| Objectives To study the role of miR-411-3p in Bleomycin(BLM)-induced skin fibrosis,and to explore whether it can regulate the mechanism of skin fibrosis by targeting myocardin related transcription factor-A(MRTF-A)/Serum response factor(SRF)signal.Methods C57BL/6 mice were given bleomycin stimulation to establish a skin fibrosis model.The experiment was divided into 4 groups: control 4W group,bleomycin model4 W group,BLM 4W + miR-411-3p agomir-NC group,BLM 4W + miR-411-3p agomir group;primary skin fibroblasts were extracted from new rats,the experimental groups were as follows:(1)control group,BLM-induced group,(2)miR-411-3p mimic-NC group,miR-411-3p mimic group,miR-411-3p inhibitor-NC group,miR-411-3p inhibitor group,(3)si RNA-NC group,si-MRTF-A group,(4)miR-411-3p mimic-NC + BLM group,miR-411-3p mimic + BLM group,miR-411-3p inhibitor-NC + BLM group,miR-411-3p nhibitor + BLM group.HE and VG staining were used to observe the pathological morphology of skin tissues,the localization and expression of miR-411-3p in skin tissues and fibroblasts were tested by in situ hybridization,and the F-actin of skin fibroblasts was detected by Phalloidin staining.Immune cell fluorescence staining was used to test the expression of type I collagen(COLI)in skin fibroblasts,RT-qPCR tests the expression of miR-411-3p,TGF-β1,α-SMA,COLI,MRTF-A and SRF in skin tissues and skin fibroblasts,western blot measures the expression of smooth muscle actin(α-SMA),COLI,MRTF-A,SRF in skin tissue and skin fibroblasts,and the expression of TGF-β1,Rho A and p-ROCK factors in skin fibroblasts.The dual luciferase reporter assay tests the regulation of miR-411-3p on MRTF-A.Results 1 Compared with the control group,the expressions of F-actin,α-SMA,and COLI were significantly up-regulated in the BLM-induced group,as well as the expression of TGF-β1,MRTF-A,SRF,Rho A and p-ROCK factors are up-regulated,but miR-411-3p mimic treatment can inhibit the expression of above changes which were induced by BLM(P(27)0.05).2 Compared with mimic-NC group,miR-411-3p mimic can downregulate the expression of F-actin,?-SMA and COLI,meanwhile,reduce the expression of TGF-β1,MRTF-A,SRF,Rho A and p-ROCK factors in skin fibroblasts(P(27)0.05).3 Compared with inhibitor-NC group,miR-411-3p inhibitor exerts produced effects on the expression of F-actin,α-SMA and COLI,and also increased the expression of TGF-β1,MRTF-A,SRF,Rho A and p-ROCK factors(P(27)0.05).4 compared with mimic-NC +BLM group,miR-411-3p mimic can F-actin,?-SMA and COLI,meanwhile,reduce the expression of TGF-β1,MRTF-A,SRF,Rho A and p-ROCK factors in BLM-induced skin fibroblasts(P(27)0.05).5 Compared with inhibitor-NC+BLM group,miR-411-3p inhibitor exerts produced effects on the expression of F-actin,α-SMA and COLI,and also increased the expression of TGF-β1,MRTF-A,SRF,Rho A and p-ROCK factors in BLM-induced skin fibroblasts(P(27)0.05).6 The dual luciferase reporter gene assay shows that miR-411-3p has a regulatory effect on the 3’URT of MRTF-A(P(27)0.05).7 The expression of α-SMA,COLI and SRF of si-MRTF-A group compared with si-NC group is down-regulated in primary skin fibroblasts(P(27)0.05).8 HE and VG staining show the thickening of skin dermis of the bleomycin stimulation group compared with the control group is increased.The gene expression level of miR-411-3p is reduced,and the expression of α-SMA and COLI is significantly increased.The expression of MRTF-A/SRF signalling factor was up-regulated(P(27)0.05).9 The expression of α-SMA,COLI and MRTF-A/SRF signalling factors was decreased of miR-411-3p agomir group compared with miR-411-3p agomirNC group in skin tissue(P(27)0.05).Conclusions 1 In skin fibroblasts,bleomycin can stimulate the secretion of TGF-β1which induce the transformation of fibroblasts.2 The signalling of TGF-β1/MRTF-A can promotes the occurrence and development of skin fibrosis.3 miR-411-3p inhibits bleomycin-induced skin fibrosis by regulating TGF-β1/Rho A signalling pathway via targeting MRTF-A.Figure28;Table33;Reference 74... |
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