Genomic Profiling of Diabetic Foot Ulcers Identifies miR-15b-5p as a Major Regulator that Leads to Suboptimal Inflammatory Response and Diminished DNA Repair Mechanisms

Diabetic foot ulcers (DFUs) are the leading cause of lower leg amputations in diabetic population. To better understand molecular pathophysiology of DFUs we used patients’ specimens and genomic profiling. We identified 3900 genes specifically regulated in DFUs. Moreover, we compared DFU to human skin acute wound (AW) profiles and found DNA repair mechanisms and regulation of gene expression among the processes specifically suppressed in DFUs, whereas essential wound healing-related processes, inflammatory/immune response or cell migration, were not activated properly. To identify potential regulators of DFU-specific genes, we used upstream target analysis. We found miR-15/16 family enriched in DFUs, but not in AW, which was confirmed by qPCR. We found that infection with the most common DFU colonizer, Staphylococcus aureus, triggers induction of miR-15-5p, which in turn, targets multiple DFU-specific genes, including genes involved in DNA repair (WEE1, MSH2 and RAD50) and the regulator of inflammatory pathway, IKBKB. Induction of miR-15b-5p, either by miR-mimic transfection in vitro or by S. aureus infection of acute wounds ex vivo, suppressed both WEE1 and IKBKB. Consequently, we detected an increase in DNA double strand breaks in DFUs. In summary, our data indicate that S. aureus infection, via induction of miR-15b-5p, may lead to suppression of DNA repair mechanisms and a sub-optimal inflammatory response, contributing to pathophysiology of DFU patients Total RNA including the miRNA fraction was extracted from the samples using the QIAGEN miRNeasy mini kit and following the manufacturer’s instructions. The RNA quality was assessed using the AGILENT bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) to estimate the RNA integrity number (RIN). Samples with a RIN higher than 5 were used for mRNA profiling. All processing and analysis of microarrays utilized standard protocols at the University of Miami Microarray Core Facility. Briefly, between 100 to 300 ng of total RNA was reverse transcribed, amplified, then the sense strand cDNA synthesized, labeled, and hybridized on arrays. The amplified, fragmented and biotin-labeled cDNAs were hybridized to the Affymetrix GeneChip Human Gene 2.0 ST microarray according to the manufacturer’s recommendations. Arrays were washed and stained using Affymetrix Fluidic stations 450 and scanned using Affymetrix GeneChip scanner 3000 7G. Image analysis was performed using the Affymetrix Command Console Software (AGCC). Resulting CEL files was imported into Expression Console™ Software (Affymetrix, Santa Clara, CA, USA) and underwent gene level normalization and signal summarization. The output files from this step were imported in Transcriptome Analysis Console (TAC) 3.0 Software (Affymetrix, Santa Clara, CA, USA) to identify differentially expressed genes and carry out clustering analysis between DFUs and FS. Genes with an FDR lower than 0.05 and a fold-change greater than 2 were considered as differentially expressed