Improved wound healing by dual inhibition of miR-146a-5p and miR-29a-3p supports a network action of dysregulated miRNAs in diabetic skin.

AIMS/HYPOTHESIS: Upregulation of miR-146a-5p and miR-29-3p is observed in chronic non-healing wounds in diabetes. Their single or combined inhibition's molecular and cellular effects were assessed in human keratinocytes (HaCaT cells) and in vivo using a mouse model of type 1 diabetes. METHODS: As primary outcomes, we screened for proteome changes in HaCaT cells by LC-MS/MS after transfection with miR-146a-5p or miR-29a-3p inhibitors individually or in combination and following stimulation with TNF-α. Moreover, as a secondary outcome, we collected the data and cryopreserved and paraffin-embedded skin biopsies to estimate the tissue response to miRNA inhibition using immunofluorescence and histological analysis. Cryopreserved biopsies were also used for the LC-MS/MS proteome profiling to identify targets and cellular pathways involved in observed tissue changes. RESULTS: We identified a panel of extracellular matrix proteins, mainly laminins, whose levels changed after transfection with miR-146a-5p or miR-29a-3p inhibitors in HaCaT cells, counteracting TNF-α effects. There was a difference in wound closure rate in vivo between the dual inhibition of miR-146a-5p and miR-29a-3p and scramble controls on day 8 (p<0.01) and day 9 (p<0.05), although not at day 10. Histological analysis at day 10 shows a loose papillary layer in the scramble inhibition group, indicating incomplete wound closure compared with dual miRNA inhibition. Moreover, the dual action of the inhibitors decreased inflammation at day 3 and day 10 (both p<0.001) and reactive oxygen species formation (p<0.01) 3 days post wounding, while increasing the angiogenesis on day 3 (p<0.01) and day 10 (p<0.001). This was consistent with cytoskeletal rearrangements and collagen alterations observed in proteome profiling. CONCLUSIONS/INTERPRETATION: These findings demonstrate that dual inhibition of miR-146a-5p and miR-29a-3p in vitro synergises in a bidirectional manner, resulting either in intermediate effects or in cancelling each other's activity for the levels of specific proteins of basal lamina that impair proliferation and cell motility, compared with the individual inhibitors. Topical supplementation of miR-146a-5p and miR-29a-3p inhibitors to diabetic mouse wounds resulted in a reduction in wound size on days 8 and 9, which correspond to the later stages of healing, but did not lead to complete healing by day 10. However, dual inhibition demonstrates favourable effects on high oxidative stress, elevated inflammation and poor angiogenesis. These effects are superior to single miRNA inhibition, suggesting that combined miRNA inhibition could be a promising therapeutic strategy for diabetic wound healing. Nevertheless, further studies in humans are warranted.