1: MicroRNAs: Potential For Molecular Modulation Of Mechanically-induced Skin Growth During TE

Abstract

Purpose: Tissue expansion (TE) is commonly utilized to promote skin growth prior to reconstructing a defect or deformity. In spite of its ubiquitous use, the role of molecular modulators during TE, such as microRNAs (miRNAs), has not previously been studied. MiRNAs are small endogenous molecules that regulate many biological processes, including cell proliferation, differentiation, and inflammatory response. Here, we investigate genome-wide changes in miRNA expression in skin during TE. Methods: Changes in miRNA expression were evaluated in a porcine TE model. Full-thickness skin biopsies were collected after 1 hour, 24 hours, 3 days, and 7 days of expansion, as well as from unexpanded skin (control). RNA extracted from biopsies was analyzed with next-generation sequencing (NGS). Differential expression analysis was performed using R software with the Bioconductor-DESeq2 package. Results were corrected for multiple testing using the Benjamini-Hochberg method. A combination of adjusted p-value 1 were used as the threshold to determine the significance of differentially expressed (DE) miRNAs. Potential target genes for DE miRNAs were identified by in silico analysis using three data bases: miRDB, miRTarBase, and DIANA Tools with Tarbase. Functional enrichment analysis of target genes was performed using the g:GOSt functional profiling tool, and results were visualized using R and Cytoscape software. Results: We identified 52 miRNAs that were differentially upregulated (n = 18) or downregulated (n = 34) during at least one of the tested timepoints during TE. At the four time points (1 hour, 24 hours, 3 days and 7 days), there were 15, 6, 22, and 20 DE miRNAs, respectively. Eight miRNAs (ssc-miR-193a-5p, ssc-miR-21, ssc-miR-9-1, ssc-miR-708-3p, ssc-miR-212, ssc-miR-196a, ssc-miR-15b, ssc-miR-184) were DE at more than one timepoint. The comparative analysis showed 9, 4, 16 and 15 unique miRNAs after 1 hour, 24, hours, 3 days and 7 days of TE, respectively. Gene Ontology and KEGG pathway analyses for predicted target genes demonstrated enrichment in cellular processes related to metabolism, transcription, translation, signal transduction, cell differentiation, migration, and angiogenesis.