Diabetic wounds are recalcitrant to healing. However, the mechanism causing this dysfunction is not fully understood. High expression of matrix metalloproteinase-9 (MMP-9) is indicative of poor wound healing. In this study, we show that specificity protein-1 (Sp1), a regulator of MMP-9, binds directly to its promoter and enhances its expression. Additionally, we demonstrated that Sp1 is the direct target of two microRNAs (miRNAs), miR-129 and -335, which are significantly downregulated in diabetic skin tissues. In vitro experiments confirmed that miR-129 or -335 overexpression inhibits MMP-9 promoter activity and protein expression by targeting Sp1, whereas the inhibition of these miRNAs has the opposite effect. The beneficial role of miR-129 or miR-335 in diabetic wound healing was confirmed by the topical administration of miRNA agomirs in diabetic animals. This treatment downregulated Sp1-mediated MMP-9 expression, increased keratinocyte migration, and recovered skin thickness and collagen content. The combined treatment with miR-129 and miR-335 induced a synergistic effect on Sp1 repression and MMP-9 downregulation both in vitro and in vivo. This study demonstrates the regulatory mechanism of Sp1-mediated MMP-9 expression in diabetic wound healing and highlights the potential therapeutic benefits of miR-129 and -335 in delayed wound healing in diabetes.

Excessive expression of matrix metalloproteinase 9 (MMP-9) impedes healing of diabetic chronic wounds, thus wound dressing that could effectively inhibit the expression of MMP-9 offers significant clinical translation for diabetic wound healing. Herein, a hybrid hydrogel dressing was developed for localized and sustained delivery of MMP-9 siRNA (siMMP-9). siMMP-9 was complexed with Gly-TETA (GT), the GT/siMMP9 complex was then loaded into a thermosensitive hydrogel based on Pluronic F-127 (PF) and methylcellulose (MC). In vitro, this hybrid hydrogel dressing exhibited negligible cytotoxicity, prolonged the release of GT/siMMP-9 for up to 7 days, and significantly reduced MMP-9 expression. In vivo assessment in diabetic rats demonstrated that hydrogel provided localized and sustained delivery via the thermosensitive controlled release of entrapped GT/siMMP-9 into wound tissues for 7 days, resulting in dramatic MMP-9 silencing which significantly improved diabetic wound closure. This hybrid hydrogel dressing exhibited excellent biocompatibility, with no observed systemic toxicity in rats. Taken together, the hybrid hydrogel dressing may constitute an effective and biocompatible means of enhancing diabetic wound healing through effective silencing of the MMP-9 gene, and this hydrogel delivery system also offers a platform for in vivo delivery of siRNA for the treatment of other diseases.

Impaired wound healing is one of the main causes of diabetic foot ulcerations. However, the exact mechanism of delayed wound healing in diabetes is not fully understood. Long noncoding RNAs (lncRNAs) are widely involved in a variety of biological processes and diseases, including diabetes and its associated complications. In this study, we identified a novel lncRNA, MRAK052872, named lncRNA UpRegulated in Diabetic Skin (lnc-URIDS), which regulates wound healing in diabetes. lnc-URIDS was highly expressed in diabetic skin and dermal fibroblasts treated with advanced glycation end products (AGEs). lnc-URIDS knockdown promoted migration of dermal fibroblasts under AGEs treatment in vitro and accelerated diabetic wound healing in vivo. Mechanistically, lnc-URIDS interacts with procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (Plod1), a critical enzyme responsible for collagen cross-linking. The binding of lnc-URIDS to Plod1 results in a decreased protein stability of Plod1, which ultimately leads to the dysregulation of collagen production and deposition and delays wound healing. Collectively, this study identifies a novel lncRNA that regulates diabetic wound healing by targeting Plod1. The findings of the current study offer some insight into the potential mechanism for the delayed wound healing in diabetes and provide a potential therapeutic target for diabetic foot.

Diabetes elevates matrix metalloproteinase-9 (MMP-9) levels in the skin and its keratinocytes, and activated MMP-9 impairs skin wound healing. Epigenetic regulation of the DNA methylation status within the MMP-9 promoter plays an important role in the alteration of MMP-9 expression. Our aim was to investigate the role and mechanism of growth arrest and DNA damage-inducible 45a (GADD45a), a well-known DNA demethylation regulatory protein that mediates DNA methylation, in the regulation of MMP-9 expression. In this study, we showed that GADD45a was markedly upregulated in skin tissues from patients with diabetic foot ulcers, in diabetic rats and in human keratinocyte (HaCaT) cells exposed to advanced glycation end products (AGEs). We observed a significant positive correlation between the levels of GADD45a and MMP-9 expression. Knockdown of GADD45a ameliorated the increase in MMP-9 transcription induced by diabetic condition by inhibiting demethylation in the MMP-9 promoter and promoted diabetic HaCaT cell migration, but GADD45a knockdown did not affect HaCaT cell proliferation or apoptosis. Additionally, we demonstrated that overexpression of GADD45a activated MMP-9 expression by inducing promoter demethylation. Moreover, we found that GADD45a binds to the promoter of MMP-9 and recruits thymine-DNA glycosylase (TDG) for base excision repair (BER)-mediated demethylation in diabetic HaCaT cells and diabetic rat skin. Our results reveal a new mechanism in which GADD45a is required for demethylation of the MMP-9 promoter and the induction of diabetic wound healing. Inhibiting GADD45a might be a therapeutic strategy for diabetic foot ulcers.