MicroRNAs are non-coding RNAs increasingly recognised as regulators of gene expression in epithelial-to-mesenchymal transition1 (EMT). Keratinocyte migration is stalled at the margin of non-healing wounds, impeding wound closure2. In acute healing, EMT allows keratinocyte migration and re-epithelialisation3. This project investigates microRNAs at the margin of an in vitro model of keratinocyte migration.
In a novel approach, a circular barrier to cell migration was used to generate constrained two-dimensional monolayers of primary human keratinocytes. By removing said barrier, peripheral keratinocytes were released to migrate. Replacing the barrier then isolated migrating keratinocytes at the monolayer periphery from the centre. RNA was extracted from peripheral and central keratinocytes. Next Generation Sequencing (NGS) was employed to profile microRNAs. Furthermore, Quantitative RT-PCR and the ΔΔCτ method were used to assay and compare, respectively, genetic EMT markers and microRNAs. Immunofluorescence allowed visualization of protein markers of EMT throughout the monolayer.
NGS and qRT-PCR has yielded microRNA targets for further investigation. Immunofluorescence revealed concurrent localisation of EMT hallmarks to the monolayer periphery.