title: MicroRNA regulation of nonmuscle myosin light chain kinase expression in human lung endothelium.
abstract: 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.
authors.affiliation: Institute for Personalized Respiratory Medicine, Department of Medicine, Section of Pulmonary, Critical Care, Sleep, and Allergy, University of Illinois at Chicago, Chicago, IL 60612, USA.
authors.forename: Djanybek M
authors.initials: DM
authors.lastname: Adyshev
authors.forename: Nurgul
authors.initials: N
authors.lastname: Moldobaeva
authors.forename: Brandon
authors.initials: B
authors.lastname: Mapes
authors.forename: Venkate
authors.initials: V
authors.lastname: Elangovan
authors.forename: Joe G N
authors.initials: JG
authors.lastname: Garcia
mesh_terms: D020413:3' Untranslated Regions; D055371:Acute Lung Injury / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D032701:Biomimetics; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002199:Capillary Permeability; D002460:Cell Line; D004727:Endothelium / Q000201:enzymology* / Q000473:pathology; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D008156:Luciferases / Q000378:metabolism; D008168:Lung / Q000201:enzymology / Q000473:pathology; D035683:MicroRNAs / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000235:genetics / Q000378:metabolism*; D011014:Pneumonia / Q000378:metabolism / Q000473:pathology; D011651:Pulmonary Artery / Q000378:metabolism / Q000473:pathology; D013314:Stress, Mechanical; D014158:Transcription, Genetic; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism / Q000494:pharmacology
chemical_list: D020413:3' Untranslated Regions; D000894:Anti-Inflammatory Agents, Non-Steroidal; D002135:Calcium-Binding Proteins; D008070:Lipopolysaccharides; D035683:MicroRNAs; D014409:Tumor Necrosis Factor-alpha; D008156:Luciferases; C499378:MYLK protein, human; D009219:Myosin-Light-Chain Kinase