miR-155在系统性红斑狼疮发生中的作用及表达调控机制

Systemic lupus erythematosus (SLE) is a prototype autoimmune disease characterized by auto-antibody production and multi-organ damage. The etiology and pathogenetic mechanisms of SLE have not been clearly understood..Previous studies demonstrated Th17 cells, a novel subset of Th cells that selectively secrete several proinflammatory cytokines, mainly IL-17 may play an important role in SLE. E26 transformation-specific-1 (Ets1) belongs to the Ets family of transcription factors, is essential to regulation of the immune system including immune cell proliferation and differentiation. Increasing data demonstrated Ets1 play a role in negative regulation of Th17 cells and B cells differentiation. Recently, association of genetic variation in Ets1 with susceptibility to systemic lupus erythematosus (SLE) have been independently identified by two genome-wide association studies,also confirmed in further replication studies, and decreased Ets1 mRNA level in peripheral blood mononuclear cells (PBMCs) of SLE patients has been reported. All these findings suggest that the transcription factor is broadly linked to the pathogenesis of this disease. However, the detailed role of miR-155 in Th17 differentiation and pathogenesis of SLE still remained unclear..In our previous studies, we found there was a much higher miR-155 expression levels in SLE patients than in normal controls and its expression level is positively correlated with IL17A levels, and negatively with ETS1. Also, methylation inhibitor 5-azacytidine can increase miR-155 expression level in cultured T cells. .Based on these findings and our preliminary results, we proposed the hypothesis that transcription factors,acetylation or hypomethylation in miR-155 promoter results in increasing its expression in SLE, which targets the negative regulator of Th17 cell such as ETS1. Subsequently, it promotes Th17 differentiation then lead to the pathogenesis of SLE. Silencing or inhibiting miR-155 expression may ameliorate the disease severity. To test the hypothesis, in vitro studies combined with the SLE patients are used to identify the upstream regulation mechanisms of miR-155 expression, miR-155 overexpression and knockout MRL/lpr lupus mice are used to investigate its role in the pathogenesis of SLE. .In this proposed study, we will elucidate the regulation mechanisms of miR-155 expression and its essential role in the progression of SLE. Our work may be helpful for finding new SLE therapy targets.

miR-155是一个在自身免疫病发生过程中起重要作用的microRNAs，但在Th17分化及系统性红斑狼疮(SLE)发生中的机制并不明确。我们前期研究发现，miR-155在SLE中高表达且与IL17A表达水平正相关，与ETS1表达负相关，过表达miR-155可下调ETS1的表达。据此，课题组提出假设：转录因子表达水平或结合能力的改变、甲基化、乙酰化等机制导致miR-155表达上调，进而抑制了Th17的负调控因子ETS1等的表达水平，从而促进Th17的分化，进而导致SLE的发生发展,抑制miR-155的表达或可缓解SLE的发生发展。为验证该假设，课题组将利用过表达miR-155与敲除miR-155的MRL/lpr狼疮小鼠模型在体内研究miR-155导致疾病发生的具体机制，体外与体内实验结合临床标本分析miR-155的上游表达调控机制，探索miR-155在SLE中的潜在诊断与治疗价值。

为了明确miR-155在SLE发生发展中的作用，我们分别选取野生对照小鼠、Faslpr/lpr狼疮小鼠和miR-155-/- Faslpr/lpr小鼠进行了分析，结果显示， Faslpr/lpr狼疮小鼠脾肿大症状明显， 40-50周龄的miR-155-/- Faslpr/lpr小鼠尿蛋白水平低于Faslpr/lpr狼疮小鼠。Faslpr/lpr小鼠的肾脏组织结构紊乱，肾小球增大，肾小球毛细血管基底膜增厚，有明显的炎性细胞浸润。肾脏中IgM，IgA和C1q的沉积减少。脾脏和淋巴结中CD4+（CD4+CD69+）T细胞比例明显增加，狼疮小鼠脾脏中有自发生发中心的形成，ELISA实验结果也证实miR-155-/- Faslpr/lpr小鼠淋巴结中Tfh细胞的比例减少，上述结果表明敲除miR-155可缓解Faslpr/lpr小鼠的狼疮样表型。利用表达谱芯片对miR-155-/-小鼠和野生小鼠的脾脏表达谱进行了差异分析。对芯片结果进行分析，发现与野生型小鼠脾脏相比，miR-155-/-小鼠脾脏存在544个基因表达上调和508个基因表达下调（基因表达存在两倍差异并且P<0.05）。利用荧光素酶报告基因技术证实了S1PR1是miR-155的一个靶基因。利用S1PR1的特异性拮抗剂W146连续三天腹腔注射miR-155-/- Faslpr/lpr小鼠，并对小鼠细胞学水平的表型进行了流式检测。结果发现与DMSO处理组小鼠相比，W146处理组miR-155-/-Faslpr/lpr小鼠的脾脏和淋巴结中CD4+/CD8+比例异常增加，活化的CD4+T细胞比例增多，并且淋巴结中Tfh细胞的比例增加，这提示S1PR1特异性拮抗剂W146可加重miR-155-/- Faslpr/lpr小鼠的狼疮样表型。以上结果证实了mir-155通过调控S1PR1参与了SLE的发生发展。

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