实验，我们发现BCL6与EZH2（一种H3K27甲基化酶）存在相互结合。通过ChIP-PCR证实，BCL6与EZH2在SLE患者MIR-142基因启动子区结合明显增多，且SLE患者MIR-142基因启动子区H3K27me3水平显著升高。我们通过过表达及抑制BCL6的表达发现，在正常人CD4+ T细胞中过表达BCL6，随着BCL6表达的升高，BCL6及EZH2与MIR-142基因启动子区的结合明显增多，其H3K27me3明显升高，miR-142-3p/5p表达明显降低；在SLE患者CD4+ T细胞中干扰BCL6表达后则出现相反结果，随着BCL6表达的降低，BCL6及EZH2与MIR-142基因启动子区的结合明显减少，其H3K27me3明显降低，miR-142-3p/5p表达明显升高。我们的研究首次证实SLE 患者CD4+ T细胞MIR-142基因启动子区BCL结合水平显著升高，招募更多的EZH2结合到此区域，导致此区域H3K27me3水平升高。升高的H3K27me3抑制了miR-142-3p/5p表达，结果致使T细胞过度活化，并促使B细胞产生抗体增多，最终促使SLE的发病。