Mir-193b-3p Suppresses Cellular Migration And Contributes To Impaired Wound Closure In Diabetic Foot Ulcers
Irena Pastar1, Ivan Jozic1, Horacio A. Ramirez1, Nkemcho Ojeh2, Rivka Stone1, Tongyu Cao Wikramanayake1, Robert S. Kirsner1, Marjana Tomic-Canic1.
1University of Miami Miller School of Medicine, Miami Beach, FL, USA, 2Faculty of Medical Sciences, the University of the West Indies, Bridgetown, Barbados.
Regulation of keratinocyte migration is indispensable for successful wound healing. We aimed to identify cellular functions that contribute to pathophysiology and lack of epidermal migration in diabetic foot ulcers (DFU) by utilizing comparative mRNA and miR profiling of DFUs and healing acute wounds (AW). Ingenuity pathway analysis of paired mRNA/miR profiles identified suppression of cellular migration as a uniquely enriched entity in DFUs and revealed miR-193b-3p as a potential master regulator of downregulated genes in DFU, but not AW. We isolated epidermal miR from DFUs and AW using laser captured microdissection and confirmed induction of miR-193b-3p in DFUs, and not in the AW. Next we validated miR-193b-3p downstream target genes (KRAS, MAP3K1, TNS1, SOS2, JAK2 and ARHGEF6) by qRT-PCR or 3’-UTR luciferase reporter assays. Functional conformation of miR-193b-3p mediated inhibition of healing was performed by miR overexpression studies in vitro and in vivo. To examine the role of miR-193b-3p in cell migration, we performed wound scratch assays and confirmed that miR-193b-3p inhibits migration, without affecting proliferation. Importantly miR-193b-3p exhibited a dominant inhibitory effect on keratinocyte migration, it suppressed wound closure in vitro even in the presence of previously established pro-migratory miRs. To validate miR-193b-3p function in vivo, we injected control or miR-193b-3p mimics into murine excisional wounds, and confirmed delayed wound closure due to miR-193b-3p overexpression. To elucidate the mechanism of miR-193b-3p mediated inhibition of cellular migration we selectively induced filopodia, lamellipodia and stress fiber formation in keratinocytes over-expressing miR-193b-3p and visualized actin cytoskeleton by phalloidin-rhodamine staining. MiR-193b-3p inhibited formation of stress fibers in keratinocytes, which is essential for proper directional cell migration. In summary, we identified miR-193b-3p as a potential therapeutic target and major regulator responsible for healing impairment in DFUs through suppression of keratinocyte migration via inhibition of stress fiber formation.
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