Impaired cell migration and structural defects in myeloid cells overexpressing miR-30b and miR-142-3p

Abstract

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.