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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

ABSTRACT: 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

ORGANISM(S): Homo sapiens

PROVIDER: GSE80178 | GEO | 2017/12/28

SECONDARY ACCESSION(S): PRJNA318248

REPOSITORIES: GEO

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Project description:Diabetic foot ulcers (DFUs) are one of the major complications of diabetes. Its molecular pathology remains poorly understood, impeding the development of effective treatments. Although it has been established that multiple cell types, including fibroblasts, keratinocytes, macrophages and endothelial cells, all contribute to inhibition of healing, less is known regarding individual contributions of each cell type. Thus, we generated primary fibroblasts from non-healing DFUs and evaluated their cellular and molecular properties in comparison to non-diabetic foot fibroblasts (NFFs). Specifically, we analyzed both micro-RNA and mRNA expression profiles of primary DFU fibroblasts. These paired genomic analyses identified a total of 331 reciprocal miRNA-mRNA pairs (21 miRNAs (FC>2.0) along with 239 predicted target genes (FC>1.5) that are significantly and differentially expressed. Of these, we focused on three miRNAs (miR-21-5p, miR-34a-5p, miR-145-5p) found to be induced in DFU fibroblasts as most differentially regulated. Their involvement in cellular functions important for wound healing was investigated by testing the expression of their downstream targets as well as by quantifying cellular behaviors in a prospectively collected and generated cell lines from 15 patients (7 DFUF and 8 NFF samples). We found large number of downstream targets of miR-21-5p, miR-34a-5p, miR-145-5p to be coordinately regulated in mRNA profiles, which was confirmed by qPCR. Genomic analysis of miR-21-5p, miR-34a-5p, miR-145-5p and their target genes indicates that these are contributing to the inhibition of cell movement and cell proliferation, together with increased cell differentiation and senescence in DFU fibroblasts, which was confirmed by cellular assays. We conclude that induction of miR-21-5p, miR-34a-5p, miR-145-5p in DFU dermal fibroblasts plays an important role in impairing multiple cellular functions, thus contributing to overall inhibition of healing in DFUs.

Project description:Infection in diabetic foot ulcers (DFU) are one of the major complications associated with diabetic patients. Staphylococcus aureus is the most common offending pathogen in patients with infected DFU. Previous studies have suggested application of species-specific antibodies against S. aureus for diagnosis and monitoring treatment response. Early and accurate identification of the main pathogen is critical for management of DFU infection. Understanding the host immune response against species-specific infection may facilitate diagnosis and suggest potential intervention options to promote healing infected DFUs. We sought to investigate evolving host transcriptome associated with surgical treatment of S. aureus infected DFU. This study compared the transcriptome profile of twenty-one patients with S. aureus infected DFU who underwent initial foot salvage therapy with irrigation and debridement followed by intravenous antibiotics therapy. Blood samples were collected at the recruitment (0-weeks) and 8-week after therapy to isolate peripheral blood mononuclear cells (PBMC). We analyzed the PBMCs expression of transcriptomes at two different time points (0 vs 8-week). Subjects were further divided into two groups at 8-week: healed (n=17, 80.95%) versus non-healed (n=4,19.05%) based on the wound healing status. DESeq2 differential gene analysis was performed. An increased expression of IGHG1, IGHG2, IGHG3 and IGLV3-21, IGLV6-57 were noted during active infection at 0-week compared to 8-week. Lysine and arginine-rich histones (HIST1H2AJ, HIST1H2AL, HIST1H2BM, HIST1H3B and HIST1H3G) were upregulated at the initial phase of active infection at 0-week. CD177 and RRM2 were also upregulated at the initial phase of active infection (0-week) compared to 8-week follow-up. Genes of heat shock protein members (HSPA1A, HSPE1, and HSP90B1) were high in not-healed compared to healed patients 8-week after therapy. The outcome of our study suggests that identification of genes evolution based on a transcriptomic profiling could be a useful tool for diagnosing infection, assessing severity and assess host-immune response to therapies.

Project description:Diabetes Mellitus (DM) is a chronic, severe disease rapidly increasing in incidence and prevalence and is associated with numerous complications. Patients with DM are at high risk of developing diabetic foot ulcers (DFU) that often lead to lower limb amputations, long term disability, and a shortened lifespan. Despite this, the effects of DM on human foot skin biology are largely unknown. Thus, the focus of this study was to determine whether DM changes foot skin biology predisposing it for healing impairment and development of DFU. Foot skin samples were collected from 20 patients receiving corrective foot surgery and, using a combination of multiple molecular and cellular approaches we performed comparative analyses of non-ulcerated non-neuropathic diabetic foot skin (DFS) and healthy non-diabetic foot skin (NFS). MicroRNA (miR) profiling of laser captured epidermis and primary dermal fibroblasts from both DFS and NFS samples identified 5 miRs de-regulated in the epidermis of DFS though none reached statistical significance. MiR-31-5p and miR-31-3p were most profoundly induced. Although none were significantly regulated in diabetic fibroblasts, miR-29c-3p showed a trend of up-regulation, which was confirmed by qPCR in a prospective set of 20 skin samples. Gene expression profiling of full thickness biopsies identified 36 de-regulated genes in DFS (>2 fold-change, unadjusted p-value ≤ 0.05). Of this group, three out of seven tested genes were confirmed by qPCR: SERPINB3 was up-regulated whereas OR2A4 and LGR5 were down-regulated in DFS. However no morphological differences in histology, collagen deposition, and number of blood vessels or lymphocytes were found. No difference in proliferative capacity was observed by quantification of Ki67 positive cells in epidermis. These findings suggest DM causes only subtle changes to foot skin. Since morphology, mRNA and miR levels were not affected in a major way, additional factors, such as neuropathy, vascular complications, or duration of DM, may further compromise tissue’s healing ability leading to development of DFUs. foot skin biopsies obtained from diabetic and non-diabetic people were thoroughly washed in DMEM supplemented with 2× Penicillin/Streptomycin/Fungizone, and gentamicin (50mg/L). After, the epidermis was removed from specimens, the dermis was finely minced and placed in 12-well plates. For the establishment of fibroblast cultures, DMEM (Life Technologies) supplemented with 10% FBS, HEPES (1.9mg/ml), streptomycin (100 μg/ml), penicillin (100 U/ml), and Fungizone Antimycotic (0.25 µg/mL) was used. RNA was isolated from cells (passage 1 or 2) using miRNeasy Mini Kit (QIAGEN). miR expression profiles of DFF and NFF fibroblasts were generated by nanoString nCounter miR Expression Assays (NanoString Technologies, Seattle, WA, USA) and analyzed using nSolver2.0 software following manufacturer's instructions. Data were normalized to gene controls included in the assays.

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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

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