miR-200c delays the process of skin wound healing by inhibiting RhoA/ROCK pathway
Objective To explore the role of miR-200c in skin wound healing and its regulatory mechanism.Methods The full-layer skin trauma model of SD rats were established,and divided into agomir-NC group,miR-200c agomir group,sh-NC group and sh-RhoA group.The expression of miR-200c in wound tissues was detected by qRT-PCR,and the expressions of MMP-9,N-cadherin,E-cadherin,RhoA and ROCK proteins in wound tissues were detected by Western blot.The wound healing rate was calculated at day 1,7,14 after trauma,and the wound healing was detected at day 5 by HE staining.The targeting relationship between miR-200c and RhoA was analyzed by double luciferase assay.Human immortalized keratinocyte cell line HaCaT cells were divided into inhibitor-NC group,miR-200c inhibitor group,miR-200c inhibitor+sh-NC group and miR-200c inhibitor+sh-RhoA group.Cell proliferation was detected by EdU assay and cell migration was detected by cell scratch assay.The expression of miR-200c in HaCaT cells was detected by qRT-PCR,and the expressions of MMP-9,N-cadherin,E-cadherin,RhoA and ROCK proteins in HaCaT cells were detected by Western blot.Results Compared with normal skin tissue,the expression of miR-200c was decreased in wound skin tissue(P＜0.01),and RhoA was increased(P＜0.01).Double luciferase assay result showed that miR-200c had a targeting binding relationship with RhoA 3'UTR.Compared with agomir-NC group,the wound healing rate of skins and the protein expressions of MMP-9,N-cadherin,RhoA and ROCK were decreased in miR-200c agomir group(P＜0.05),and the protein expression of E-cadherin was increased(P＜0.05).Compared with sh-NC group,the wound healing rate of skins and the protein expressions of MMP-9,N-cadherin,RhoA and ROCK were decreased in sh-RhoA group(P＜0.05),and the protein expression of E-cadherin was increased(P＜0.05).Compared with inhibitor-NC group,the cell proliferation level,the cell mobility,and the protein expressions of MMP-9 and N-cadherin in HaCaT cells were increased in miR-200c inhibitor group(P＜0.05),and the expression of E-cadherin protein was decreased(P＜0.05).Compared with miR-200c inhibitor+sh-NC group,the cell proliferation level,the cell mobility,and the protein expressions of MMP-9 and N-cadherin in HaCaT cells were decreased in miR-200c inhibitor+sh-RhoA group(P＜0.05),and the expression of E-cadherin protein was increased(P＜0.05).Conclusion miR-200c targets RhoA.The overexpression of miR-200c can inhibit the re-epithelialization process of skin wound healing by regulating RhoA/ROCK pathway,thus delaying the process of skin wound healing.
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