Use of a Novel MicroRNA-519c Construct for Targeted Downregulation of Hypoxia Inducible Factor 1 (Hif-1α): A New Therapeutic Strategy Against Heterotopic Ossification
Joe Habbouche, BS, Kavitha Ranganathan, MD, David Cholok, BS, Ming Dang, PhD, Arminder Kaura, BS, Michael Chung, MD, Jonathan Butts, BS, John Li, MD, Chirstopher Brueler, BS, Caitlin Priest, BS, Hsiao Hsin Sung Hsieh, DDS, Peter Ma, PhD, Shuli Li, PhD, Serra Ucer, PhD, Steven R. Buchman, MD, Benjamin Levi, MD.
University of Michigan, Ann Arbor, MI, USA.
PURPOSE: : Heterotopic Ossification (HO) is the abnormal formation of ectopic bone after trauma or thermal injury. Hypoxia-inducible factor-1 alpha (Hif-1α) is a critical mediator of this process. We have previously demonstrated that loss of Hif-1α in a transgenic mouse model decreases HO. Building on these findings, the goal of the current study is to use targeted inhibition of Hif-1α to decrease HO formation. MicroRNAs (miRNA) are emerging in the field of cancer research as an important method of specific and targeted intervention given their unique ability to modulate protein translation without the severe side effects common to more ubiquitous modalities of treatment. In this study, we introduce the novel formulation of miRNA within a polymer sphere vehicle as a therapeutic option for HO that improves delivery and selectivity compared to other available alternatives. We validate the efficacy of this novel polymer-microRNA polyplex to downregulate Hif-1α and vascular endothelial growth factor (VEGF) to serve as a potential pharmacologic antagonist against HO in a targeted fashion.
METHODS: A polymer-microRNA polyplex was designed by incubating a mixture of the polymer transfection reagent with fluorescently labeled miR-519c for 30 minutes. The transfection reagent formed a polymeric sphere around each microRNA particle to promote uptake into the nucleus of each cell. MC3T3 cells were then transfected with this agent (3.25 ug/mL) or fluorescently conjugated control transfection vector. Flow cytometry was performed 48 hours after transfection to sort cells based on uptake of miR-519c or the control vector. These cells were subsequently harvested for Western blot and immunocytochemical analysis. Additionally, mice (n=3; C57BL/6) underwent a 30% total body surface area burn and Achilles’ tenotomy. miR-519c was injected into the tenotomy site 24 hours after injury. MicroCT was performed 9 weeks after injury to evaluate HO formation.
RESULTS: The polymer-miR-519c complex demonstrated a transfection efficiency of 70% in vitro, and the vector alone demonstrated an efficiency of 100% (Fig.1A-C). Although there was no significant difference in expression of Hif-1α on qPCR analysis (0.91 v. 0.92, p=0.41) given the post-transcriptional effect of miRNA519c, VEGF, a downstream target of Hif-1α, was significantly decreased (1.19 v. 1.23, p=0.004) (Fig.1D). As downregulation of Hif-1α and VEGF significantly decreased HO formation in vivo, mice were treated with miR-519c and experienced no complications with regards to wound healing or systemic side effects attributable to local downregulation of Hif-1α.
CONCLUSION: Hif-1α is effectively downregulated in a direct, targeted fashion by miR-519c. The ability to decrease Hif-1α expression in this manner represents a significant advancement over currently available forms of pharmacologic prophylaxis against HO by minimizing the side effects and burdensome dosing strategies required by systemic methods of administration.
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