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miR429 is Upregulated in Inflammatory Monocytes and Regulates Monocyte Migration
Author(s) -
Barry Zachary R,
Kynast Ross W,
Stinson W Alexander,
Fox David A,
Amin M Asif,
Rabquer Bradley James
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1028.13
Subject(s) - monocyte , inflammation , gene expression , biology , immunology , immune system , downregulation and upregulation , regulation of gene expression , microrna , microbiology and biotechnology , gene , cancer research , genetics
Purpose Rheumatoid Arthritis (RA) is an autoimmune disease characterized by chronic inflammation and joint destruction. Inflammation is characteristic of an immune response, brought about by increased migration of leukocytes to the damaged area. Monocytes migrate from the blood into the tissue to exert their effects. A multitude of genes involved in inflammatory pathways regulate monocyte migration. miRNAs are post transcriptional regulators that effect gene expression in a variety of cell types and in response to various stimuli. We hypothesized that inflammation regulates miRNA expression, including miR429, which then plays a specific role in regulating the expression of monocyte genes involved in monocyte migration. Methods and Results Monocytes were isolated from the blood of normal subjects (n=5) and patients with RA (n=8). miRNA was isolated and cDNA prepared. qPCR was performed using primers specific for miR429. There was a 2.6 fold increase in the expression of miR429 in the RA patients compared to the normal subjects. The miRNA databases, miRanda and MiRwalk, were used to identify gene targets for miR429 that are involved in monocyte and inflammation pathways. This analysis identified 38 gene targets with possible relevance to monocyte inflammatory processes, including MAP4K4, PIKfyve, and PAK2. miR429 mimics and inhibitors were then used to assess the role of miR429 in regulating gene expression and migration. After confirming mimic and inhibitor specificity and efficiency in U937 monocytes, we found miR429 had an effect on PAK2 expression. The mimic reduced the expression of PAK2 by a fold change of 0.57, and the inhibitor increased the expression of PAK2 by a fold change of 28.6. Chemotaxis was performed to test the effect of miR429 on monocyte migration in response to monocyte chemoattractant protein (MCP‐1). The miR429 mimic treated U937 cells (n=11 replicates) had similar migration to MCP‐1 when compared to control cells treated with only transfection reagent (n=10). However, migration was decreased by 47% in U937 cells treated with the miR429 inhibitor (n=10 replicates) compared to the control. Conclusion Our results suggest miR429 is upregulated in RA monocytes, and that miR429 plays a role in monocyte migration by regulating genes such as the cytoskeletal regulator, PAK2. Support or Funding Information Hewlett‐Mellon Fund for Faculty Development at Albion College