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 in vivo 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（miRNA）是小的非编码RNA分子，可调节重要的细胞内生物学过程。在重症肌无力（MG）中，发现了一种疾病特异性模式的循环miRNA升高，并被提议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血清阳性（AChR +）MG中的miR-150-5p，miR-21-5p和miR-30e-5p，以及miR-151a-3p，miR-423-5p，let-7a-5p，肌肉特异性酪氨酸激酶抗体血清阳性（MuSK +）MG中的let-7f-5p。在这项研究中，我们使用来自DNA元素百科全书（ENCODE）的染色质免疫沉淀测序（ChIP-seq）数据检查了这些miRNA的调控，以深入了解驱动其在MG中表达的转录因子途径。体内具有充分表达这些转录因子的细胞系的分析此分析揭示了几个调控MG特异性miRNA的转录因子家族，包括叉头盒或FOXO蛋白（FoxA1，FoxA2，FoxM1，FoxP2），AP-1，干扰素调节因子（ IRF1，IRF3，IRF4）以及转录蛋白的信号转导和激活剂（Stat1，Stat3，Stat5a）。我们还发现了活化T细胞核因子（NFATC1），活化B细胞核因子κ-轻链增强子（NF-κB），早期生长反应因子（EGR1）和雌激素受体1（ ESR1）。AChR + MG miRNAs受FOXO转录因子显示出更强的整体调控能力，其中miR-21-5p，let-7a和let 7f具有ESR1结合位点。使用鼠巨噬细胞系，我们发现LPS诱导的miR-21-5p，miR-30e-5p，miR-423-5p，let-7a和let-7f的表达激活了NF-κB介导的炎症。用抗炎药泼尼松或deflazacort预处理细胞会减弱炎症诱导的miRNA的诱导。有趣的是，炎症的激活诱导了AChR +特异的miRNA miR-21-5p和miR-30e-5p包装入外泌体，提示在MG患者血清中这些miRNA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表达的调控转录因子。我们发现炎症刺激后免疫细胞中miR-21-5p和miR-30e-5p表达升高，以及皮质类固醇的抑制作用增强了这些miRNA在MG自身免疫反应中的假定作用。