View Abstract

A1219 - Cytokine-Induced microRNA Dysregulation Mediates Targets in Asthmatic Airway Smooth Muscle to Enhance Contractile Signaling
Author Block: J. Evasovic, S. Favela, M. A. Hernandez, M. A. Ba, C. A. Singer; Pharmacology, University of Nevada, Reno School of Medicine, Reno, NV, United States.
Rationale: Asthma is a chronic inflammatory disease of the lower airway characterized by immoderate hypercontractility mediated by airway smooth muscle (ASM). Fundamental ASM contractile dynamics are implicated in airway obstruction; however, underlying mechanisms intrinsic to hypercontractility remain ambiguous. Calcium-dependent pathways are key regulators of ASM contractility and airway obstruction associated with asthma. Inositol 1,4,5-Trisphosphate Receptor Type 1 (ITPR1), Protein Phosphatase 1 Regulatory Subunit 12A (PPP1R12A) and Protein Kinase C Epsilon (PRKCE) are pivotal effectors within Ca2+ signaling pathways and are predicted targets of the miR-106b~25 cluster. Previous experiments identified downregulation of this cluster in human ASM stimulated with early-phase IL-1β, TNF-α and IFN-γ. We further hypothesize that stimulation with late-phase IL-13 and TGF-β promotes microRNA dysregulation and subsequent expression of contractile signaling targets within asthmatic ASM cells, resulting in increased intracellular Ca2+ and contractile output.
Methods: Total RNA and whole cell protein lysates were extracted from proliferating and 7-day differentiated asthmatic and non-asthmatic ASM cells treated with 10ng/mL IL-13 or TGF-β. Quantitative PCR was performed to assess expression of miR-25, -93, and -106b relative to U6 snRNA and expression of ITPR1, PPP1R12A and PRKCE relative to 18S rRNA. Total protein of these targets will be separated and transferred to nitrocellulose for western-blot detection using appropriate antibodies and normalized to GAPDH. Luciferase reporter assays will be used to verify microRNA binding to their respective 3’ UTRs of predicted mRNA targets.
Results:
Proliferating and 7-day differentiated asthmatic ASM cells stimulated with IL-13 and TGF-β showed significant downregulation of the miR-106b~25 cluster relative to non-treated non-asthmatic ASM cells. Conversely, ITPR1 expression in 7-day differentiated asthmatic ASM was increased 18.37-fold (± 0.06) compared to non-asthmatic controls in IL-13 treated cells and 6.70 (± 0.04) in TGF-β treated cells. PPP1R12A expression in 7-day differentiated asthmatic ASM was decreased 1.62-fold (± 0.07) compared to non-asthmatic controls in IL-13 treated cells and 2.44-fold (± 0.75) in TGF-β treated cells. Furthermore, PRKCE expression in 7-day differentiated asthmatic ASM was increased 0.21-fold (± 0.04) compared to non-asthmatic controls in IL-13 treated cells yet decreased 3.81-fold (± 0.04) in TGF-β treated cells.
Conclusions:
Together, these data indicate significant dysregulation of microRNAs and Ca2+ signaling-related genes in cytokine-stimulated asthmatic ASM cells. These results suggest a dynamic mechanistic relationship between microRNAs and their mRNA targets and support the continued investigation of the miR-106b~25 cluster as a mediator of Ca2+-dependent pathways underlying dysfunction of airway hypercontractility in asthma.