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Impaired cell migration and structural defects in myeloid cells overexpressing miR-30b and miR-142-3p
Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms ( IF 4.7 ) Pub Date : 2020-09-24 , DOI: 10.1016/j.bbagrm.2020.194628
Araceli Valverde 1 , Salvador Nares 1 , Afsar Raza Naqvi 1
Affiliation  

Macrophages (MΦ) and dendritic cells (DC) play a fundamental role in shaping immune responses by sensing a plethora of Pathogen Associated Molecular Patterns (PAMPs), phagocytosis and antigen presentation to T lymphocytes. These important biological processes require efficient cell movement and an intact cellular morphology for dynamic interaction. The role of microRNAs (miRs) in this regard, however, is not well understood. In the present study, we show that miR-30b and miR-142-3p regulate migration and morphology of MΦ and DC. Transient overexpression of miR-30b and miR-142-3p attenuates migration and these cells display unique morphological deformities observed under electron microscopy. In addition, miR-142-3p overexpression in MΦ impaired phagocytosis of FITC-conjugated latex beads using live microscopy imaging. Interestingly, live cell imaging and F-actin staining revealed marked changes in the cell polarity and actin polymerization status, respectively. To identify miR-142-3p regulated pathways, we profiled global transcriptome changes in miR-142-3p or control mimic transfected DC. Expression of several genes were differentially altered by miR-142-3p and were associated with pathways related to cell movement, cell adhesion, and cytoskeletal rearrangement. Bioinformatics analysis identified a significant subset of downregulated genes with one or more predicted miR-142-3p binding sites in their 3′UTR strongly suggesting direct post-transcriptional impact of these miRNAs on multiple transcripts. Using dual luciferase assays, novel miR-142-3p binding sites were validated for three genes (Vinculin, Dab2 and Skap2) directly associated with cytoskeletal rearrangement and cell movement. In summary, our results show that miR-30b and miR-142-3p are regulators of myeloid cell cytoskeletal homeostasis and morphology.



中文翻译:

过表达 miR-30b 和 miR-142-3p 的骨髓细胞中的细胞迁移受损和结构缺陷

巨噬细胞 (MΦ) 和树突状细胞 (DC) 通过感知大量病原体相关分子模式 (PAMP)、吞噬作用和抗原呈递给 T 淋巴细胞,在形成免疫反应方面发挥着重要作用。这些重要的生物过程需要有效的细胞运动和完整的细胞形态以进行动态相互作用。然而,microRNA (miR) 在这方面的作用尚不清楚。在本研究中,我们表明 miR-30b 和 miR-142-3p 调节 MΦ 和 DC 的迁移和形态。miR-30b 和 miR-142-3p 的瞬时过表达减弱了迁移,这些细胞在电子显微镜下显示出独特的形态畸形。此外,使用实时显微镜成像,MΦ 中的 miR-142-3p 过表达会损害 FITC 偶联的乳胶珠的吞噬作用。有趣的是,活细胞成像和 F-肌动蛋白染色分别显示细胞极性和肌动蛋白聚合状态的显着变化。为了鉴定 miR-142-3p 调节的通路,我们分析了 miR-142-3p 或对照模拟转染 DC 中的全局转录组变化。miR-142-3p 差异性改变了几个基因的表达,并且与细胞运动、细胞粘附和细胞骨架重排相关的通路相关。生物信息学分析确定了一个重要的下调基因子集,在其 3'UTR 中具有一个或多个预测的 miR-142-3p 结合位点,强烈表明这些 miRNA 对多个转录本的直接转录后影响。使用双荧光素酶测定法,验证了三个基因(长春花蛋白、Dab2 和 Skap2) 与细胞骨架重排和细胞运动直接相关。总之,我们的结果表明 miR-30b 和 miR-142-3p 是髓样细胞细胞骨架稳态和形态的调节剂。

更新日期:2020-10-05
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