miR-200c通过抑制RhoA/ROCK通路延缓皮肤创伤愈合进程

miR-200c delays the process of skin wound healing by inhibiting RhoA/ROCK pathway

目的探究miR-200c在皮肤创伤愈合中的作用及其调控机制。方法构建SD大鼠背部全层皮肤创伤模型,将创伤后大鼠分为agomir-NC组、miR-200c agomir组、sh-NC组和sh-RhoA组。采用qRT-PCR检测创面组织中miR-200c表达,Western blot检测创面组织中MMP-9、N-cadherin、E-cadherin和RhoA蛋白表达。创伤后第1,7,14天计算大鼠创面愈合率。采用双荧光素酶报告实验分析miR-200c和RhoA靶向关系。将人永生化角质形成细胞系HaCaT细胞分为inhibitor-NC组、miR-200c inhibitor组、miR-200c inhibitor+sh-NC组和miR-200c inhibitor+sh-RhoA组。采用EdU法检测细胞增殖,细胞划痕实验检测细胞迁移,qRT-PCR检测HaCaT细胞中miR-200c表达,Western blot检测HaCaT细胞中MMP-9、N-cadherin、E-cadherin、RhoA和ROCK蛋白表达。结果与正常皮肤比较,创面组织中miR-200c表达降低(P<0.01),RhoA表达升高(P<0.01)。miR-200c与RhoA 3′UTR存在靶向结合关系。与agomir-NC组比较,miR-200c agomir组大鼠创面愈合率以及MMP-9、N-cadherin、RhoA和ROCK蛋白表达均降低(P<0.05),E-cadherin蛋白表达升高(P<0.05);与sh-NC组比较,sh-RhoA组大鼠创面愈合率以及MMP-9、N-cadherin、RhoA和ROCK蛋白表达均降低(P<0.05),E-cadherin蛋白表达升高(P<0.05)。与inhibitor-NC组比较,miR-200c inhibitor组HaCaT细胞增殖水平、细胞迁移率以及MMP-9和N-cadherin蛋白表达均升高(P<0.05),E-cadherin蛋白表达降低(P<0.05);与miR-200c inhibitor+sh-NC组比较,miR-200c inhibitor+sh-RhoA组HaCaT细胞增殖水平、细胞迁移率以及MMP-9和N-cadherin蛋白表达均降低(P<0.05),E-cadherin蛋白表达升高(P<0.05)。结论miR-200c靶向RhoA,过表达miR-200c可以通过调控RhoA/ROCK通路抑制皮肤创伤愈合的再上皮化过程,进而延缓皮肤创伤愈合进程。

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.