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Abstract Diabetic foot ulcer (DFU) is a serious complication of diabetic patients which negatively affects their foot health. This study aimed to estimate the role and mechanism of the miR‐200 family in DNA damage of diabetic wound healing. Human foreskin fibroblasts (HFF‐1 cells) were stimulated with high glucose (HG). Db/db mice were utilized to conduct the DFU in vivo model. Cell viability was evaluated using 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide assays. Superoxide dismutase activity was determined using detection kits. Reactive oxygen species determination was conducted via dichlorodihydrofluorescein‐diacetate assays. Enzyme‐linked immunosorbent assay was used to evaluate 8‐oxo‐7,8‐dihydro‐2′deoxyguanosine levels. Genes and protein expression were analyzed by quantitative real‐time polymerase chain reaction, western blotting, or immunohistochemical analyses. Luciferase reporter gene and RNA immunoprecipitation assays determined the interaction with miR‐200a/b/c‐3p and GLI family zinc finger protein 2 (GLI2) or ataxia telangiectasia mutated (ATM) kinase. HG repressed cell proliferation and DNA damage repair, promoted miR‐200a/b/c‐3p expression, and suppressed ATM and GLI2. MiR‐200a/b/c‐3p inhibition ameliorated HG‐induced cell proliferation and DNA damage repair repression. MiR‐200a/b/c‐3p targeted ATM. Then, the silenced ATM reversed the miR‐200a/b/c‐3p inhibition‐mediated alleviative effects under HG. Next, GLI2 overexpression alleviated the HG‐induced cell proliferation and DNA damage repair inhibition via miR‐200a/b/c‐3p. MiR‐200a/b/c‐3p inhibition significantly promoted DNA damage repair and wound healing in DFU mice. GLI2 promoted cell proliferation and DNA damage repair by regulating the miR‐200/ATM axis to enhance diabetic wound healing in DFU.

ABSTRACT Background Umbilical cord‐derived mesenchymal stem cells (UCMSCs) could alleviate diabetes‐induced injury. Hence, this investigation aimed to explore the role and mechanism of UCMSCs‐derived exosomal circHIPK3 (exo‐circHIPK3) in diabetes mellitus (DM). Methods HFF‐1 cells were cultured in high glucose (HG) medium or normal medium, and treated with UCMSCs‐derived exo‐circHIPK3 or miR‐20b‐5p mimics or Unc‐51‐like autophagy activating kinase 1 (ULK1) overexpression vector. The surface markers of UCMSCs were analyzed using a flow cytometer. The differentiation potential of UCMSCs was evaluated using oil red O staining, alizarin red staining and alkaline phosphatase (ALP) staining. Cell proliferation was determined by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. The miRNA expressions were analyzed by reverse transcription‐quantitative polymerase chain reaction (qRT‐PCR). Protein levels were quantified by western blot. An immunofluorescence staining was used for observing LC3 expression. The interaction between miR‐20b‐5p and circHIPK3, and between miR‐20b‐5b and ULK1 were identified by a RNA immunoprecipitation (RIP) assay and a luciferase reporter assay. Results Up‐regulation of circHIPK3 was found in UCMSCs‐derived exosomes. Exo‐circHIPK3 decreased the miR‐20b‐5p level while increasing the contents of ULK1 and autophagy‐related gene 13 (Atg13) in HG‐induced fibroblasts. In addition, exo‐circHIPK3 activated HG‐induced fibroblast autophagy and proliferation. Overexpressed miR‐20b‐5p promoted fibroblast injury by inhibiting cell autophagy via the ULK1/Atg13 axis in HG conditions of high glucose. Moreover, exo‐circHIPK3 enhanced autophagy and cell viability in HG‐induced fibroblasts through the miR‐20b‐5p/ULK1/Atg13 axis. Conclusion UCMSCs‐derived exosomal circHIPK3 promoted cell autophagy and proliferation and accelerated the fibroblast injury repair by the miR‐20b‐5p/ULK1/Atg13 axis.

Abstract Background Diabetic foot ulcer (DFU) is a chronic infectious disease caused by diabetes mellitus (DM). Angiogenesis plays the decisive role in wound healing of DFU. Adipose-derived stem cells (ADSCs) can ameliorate angiogenesis in DFU by exosomes. This study aims to determine the mechanism of exosomes from mmu_circ_0001052-modified ADSCs in angiogenesis of DFU. Methods HUVECs were induced by high glucose and mice stimulated using STZ injection during high-fat feeding, which were treated with exosomes derived from mmu_circ_0001052-modified ADSCs. Real-time PCR determined the expression of gene and western blot determined protein levels. Proliferation, migration, apoptosis and angiogenesis of HUVECs were studied by MTT assay, transwell test, flow cytometry and tube formation experiment, respectively. Histological lesion of wound was determined by HE staining. Results The expression of circ_0001052 was upregulated in ADSCs and miR-106a-5p elevated in high glucose-induced HUVECs. Exosomal mmu_circ_0001052 significantly accelerated wound healing in mice with DFU. Also, exosomal mmu_circ_0001052 evoked the reduction of miR-106a-5p and the elevation of FGF4 in high glucose-induced HUVECs and wound tissue of DFU mice. Exosomal mmu_circ_0001052 was determined to sponge miR-106a-5p that targeted FGF4 in DFU. In high glucose-induced HUVECs, exosomal mmu_circ_0001052 inhibited apoptosis and miR-106a-5p expression, and meanwhile promoted proliferation, migration, angiogenesis and expressions of FGF4, VEGF and p-p38/p38, which were reversed by miR-106a-5p elevation. Conclusion Mmu_circ_0001052 in ADSCs-derived exosomes promote angiogenesis of DFU via miR-106a-5p and FGF4/p38MAPK pathway. Graphical Abstract

Background: The process of wound healing is complex. Increasing evidences have shown that lncRNA MALAT1 is abundant in fibroblasts and may be engaged in wound healing process. Therefore, we explored the mechanism of MALAT1 affecting wound healing. Methods: The expression levels of MALAT1, miR-141-3p as well as ZNF217 in human fibroblast cells (HFF-1) were quantified by qRT-PCR. HFF-1 proliferation was measured by MTT, while migration was detected by wound healing assay. SMAD2 activation and matrix proteins expression were detected by western blotting. The interaction between miR-141-3p and MALAT1 or ZNF217 was further confirmed using the luciferase reporter gene assay. In vivo wound healing was assessed by full-thickness wound healing model on C57BL/6 mice. Result: Knockdown of MALAT1 as well as overexpression miR-141-3p remarkably inhibited the proliferation, migration and matrix protein expression in HFF-1 cells. MALAT1 directly targeted and inhibited the expression of miR-141-3p. MiR-141-3p suppressed the activation of TGF-β2/SMAD2 signaling pathway by targeting ZNF217. Knockdown of MALAT1 inhibited wound healing process in mice. Conclusions: MALAT1 up-regulates ZNF217 expression by targeting miR-141-3p, thus enhances the activity of TGF-β2/SMAD2 signaling pathway and promotes wound healing process. This investigation shed new light on the understanding of the role of MALAT1 in wound healing, and may provide potential target for the diagnosis or therapy of chronic wounds.