Abstract

Microphthalmia-associated transcription factor (MITF) is a master regulatory factor for melanocytes. MITF regulates multiple pigmentary genes for maintaining cellular homeostasis. MicroRNAs (miRNAs) play crucial roles in numerous biological processes however their molecular/cellular mechanisms to regulate pigmentation have not been fully explored. This study was undertaken to investigate the role of miRNAs in skin depigmentation via regulation of MITF gene. Depigmentation in C57BL/6 black mice was induced by an autoimmune response against tyrosinase. Bioinformatics approach was used to detect miRNAs conserved in 3′untraslated region (3′UTR) of MITF mRNA. The iMC23 mouse melanocytes were used for transfection experiments. The data demonstrated that the MITF mRNA/protein was markedly low in lesional skin of depigmented mice (p < 0.05). Targetscan genomic database determined that 3′UTR of mouse MITF constitutes 4819 nucleotide bases and has 23 conserved sites for different miRNAs To validate the pairing of these predicted miRNAs with MITF mRNA, five miRNAs were deregulated in lesional skin (p < 0.05). Among them, mmu-miR-181a-5p and mmu-miR-183-5p were up-regulated, whereas mmu-miR-26a-5p, mmu-miR-26b-5p and mmu-miR-32-5p were down-regulated (p < 0.05). To verify these results, the iMC23 mouse melanocytes were used. Transfection of iMC23 cells with specific miRNAs mimics or inhibitors or with 3’UTR reporter clone of MITF, showed only mmu-miR-183-5p binds to 3’UTR of MITF mRNA and regulates its expression in iMC23 melanocytes. In conclusions, this is the first study shows that miR-183-5p is a direct regulator of MITF in iMC23 melanocytes. Thus, miR-183-5p is an important regulator of melanocytes homeostasis and may be a novel target for autoimmune depigmentation therapy.

摘要

小眼症相关转录因子 (MITF) 是黑色素细胞的主要调节因子。MITF 调节多种色素基因以维持细胞稳态。MicroRNAs (miRNAs) 在许多生物过程中发挥着至关重要的作用，但是它们调节色素沉着的分子/细胞机制尚未得到充分探索。本研究旨在通过调节 MITF 基因来研究 miRNA 在皮肤脱色中的作用。C57BL/6 黑色小鼠的色素脱失是由针对酪氨酸酶的自身免疫反应引起的。生物信息学方法用于检测 MITF mRNA 的 3'非翻译区 (3'UTR) 中保守的 miRNA。iMC23 小鼠黑素细胞用于转染实验。数据表明 MITF mRNA/蛋白质在脱色小鼠的损伤皮肤中显着降低 (p < 0.05)。Targetscan 基因组数据库确定小鼠 MITF 的 3'UTR 构成 4819 个核苷酸碱基，并具有 23 个不同 miRNA 的保守位点。为了验证这些预测的 miRNA 与 MITF mRNA 的配对，5 个 miRNA 在病变皮肤中失调（p < 0.05）。其中，mmu-miR-181a-5p和mmu-miR-183-5p上调，而mmu-miR-26a-5p、mmu-miR-26b-5p和mmu-miR-32-5p下调调节（p < 0.05）。为了验证这些结果，使用了 iMC23 小鼠黑色素细胞。用特定 miRNA 模拟物或抑制剂或用 MITF 的 3'UTR 报告克隆转染 iMC23 细胞，显示只有 mmu-miR-183-5p 与 MITF mRNA 的 3'UTR 结合并调节其在 iMC23 黑素细胞中的表达。总之，这是第一项研究表明 miR-183-5p 是 iMC23 黑素细胞中 MITF 的直接调节因子。因此，