Asthmatic human airway smooth muscle cells (hASMCs) exhibit enhanced expression of numerous cytokine-responsive genes but this trend has not been observed for cyclooxygenase-2 (COX-2) expression despite knowledge that conserved regulatory mechanisms exist for cytokine-responsive gene expression. Enhanced expression of cytokine-responsive genes in asthmatic hASMCs has been attributed to differences in histone post-translational modifications and microRNA (miR or miRNA) expression. COX-2 expression is of interest because it serves as a model cytokine-responsive gene and is regulated by epigenetic mechanisms. In other cell types, miR-146a represses COX-2 and Interleukin (IL)-1β expression, directly and indirectly, respectively. Due to sequence homology, miR-146b is predicted to repress the expression of COX-2 and IL-1β. I investigated COX-2 expression in asthmatic and non-asthmatic hASMCS treated with cytomix (IL-1β, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ). Also, I chose to compare histone acetylation, transcription factor binding, and miR-146a/b expression in asthmatic and non-asthmatic hASMCs to identify any correlations with COX-2 expression. A major goal of this project was to help identify new treatment targets for asthma therapeutics . I hypothesized that asthmatic hASMCs treated with cytomix express more COX-2 and secrete more prostaglandinE2 (PGE2) than non-asthmatic hASMCs due to differences in COX-2 epigenetic regulation. It is reported here that asthmatic hASMCs treated with cytomix expressed more COX-2 (mRNA/protein), and secreted more PGE2 than non-asthmatic hASMCs. Histone H3/H4 pan-acetylation at the COX-2 promoter did not increase with cytomix treatment and was not different in asthmatic and non-asthmatic hASMCs. Treatment of hASMCs with cytomix increased RNA Polymerase II and nuclear factor-κB binding at the COX-2 promoter with no difference between asthmatic and non-asthmatic hASMCs. Treatment of hASMCs with cytomix increased miR-146a and miR-146b expression with greater miR-146a expression in asthmatic. MiR-146a/b expression in asthmatic hASMCs treated with cytomix did not negatively correlate with COX-2 expression. These results led me to investigate whether miR-146a/b were capable of negatively regulating COX-2 and IL-1β expression in hASMCs. MiR-146a and miR-146b mimics reduced COX-2 and IL-1β mRNA/protein, and PGE2 secretion in hASMCs. MiR-146a and miR-146a/b combination inhibition increased COX-2 and pro-IL-1β protein in hASMCs but not miR-146b inhibition alone. In conclusion, elevated miR-146a expression and histone acetylation are not responsible for increased COX-2 expression in asthmatic hASMCs. MiR-146a is a minor negative regulator of COX-2 and IL-1β expression in hASMCs at physiological expression levels but mimics are capable of antagonizing cytokine-responsive gene expression profoundly. These results coupled with other evidence from the literature indicate that miR-146a/b should be investigated in animal models of asthma to determine if they are relevant asthma drug target in patients that do not respond to current anti-inflammatory therapies.
|Advisor:||Gerthoffer, William T.|
|Commitee:||Cioffi, Donna L., Honkanen, Richard E., Lincoln, Thomas M., McMurtry, Ivan F.|
|School:||University of South Alabama|
|Department:||College of Medicine|
|School Location:||United States -- Alabama|
|Source:||DAI-B 75/05(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Cellular biology, Biochemistry|
|Keywords:||Airway smooth muscle cell, Asthma, Cyclooxygenase-2, Interleukin-1beta, microRNA|
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