MicroRNA-122-5p is upregulated in diabetic foot ulcers and decelerates the transition from the inflammatory to the proliferative stage

Figure 1 Sequencing and bioinformatics analysis of MicroRNAs. A: In diabetic ulcer mice wounds, wound tissues were harvested 14 days postinjury, and total RNA was extracted. Subsequently, MicroRNAs (miR)-seq was performed, followed by the generation of a volcano plot; B: Representative heatmap of differentially expressed miRs in the skin tissues of diabetic ulcer mice; C: The top 10 miRs with the highest expression levels in each group; D: Gene ontology annotations and enrichment analysis of the target genes of miRs; E: Kyoto encyclopedia of genes and genomes annotations and enrichment analysis of the target genes of miRs; F: Relative expression levels of miR-122-5p in wound tissue from streptozotocin-induced diabetic mice [2^-∆∆Ct = 2.30, 95% confidence interval (CI): 1.81-2.72] showed a significant increase compared to normal mice (2^-∆∆Ct = 1.12, 95%CI: 0.64-1.31, P < 0.001) by quantitative real-time polymerase chain reaction; Further experiments demonstrated that the miR-122-5p overexpression group (2^-∆∆Ct = 3.82, 95%CI: 3.45-4.37, P < 0.001) exhibited significantly higher expression compared to the diabetic ulcer group; miR-122-5p levels were significantly increased in patients with diabetic foot ulcer (2^-∆∆Ct = 1.86, 95%CI: 2.14-3.51) compared with healthy individuals (2^-∆∆Ct = 1, 95%CI: 0.96-1.23, P < 0.001). ^cP < 0.001. miR: MicroRNA; FC: Fold change; DU: Diabetic ulcer; GO: Gene ontology; KEGG: Kyoto encyclopedia of genes and genomes; DFU: Diabetic foot ulcer; AAVDJ: Adeno-associated virus-DJ.

Figure 2 Adeno-associated virus-DJ-microRNA-122-5p up-decelerated wound healing in diabetic mice. A: Schematic diagram of the timeline of mice tests on the therapeutic effect of wound; B-D: A full-thickness skin wound was created on the dorsal area of the mice (1 cm × 1 cm). Animals were randomized into three groups and treated with phosphate-buffered saline. Optical pictures and related quantification of the wound closure rate in the control, diabetic ulcer (DU), and adeno-associated virus (AAV)-DJ-microRNA (miR)-122-5p groups at days 0, 3, 7, and 14 after the skin operation (n = 5); E: Gross view of wounds and wound area among the three groups of mice; F: Hematoxylin-eosin staining images of wound tissues in the control, DU, and AAVDJ-miR-122-5p groups at day 14 (n = 5; scale bar = 500 μm for 10 × and 100 μm for 40 ×); G: Masson trichrome staining at day 14 post operation (n = 5, scale bar = 500 μm for 10 × and 100 μm for 40 ×). ^aP < 0.05. ^bP < 0.01. DU: Diabetic ulcer; miR: MicroRNA; AAVDJ: Adeno-associated virus-DJ; STZ: Streptozotocin; HE: Hematoxylin-eosin.

Figure 3 MicroRNA-122-5p upregulated matrix metalloproteinase 9 to target inflammatory factors during the transition from the inflammatory phase to the proliferative phase. A and B: Levels of matrix metalloproteinase (MMP) 9, tumor necrosis factor (TNF)-α, and hypoxia inducible factor (HIF)-1α in wound tissues of mice after 14 days were detected using immunohistochemistry (20 ×); C: The expression of MMP9, TNF-α, and HIF-1α in wound tissues of mice was assessed using quantitative real-time polymerase chain reaction; D and E: Expressions of MMP9, TNF-α, and HIF-1αproteins were tested in wound tissues (n = 3); F and G: Expressions of MMP9, TNF-α, and HIF-1α proteins were tested in NIH3T3 cells in different groups (n = 3). ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. DU: Diabetic ulcer; miR: MicroRNA; AAVDJ: Adeno-associated virus-DJ; TNF-α: Tumor necrosis factor-α; HIF-1α: Hypoxia inducible factor-1α; MMP9: Matrix metalloproteinase 9; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; NC: Normal control.

Figure 4 MicroRNA-122-5p reduced vascular endothelial growth and delayed fibrosis during the transition from the inflammatory phase to the proliferative phase. A and B: Levels of vascular endothelial growth factor (VEGF), fibronectin (FN) 1, and α-smooth muscle actin (α-SMA) in wound tissues of mice after 14 days were detected using immunohistochemistry (20 ×); C: The expression of VEGF, FN1, and α-SMA in wound tissues of mice was assessed using quantitative real-time polymerase chain reaction; D and E: Expressions of VEGF, FN1, and α-SMA proteins were tested in wound tissues (n = 3); F and G: Expressions of VEGF, FN1, and α-SMA proteins were tested in NIH3T3 cells in different groups (n = 3). ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. DU: Diabetic ulcer; miR: MicroRNA; AAVDJ: Adeno-associated virus-DJ; SMA: Smooth muscle actin; VEGF: Vascular endothelial growth factor; FN: Fibronectin; NC: Normal control.

Figure 5 Adeno-associated virus-DJ-microRNA-122-5p up-decelerated the transition from the inflammatory to the proliferative stage. A: In situ hybridization was performed using a microRNA (miR)-122-5p-specific probe. Green indicates miR-122-5p expression, and blue indicates 4’,6-diamidino-2-phenylindole (20 ×); B: Tissue immunofuorescence staining of F4/80, ARG1, and inducible nitric oxide synthase (20 ×); C and D: The migration capacity and quantitative analysis of NIH3T3 cultured with RAW264.7 media in different groups were assessed using a cell scratch assay (n = 3); E and F: Matrix metalloproteinase 9, vascular endothelial growth factor, inflammation factors, and fibrosis factors were detected in vivo by enzyme-linked immunosorbent assay assays (n = 3). ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. miR: MicroRNA; DU: Diabetic ulcer; AAVDJ: Adeno-associated virus-DJ; DAPI: 4’,6-diamidino-2-phenylindole; iNOS: Inducible nitric oxide synthase; TNF-α: Tumor necrosis factor-α; HIF-1α: Hypoxia inducible factor-1α; MMP9: Matrix metalloproteinase 9; SMA: Smooth muscle actin; VEGF: Vascular endothelial growth factor; FN: Fibronectin; NC: Normal control.