The Mechanism Of MicroRNA-200b/c Regulation In Cutaneous Wound Healing By Targeting Rac1

BackgroundSkin,as the largest organ of human body,has important barrier protection function.Proliferation is an important phase of skin wound healing,during which epidermal re-epithelialization plays a key role in restoring the integrity of damaged skin.The essential role in re-epithelialization is the migration of keratinocytes to complete wound coverage.The migration of keratinocytes requires cytoskeleton to provide motility.Rho GTPase family is a class of proteins which regulate actin cytoskeleton.Rho GTPase family members RhoA,Rac1 and Cdc42 regulate actin stress fibers,lamellipodia and filopodia,respectively.As a class of non-coding small RNAs,microRNAs(miRNAs)play an important role in the healing process of skin wound.Preliminary prediction by Targetscan showed that Racl and RhoA of Rho GTPase family members were potential target genes for miR-200b/c which were miR-200 family members.The miR-200 family is involved in the hair formation of skin.The expression of miR-200c is down-regulated in the normal skin and is highly expressed in the elderly skin.However,the regulatory mechanism of miR-200 on skin wound healing has not been clarified.As an important cytokine involved in wound healing,TGF-β has the effect of regulating EMT-like phenotype of wound re-epithelialization.Racl-related signaling pathway is an important medium for TGF-β-induced EMT(epithelial-mesenchymal transition)of keratinocytes,but whether miR-200 participates in TGF-β-related EMT-like process of skin wound healing through targeting Rho GTPase family remains unclear.ObjectiveThis study focused on miR-200 family members miR-200b/c and Rho GTPase family,explored the mechanism of miR-200b/c regulating skin wound healing,and provided the possibility for clinical searching for microRNA-mediated therapeutic targets for skin wound.MethodsIn this study,the mouse dorsal skin full-thickness wound healing model was used to analyze whether miR-200b/c and Rho GTPase had potential targeting sites by bioinformatics.RT-qPCR and Western Blot were used to detect the correlation between miR-200b/c and Rho GTPase family in normal skin wounded tissues.The direct targeting of miR-200b/c to target genes was verified by dual luciferase reporter assay.MiR-200b/c mimics were injected into the wound site of mice to observe the effect of miR-200b/c on skin wound healing.H&E,K14 staining to observe the wound re-epithelialization.HaCaT cells scratch assay was used to simulate wound healing in vitro,and RT-qPCR and Western Blot were used to detect the correlation between miR-200b/c and Rho GTPase expression in keratinocytes.Western Blot was used to detect the change of Rho GTPase protein expression after overexpression of miR-200b/c in keratinocytes.RT-qPCR was used to detect the correlation between TGF-β and miR-200b/c in vivo and in vitro.The TGF-β-mediated EMT-like phenotype after overexpression of miR-200b/c in keratinocytes was detected by immunofluorescence and phalloidin staining.Results1.In this study,we found that the miR-200b/c enriched in the epidermis was negatively correlated with the expression of Racl,a key protein regulating the cytoskeleton lamellipodia,in the process of mouse skin wound healing.miR-200b/c directly targeted Racl to inhibit its protein expression.2.Overexpression of miR-200b/c significantly delayed wound healing in mouse model;miR-200b/c effectively inhibits Rac1 expression and migration in keratinocytes.3.The expression of TGF-β1 increased during re-epithelialization of mouse skin wound tissue;TGF-β1 effectively inhibited miR-200b/c expression;TGF-β1 induces EMT-like phenotype changes in keratinocytes by inhibiting the signal of miR-200b/c targeting Rac1,thereby maintaining re-epithelialization in wound healing.ConclusionsThe results showed that miR-200b/c directly targeting Rac1 3’UTR inhibited Racl expression in keratinocytes and inhibited skin wound healing.TGF-β1 regulates EMT-like process of wound re-epithelialization by alleviating targeted inhibition of miR-200b/c on Racl,thereby maintaining homeostasis of skin wound healing.