De-regulation of microRNA and gene expression in diabetic foot ulcers lead to a dampened inflammatory response, inefficient DNA repair and inhibition of cell migration

of a dissertation at the University of Miami. Dissertation supervised by Professor Marjana Tomic-Canic. No. of pages in text. (104) Diabetic foot ulcers (DFUs) are a common and severe type of chronic wound of which molecular pathophysiology is poorly understood, resulting in slow development of new and efficacious treatments. The aim of this study was to identify cellular functions that contribute to pathophysiology of DFUs as compared to the acute wounds, by using genomic profiling. I postulated that de-regulation of DFU-specific genes and microRNAs (miRs) play an important role in the development of a chronic wound phenotype. I utilized a bedside-to-bench approach in which DFU tissue samples from patients, non-neuropathic diabetic (DFS) and non-diabetic foot skin (NFS) biopsies were used to generate microarray expression profiles and miR PCR expression profiles. Comparison of the expression profiles of DFS and NFS revealed only minor miR and gene expression changes between these two sets of samples, which was further supported by histo-morphometric assessments, suggesting that diabetes causes subtle changes to foot skin itself. Next, I compared the DFU gene expression profiles to the foot skin (FS) profiles (NFS and DFS) identifying a set of differentially expressed genes in DFUs. To select specific genes unique for DFUs I performed additional comparison of DFU list of genes to publically available human skin acute wound (AW) profiles. I used Ingenuity Pathway Analysis (IPA) software to do functional enrichment and comparative analysis between them and found down-regulation of gene expression and suppressed DNA repair mechanisms among processes uniquely enriched in DFUs. Genes involved in DNA-repair mechanisms were down-regulated in DFUs, suggesting impairment in this process, which was confirmed by increased presence of phospho-histone H2A.X (γ-H2AX), a known marker for double strand DNA damage. In contrast, the inflammatory response that was highly induced in AW was not effectively up-regulated in DFUs. Furthermore, I examined potential upstream-regulators of these DFU-specific genes and identified miR-15b-5p as predicted to be up-regulated in DFUs but not in AWs. I tested miR-15b-5p expression in DFUs by qPCR and confirmed its up-regulation in all samples of DFUs, but not in AW. Moreover, miR-15b-5p was identified as an upstream regulator of multiple genes suppressed in DFUs including IKBKB, PIK3R1, and WEE1. Further, its induction can be triggered by infection with Staphylococcus aureus, one of the most common pathogens found in DFUs. Lastly, I compared DFU miR profiles to AW miR profiles generated from an ex-vivo acute wound model. miR-193b-3p was found to be induced in DFUs, but down-regulated in AWs. Transfection of miR-193b-3p in the human keratinocytes, resulted in inhibition of scratch wound assay in vitro, even in the presence of the pro-migratory hsa-miR-31-5p or hsa-miR-15b-5p. These results imply that miR-193b-3p has a major role in inhibiting keratinocyte migration in DFUs. Surprisingly, S. aureus infection was also found to induce miR-193b-3p in human ex-vivo wounds. In summary, I discovered novel mechanisms that contribute to the pathogenesis of DFUs, which may be triggered by the presence of S. aureus. On one hand, up-regulation of miR15b-5p may lead to suppression of DNA repair mechanisms and dampened inflammatory response through suppression of WEE1 and IKBKB among other genes, whereas on the other hand, miR-193b-3p dominantly inhibits keratinocyte migration resulting in nonmigratory epidermal edge characteristic of DFUs. These mechanisms identify potential targets for development of novel therapeutic and diagnostic approaches.