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R. M. Lavker, H. Peng, S. Getsios, J. Yu; Antagonistic Effects of MicroRNAs on Keratinocyte Migration: Implications for Corneal Epithelial Wound Healing. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4782.
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Previously, we reported that microRNA-184 (miR-184), the most abundant corneal epithelial miRNA, antagonizes miR-205 to maintain levels of the lipid phosphatase, SHIP2, in corneal epithelium. SHIP2 dephosphorylates PIP3, a critical second messenger in several cell signaling pathways including Akt. Our studies also indicated that miR-205 enhanced the Akt signaling pathway via SHIP2 suppression leading to increased cell survival. Since the Akt pathway has also been implicated in regulating the actin cytoskeleton and keratinocyte migration, we investigated whether the upstream regulation of SHIP2 levels by miRs-205 and -184 plays a role in keratinocyte migration.
We used antagomirs (Antago) to reduce the levels of miR-184 and miR-205 in corneal epithelial (HCEKs) and epidermal (HEKs) keratinocytes. Such loss of miRNA function studies were combined with immunohistochemical, immunoblotting and linear scratch wound assays to study keratinocyte cytoskeletal organization and migratory activity. These investigations were complemented by siRNA studies for SHIP2.
Suppression of miR-205 in HEKs and HCEKs following a 48 hr treatment with Antago-205 resulted in major cytoskeletal reorganization events, including a marked diminution of filamentous actin and a corresponding increase in phosphorylation of the actin severing protein, cofilin. Despite the loss of filamentous actin, an increase in focal contacts (FC) and the biomarker p-paxillin were observed. Collectively, this led to a decrease in keratinocyte migration. Conversely, treatment of HeLa cells with a miR-205 mimic reduced SHIP2 levels and increased peripheral actin. Suppression of miR-184 in HCEKS, which enables miR-205 to inhibit SHIP2, or direct SHIP2 silencing using siRNA oligos, both of which lead to an enhancement in Akt signaling, resulted in a more rapid sealing of linear scratch wounds.
These findings indicate that miR-205 suppresses SHIP2 to regulate actin-based keratinocyte motility pathways suggesting that miRNAs could be used to promote corneal epithelial wound repair. Specifically, topical application of an Antago that will down-regulate miR-184 thereby enabling miR-205 to inhibit SHIP2, should increase motility and speed the sealing of wounds.
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