MiR-221 Influences Effector Functions and Actin Cytoskeleton in Mast Cells

Mast cells have essential effector and immunoregulatory functions in IgE-associated allergic disorders and certain innate and adaptive immune responses, but the role of miRNAs in regulating mast cell functions is almost completely unexplored. To examine the role of the activation-induced miRNA miR-221 in mouse mast cells, we developed robust lentiviral systems for miRNA overexpression and depletion. While miR-221 favored mast cell adhesion and migration towards SCF or antigen in trans-well migration assays, as well as cytokine production and degranulation in response to IgE-antigen complexes, neither miR-221 overexpression, nor its ablation, interfered with mast cell differentiation. Transcriptional profiling of miR-221-overexpressing mast cells revealed modulation of many transcripts, including several associated with the cytoskeleton; indeed, miR-221 overexpression was associated with reproducible increases in cortical actin in mast cells, and with altered cellular shape and cell cycle in murine fibroblasts. Our bioinformatics analysis showed that this effect was likely mediated by the composite effect of miR-221 on many primary and secondary targets in resting cells. Indeed, miR-221-induced cellular alterations could not be recapitulated by knockdown of one of the major targets of miR-221. We propose a model in which miR-221 has two different roles in mast cells: in resting cells, basal levels of miR-221 contribute to the regulation of the cell cycle and cytoskeleton, a general mechanism probably common to other miR-221-expressing cell types, such as fibroblasts. Vice versa, upon induction in response to mast cell stimulation, miR-221 effects are mast cell-specific and activation-dependent, contributing to the regulation of degranulation, cytokine production and cell adherence. Our studies provide new insights into the roles of miR-221 in mast cell biology, and identify novel mechanisms that may contribute to mast cell-related pathological conditions, such as asthma, allergy and mastocytosis.

肥大细胞（mast cells）在免疫球蛋白E（IgE）相关过敏性疾病以及部分固有免疫与适应性免疫应答中发挥关键的效应功能与免疫调节作用，但目前针对微小RNA（microRNA, miRNA）调控肥大细胞功能的研究几乎仍是空白。为探究激活诱导型微小RNA miR-221在小鼠肥大细胞中的功能，我们构建了稳定可靠的慢病毒载体系统，用于实现miRNA的过表达与敲低。尽管miR-221可在Transwell迁移实验中促进肥大细胞向干细胞因子（stem cell factor, SCF）或抗原迁移并增强其黏附能力，同时可提升肥大细胞在IgE-抗原复合物刺激下的细胞因子产生与脱颗粒水平，但无论是miR-221过表达还是其基因沉默，均不会干扰肥大细胞的分化过程。对miR-221过表达肥大细胞的转录谱分析显示，大量转录本的表达受到调控，其中包括多个与细胞骨架相关的转录本；进一步研究发现，miR-221过表达可使肥大细胞的皮层肌动蛋白水平出现可重复的升高，同时还会改变小鼠成纤维细胞的细胞形态与细胞周期。我们的生物信息学分析表明，该效应可能由miR-221对静息细胞中多个一级靶基因与二级靶基因的综合调控效应所介导。事实上，仅敲低miR-221的一个核心靶基因，无法重现miR-221诱导的细胞表型改变。我们提出如下模型：miR-221在肥大细胞中发挥两种截然不同的功能：在静息状态下，miR-221的基础表达水平参与调控细胞周期与细胞骨架，这一通用机制可能同样适用于其他表达miR-221的细胞类型（如成纤维细胞）；反之，当肥大细胞受到刺激而被激活诱导miR-221表达后，miR-221的功能则呈现肥大细胞特异性与激活依赖性，参与调控脱颗粒、细胞因子产生与细胞黏附过程。本研究为阐明miR-221在肥大细胞生物学中的功能提供了全新视角，并揭示了可能参与哮喘、过敏及肥大细胞增多症等肥大细胞相关病理过程的新型调控机制。