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.
Overall design: samples were fixed in formalin and paraffin embedded. Between 16 and 20 8-10µm sections were cut from the formalin-fixed paraffin embedded tissue blocks of non-diabetic and diabetic foot skin, placed on Arcturus PEN-membrane glass slides (Life Technologies, Carlsbad, CA, USA) and dried at 37°C for 1-2 hours. LCM was carried out on a Arcturus Veritas laser capture microdissection instrument and the epidermis was collected on CapSure® Macro LCM Caps (Life Technologies). The caps were transferred to a tube containing 60µl of deparaffinization buffer (QIAGEN Inc., Valencia, CA, USA) and total RNA, including the microRNA fraction, was extracted using the FFPE miRNeasy kit (QIAGEN Inc.) according to the manufacturer’s instructions. Total RNA concentration of the samples was quantified using NanoDrop 2000 (NanoDrop products, Wilmington, DE) and the RNA quality of these samples was determined by RT-qPCR of SNORD48 and miR-21 using the commercially available platforms miRCURY LNA™ (Exiqon, Woburn, MA, USA) or Quanta qScript™ microRNA Quantification System (Quanta BioSciences, Inc., Gaithersburg, MD, USA). The miR profiles for the epidermis of 3 NFS and 3 DFS, were generated using the miR Ready-to-Use PCR panels V3 (Exiqon) following the manufacturer’s specifications. The Ct values were normalized to the stably expressed reference gene SNORD49 using the Exiqon GenEX software and the expression levels in the NFS and DFS were compared.
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