Center for Genetic Medicine Research, Children's Research Institute, Washington, DC, United States.
Genomics & Precision Medicine, The George Washington University, Washington, DC, United States.
Front Immunol. 2020 Feb 21;11:151. doi: 10.3389/fimmu.2020.00151. eCollection 2020.
MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate important intracellular biological processes. In myasthenia gravis (MG), a disease-specific pattern of elevated circulating miRNAs has been found, and proposed as potential biomarkers. These elevated miRNAs include miR-150-5p, miR-21-5p, and miR-30e-5p in acetylcholine receptor antibody seropositive (AChR+) MG and miR-151a-3p, miR-423-5p, let-7a-5p, and let-7f-5p in muscle-specific tyrosine kinase antibody seropositive (MuSK+) MG. In this study, we examined the regulation of each of these miRNAs using chromatin immunoprecipitation sequencing (ChIP-seq) data from the Encyclopedia of DNA Elements (ENCODE) to gain insight into the transcription factor pathways that drive their expression in MG. Our aim was to look at the transcription factors that regulate miRNAs and then validate some of those with cell lines that have sufficient expression of these transcription factors This analysis revealed several transcription factor families that regulate MG-specific miRNAs including the Forkhead box or the FOXO proteins (FoxA1, FoxA2, FoxM1, FoxP2), AP-1, interferon regulatory factors (IRF1, IRF3, IRF4), and signal transducer and activator of transcription proteins (Stat1, Stat3, Stat5a). We also found binding sites for nuclear factor of activated T-cells (NFATC1), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), early growth response factor (EGR1), and the estrogen receptor 1 (ESR1). AChR+ MG miRNAs showed a stronger overall regulation by the FOXO transcription factors, and of this group, miR-21-5p, let-7a, and let 7f were found to possess ESR1 binding sites. Using a murine macrophage cell line, we found activation of NF-κB -mediated inflammation by LPS induced expression of miR-21-5p, miR-30e-5p, miR-423-5p, let-7a, and let-7f. Pre-treatment of cells with the anti-inflammatory drugs prednisone or deflazacort attenuated induction of inflammation-induced miRNAs. Interestingly, the activation of inflammation induced packaging of the AChR+-specific miRNAs miR-21-5p and miR-30e-5p into exosomes, suggesting a possible mechanism for the elevation of these miRNAs in MG patient serum. In conclusion, our study summarizes the regulatory transcription factors that drive expression of AChR+ and MuSK+ MG-associated miRNAs. Our findings of elevated miR-21-5p and miR-30e-5p expression in immune cells upon inflammatory stimulation and the suppressive effect of corticosteroids strengthens the putative role of these miRNAs in the MG autoimmune response.
微小 RNA（miRNAs）是调节重要细胞内生物过程的小型非编码 RNA 分子。在重症肌无力（MG）中，已经发现了一种特定于疾病的循环 miRNA 升高模式，并被提出作为潜在的生物标志物。这些升高的 miRNA 包括乙酰胆碱受体抗体阳性（AChR+）MG 中的 miR-150-5p、miR-21-5p 和 miR-30e-5p，以及肌肉特异性酪氨酸激酶抗体阳性（MuSK+）MG 中的 miR-151a-3p、miR-423-5p、let-7a-5p 和 let-7f-5p。在这项研究中，我们使用来自 DNA 元件百科全书（ENCODE）的染色质免疫沉淀测序（ChIP-seq）数据来检查这些 miRNA 的调节，以深入了解驱动它们在 MG 中表达的转录因子途径。我们的目的是研究调节 miRNA 的转录因子，然后用这些转录因子表达充足的细胞系来验证其中一些。这项分析揭示了几个调节 MG 特异性 miRNA 的转录因子家族，包括叉头框或 FOXO 蛋白（FoxA1、FoxA2、FoxM1、FoxP2）、AP-1、干扰素调节因子（IRF1、IRF3、IRF4）和信号转导和转录激活蛋白（Stat1、Stat3、Stat5a）。我们还发现了核因子活化 T 细胞（NFATC1）、核因子 kappa-轻链增强子活化 B 细胞（NF-κB）、早期生长反应因子（EGR1）和雌激素受体 1（ESR1）的结合位点。AChR+ MG miRNAs 表现出更强的 FOXO 转录因子整体调节作用，在该组中，miR-21-5p、let-7a 和 let 7f 被发现具有 ESR1 结合位点。使用鼠巨噬细胞系，我们发现 LPS 诱导的 NF-κB 介导的炎症激活导致 miR-21-5p、miR-30e-5p、miR-423-5p、let-7a 和 let-7f 的表达。用抗炎药物泼尼松龙或地夫可特预处理细胞可减弱炎症诱导的 miRNA 的诱导。有趣的是，炎症激活将 AChR+-特异性 miRNA miR-21-5p 和 miR-30e-5p 包装到外泌体中，这表明了这些 miRNA 在 MG 患者血清中升高的可能机制。总之，我们的研究总结了驱动 AChR+和 MuSK+ MG 相关 miRNA 表达的调节转录因子。我们发现免疫细胞在炎症刺激下 miR-21-5p 和 miR-30e-5p 表达升高，以及皮质类固醇的抑制作用，这增强了这些 miRNA 在 MG 自身免疫反应中的潜在作用。
