P53调控B细胞MicroRNA-1246表达及其在系统性红斑狼疮发病中的作用

The pathogenesis of systemic lupus erythematosus (SLE) has not yet been completely investigated, one of the hallmarks of SLE is the production of autoantibodies by uncontrolled overactivated B cells. Accumulating evidence has demonstrated that several miRNAs modulate the pathogenesis of autoimmune diseases such as SLE through their effects on B cell functions, at the mean time, early B cells factor 1(EBF1) is found contributing to the development, activation and proliferation of B cells through activiting BCR signaling pathway. Previous studies by our group revealed that microRNA-1246 was reduced to less than half in B cells of SLE patients compared to B cells of healthy controls, we also discovered that microRNA-1246 can specifically regulate the expression of EBF1 and alter BCR signal transduction pathway, which leads to B cell over-activation in SLE. P53 can induce microRNA-1246 expression and change the status of chromatin through regulating the expression of histone acetyltransferases (HATs) and control the expression of miRNAs. So here comes to our brand new hypothesis that P53 contributes to the low expression of microRNA-1246 via its regulation on the acetylation status in the promoter region of microRNA-1246, therefore results in the upregulation of EBF1, which can be an important molecular mechanism that leads to B cell over-activation and the onset as well as the progression of SLE. We thus provide a theoretical framework towards the search of new and effective biological targes in SLE treatment.

系统性红斑狼疮（SLE）发病机制尚不明确，B细胞过度活化产生大量自身抗体在SLE发病中起重要作用。microRNAs可通过影响B细胞功能导致自身免疫性疾病的发生，而早期B细胞因子1（EBF1）可通过激活B细胞受体（BCR）信号通路促进B细胞发育、活化及增殖。我们前期研究发现SLE患者B细胞中microRNA-1246可通过调控其靶基因EBF1表达，激活BCR信号，从而参与SLE发病机制。P53可正向调控microRNA-1246表达，且P53可调控多种组蛋白乙酰转移酶表达进而调控microRNAs表达。因此，我们提出SLE B细胞中P53通过改变microRNA-1246基因启动子区乙酰化状态，导致microRNA-1246低表达，从而使EBF1表达增高，最终促使B细胞过度活化及SLE发生与发展这一全新假说。本项目将有助于进一步阐明SLE发病的分子机制，并有望为SLE治疗提供新的有效靶点。

我们的前期研究证实SLE患者B细胞中miR-1246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B细胞中miR-1246低表达的分子机制目前尚不清楚。我们通过real time PCR和WB检测发现SLE患者B细胞中P53表达水平明显低于正常对照，且P53表达水平与miR-1246表达水平呈显著正相关。ChIP-PCR及荧光素报告载体实验证实P53与miR-1246启动子存在结合作用。随后通过Co-IP实验，我们发现P53与P300、CBP、PCAF存在相互结合。SLE患者B细胞中P300、CBP表达水平明显降低，PCAF无显著差异。通过ChIP-qPCR证实，P53、P300、CBP、PCAF在SLE患者B细胞中miR-1246启动子区结合明显减少，且SLE患者B细胞中miR-1246启动子区H3K9、H3K14乙酰化水平显著降低。我们通过上调及下调P53的表达发现，在SLE患者B细胞中过表达P53，P53、P300、CBP 及PCAF与miR-1246启动子区的结合明显增多； miR-1246启动子区H3K9/K14乙酰化明显升高，miR-1246表达显著增加；EBF1、CD40、CD80、CD86 表达水平明显减少；转染后的B细胞与自身CD4+ T 细胞孵育后分泌IgG水平明显减少。在正常B细胞中干扰P53表达，随着P53表达的降低，P53、P300、CBP 及PCAF与miR-1246启动子区的结合明显减少；miR-1246启动子区H3K9/K14乙酰化明显降低，miR-1246表达显著减少；EBF1、CD40、CD80、CD86 表达水平明显增多；转染后的B细胞与自身CD4+ T 细胞孵育后分泌IgG水平明显增加。我们的研究首次证实SLE 患者B细胞miR-1246启动子区P53结合水平显著减少，影响P300、CBP、PCAF结合到此区域，导致此区域H3K9/K14乙酰化水平降低，抑制了miR-1246的表达，并促使B细胞过度活化，产生抗体增多，从而参与SLE的发病。