MicroRNA regulation of nonmuscle myosin light chain kinase expression in human lung endothelium.

TitleMicroRNA regulation of nonmuscle myosin light chain kinase expression in human lung endothelium.
Publication TypeJournal Article
Year of Publication2013
AuthorsAdyshev DM, Moldobaeva N, Mapes B, Elangovan V, Garcia JGN
JournalAm J Respir Cell Mol Biol
Volume49
Issue1
Pagination58-66
Date Published2013 Jul
ISSN Number1535-4989
Keywords3' Untranslated Regions, Acute Lung Injury, Anti-Inflammatory Agents, Non-Steroidal, Biomimetics, Calcium-Binding Proteins, Capillary Permeability, Cell Line, Endothelium, Gene Expression Regulation, Genes, Reporter, Humans, Lipopolysaccharides, Luciferases, Lung, MicroRNAs, Myosin-Light-Chain Kinase, Pneumonia, Pulmonary Artery, Stress, Mechanical, Transcription, Genetic, Transfection, Tumor Necrosis Factor-alpha
Abstract

<p>Increased lung vascular permeability, the consequence of endothelial cell (EC) barrier dysfunction, is a cardinal feature of inflammatory conditions such as acute lung injury and sepsis and leads to lethal physiological dysfunction characterized by alveolar flooding, hypoxemia, and pulmonary edema. We previously demonstrated that the nonmuscle myosin light chain kinase isoform (nmMLCK) plays a key role in agonist-induced pulmonary EC barrier regulation. The present study evaluated posttranscriptional regulation of MYLK expression, the gene encoding nmMLCK, via 3' untranslated region (UTR) binding by microRNAs (miRNAs) with in silico analysis identifying hsa-miR-374a, hsa-miR-374b, hsa-miR-520c-3p, and hsa-miR-1290 as miRNA candidates. We identified increased MYLK gene transcription induced by TNF-α (24 h; 4.7 ± 0.45 fold increase [FI]), LPS (4 h; 2.85 ± 0.15 [FI]), and 18% cyclic stretch (24 h; 4.6 ± 0.24 FI) that was attenuated by transfection of human lung ECs with mimics of hsa-miR-374a, hsa-miR-374b, hsa-miR-520c-3p, or hsa-miR-1290 (20-80% reductions by each miRNA). TNF-α, LPS, and 18% cyclic stretch each increased the activity of a MYLK 3'UTR luciferase reporter (2.5-7.0 FI) with induction reduced by mimics of each miRNA (30-60% reduction). MiRNA inhibitors (antagomirs) for each MYLK miRNA significantly increased 3'UTR luciferase activity (1.2-2.3 FI) and rescued the decreased MLCK-3'UTR reporter activity produced by miRNA mimics (70-110% increases for each miRNA; P < 0.05). These data demonstrate that increased human lung EC expression of MYLK by bioactive agonists (excessive mechanical stress, LPS, TNF-α) is regulated in part by specific miRNAs (hsa-miR-374a, hsa-miR-374b, hsa-miR-520c-3p, and hsa-miR-1290), representing a novel therapeutic strategy for reducing inflammatory lung injury.</p>

DOI10.1165/rcmb.2012-0397OC
Alternate JournalAm. J. Respir. Cell Mol. Biol.
PubMed ID23492194
PubMed Central IDPMC3727884
Grant ListP01 HL98050 / HL / NHLBI NIH HHS / United States
P01 HL58064 / HL / NHLBI NIH HHS / United States
R01 HL91889 / HL / NHLBI NIH HHS / United States
P01 HL098050 / HL / NHLBI NIH HHS / United States
P01 HL058064 / HL / NHLBI NIH HHS / United States
R01 HL091889 / HL / NHLBI NIH HHS / United States