microRNA expression and functions in human airway smooth muscle cells
AuthorKuhn, Andrew R.
AdvisorSinger, Cherie A.
Biochemistry and Molecular Biology
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Mechanisms by which differentiated, contractile smooth muscle cells become proliferative and secretory is one way airway smooth muscle (ASM) cells respond to mechanical and environmental stress and contributes to inflammatory responses in the lung 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. The overall hypothesis of this thesis is that ASM expresses a specific miRNA signature and the miRNA contained within participates in the regulation of smooth muscle phenotype. In order to study miRNA expressed in ASM, a survey was conducted using miRNA arrays consisting of all 385 known human and mouse miRNAs at the time of the experiment. We consistently detected 22 miRNA in 4 different human ASM tissue samples. Further experiments were performed to determine miRNA expressed in cultured human ASM cells and how these might be altered by a pro-inflammatory stimulus relevant to inflammatory airway disease. In ASM cells exposed to IL-1β, TNFα, and IFNγ, we found 11 miRNA significantly down-regulated and further verified decreased expression of miR-25, miR-140*, mir-188 and miR-320 by quantitative PCR. Further 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 TNFα; 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 in vascular smooth muscle cells. Experiments were designed to test and support the hypothesis that the inhibition of miR-25 in cytokine-stimulated ASM cells up-regulates KLF4. Further experiments were performed to test targeting of the KLF4 3'-untranslated region (UTR) by miR-25. Luciferase assay experiments were designed in which the two predicted miR-25 binding sites were mutated. Decreased expression of luciferase from site 1, site 2, and to a greater degree double mutant support the binding of miR-25 to these sites. Functional assays were also performed in miR-25 and KLF4 overexpressing ASM cells. miR-25 overexpressing cells showed a decrease in proliferation and increase in contractile protein expression. Collectively, this data provides novel evidence that miR-25 targets KLF4 in ASM cells and proposes that miR-25 is an important mediator of ASM phenotype. In addition we decided to look at another possible transcription factor/miRNA relationship. T-bet is a transcription factor shown to play a role in Th1/Th2 phenotype determination in T-cells which shifts T cells to the Th1 type. A role for T-bet control of miRNA and the significance of this role in Th1/Th2 differentiation was explored.