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MicroRNA Expression in Human Airway Smooth Muscle Cells

Role of miR-25 in Regulation of Airway Smooth Muscle Phenotype

¹ Department of Pharmacology, and ² Department of Biochemistry, UNR Bioinformatics Core, University of Nevada School of Medicine, Reno, Nevada; ³ Department of Physiology and Section of Respiratory Diseases, University of Manitoba, Winnipeg, Manitoba, Canada; and ⁴ Department of Biochemistry and Molecular Biology, University of South Alabama, Mobile, Alabama

Correspondence and requests for reprints should be addressed to Cherie A. Singer, Ph.D., Department of Pharmacology/318, University of Nevada School of Medicine, Reno, NV 89557-0046. E-mail: csinger{at}medicine.nevada.edu

Defining mechanisms by which differentiated, contractile smooth muscle cells become proliferative and secretory in response to mechanical and environmental stress is crucial for determining the contribution of airway smooth muscle (ASM) to inflammatory responses that result in airway disease. Regulation by microRNAs (miRNAs) has emerged as an important post-transcriptional mechanism regulating gene expression that may modulate ASM phenotype, but little is known about the expression and functions of miRNA in smooth muscle. In the present study we used microarrays to determine whether miRNAs in human ASM cells are altered by a proinflammatory stimulus. In ASM cells exposed to IL-1β, TNF-, and IFN-, we found 11 miRNAs to be significantly down-regulated. We verified decreased expression of miR-25, miR-140*, mir-188, and miR-320 by quantitative PCR. Analysis of miR-25 expression indicates that it has a broad role in regulating ASM phenotype by modulating expression of inflammatory mediators such as RANTES, eotaxin, and TNF-; genes involved in extracellular matrix turnover; and contractile proteins, most notably myosin heavy chain. miRNA binding algorithms predict that miR-25 targets Krüppel-like factor 4 (KLF4), a potent inhibitor of smooth muscle–specific gene expression and mediator of inflammation. Our study demonstrates that inhibition of miR-25 in cytokine-stimulated ASM cells up-regulates KLF4 expression via a post-transcriptional mechanism. This provides novel evidence that miR-25 targets KLF4 in ASM cells and proposes that miR-25 may be an important mediator of ASM phenotype.

Key Words: smooth muscle • gene expression • miRNA • inflammation • remodeling

CLINICAL RELEVANCE The study of miRNA in airway smooth muscle is an emerging area of interest in lung biology. Our studies have identified a novel miRNA target that may profoundly affect airway smooth muscle phenotype and contribute significantly to the knowledge of phenotypic plasticity in smooth muscle. This may lead to new therapeutics for lung disease such as asthma.