The Effect And Mechanism Of Syndecan-1 In Keloid Formation

Background:Keloid is caused by cutaneous injury and characterized by the abnormal hyperplasia of the fibroblasts and excessive extracellular matrix(ECM)secretion.Keloid is a skin fibrosis disease associated with abnormal wound healing.It can not be treated satisfactorily and have a high recurrence rate.Keloid can be functionally disabling with violent pruritus and pain and may lead to psychological distress to patients.Although the pathogenesis of keloids has not been fully elucidated,it is currently believed to be related to genetic predisposition,inflammation,tension alignment,and immunological conditions.During keloid,the fibroblasts are activated and differentiated into myofibroblasts.Then,they start to proliferate and produce ECM protein,and have tumor-like characteristics that invade adjacent normal skin tissues.Recent studies have shown that the expression of syndecan-1(SDC-1)is play a vital role in cell invasion,proliferation and migration in tumor.Moreover,the expression of SDC-1 is associated with various fibrotic diseases(such as heart,lung,liver),suggesting that SDC-1 may be closely related to the keloid fibroblasts proliferation and excessive deposition of ECM.In this study,we investigate the expression of SDC-1 in keloid tissues and fibroblasts,and used the small interfering RNA(siRNA)method to knock down the SDC-1 protein in KFs.To explore SDC-1 in cell proliferation and expression of ECM.In addition,we further clarify the knockdown of SDC-1 on ECM protein induced by Transforming growth factor-β1(TGF-β1)and its mechanism of action.Objective:To clarify the expression of SDC-1 in human keloid tissues and fibroblasts.To explore the effect of SDC-1 knockdown on KFs cell proliferation,apoptosis,ECM proteins and fibrosis-related signaling pathways related proteins expression.To further investigate the knockdown of SDC-1 on TGF-β1-induced ECM proteins and its mechanism of action in KFs.Methods:1.Keloids(n=10)and their adjacent normal skin tissues(n=10)were collected,H&E staining,immunohistochemistry and Western blot were performed to investigate the difference of SDC-1 expression in keloid tissues.The primary keloid fibroblasts and normal fibroblasts cell culture was performed by collagenase Ⅱ digestion.And the expression of ECM protein was detected by Western blot.In addition,qRT-PCR and Western blot were used to detect the expression of SDC-1 in keloid fibroblasts.2.Primary cultured keloid fibroblasts were transfected with SDC-1 siRNA.After verifying the successful knockdown of SDC-1,the function of SDC-1 on the cell proliferation,cell cycle and cell apoptosis in KFs was investigated through Cell Counting Kit-8(CCK-8)and flow cytometry.ECM,TGF-β1/Smad,MAPK and PI3K/AKT related proteins were evaluated by Western blot.3.The KFs were treated with 0,5,10 and 20 ng/mL TGF-β1 for 48 hours.Western blot was used to detect the expression of ECM proteins,and the most effective concentration was screened for downstream studies.After 24 hours of SDC-1 siRNA treatment,5 ng/mL TGF-β1 treatment for 48 hours,CCK-8 was detected the cell proliferation in KFs,Western blot was detected the expression of ECM proteins,TIMP-1 and MMP-2 in KFs.After successfully knockdown of SDC-1,5 ng/mL TGF-β1 was treated for 1 hour,the expression of TGF-β receptor I,TGF-β1/Smad,MAPK and PI3K/AKT signaling pathways related proteins in KFs were detected by Western blot.Results:1.H&E staining results showed that more inflammation and excessive collagen deposition appeared in keloids.The level of SDC-1 was markedly higher in keloid tissues as compared to the adjacent normal skin tissues(**P<0.01).Western blot was used to detect the expression of ECM proteins in primary cultured keloid fibroblasts(KFs)and normal fibroblasts(NFs).The results showed that compared with NFs,the expression of ECM proteins was increased in KFs(*P<0.05,**P<0.01).qRT-PCR and Western blot results also showed that SDC-1 was upregulated in the KFs compared with NFs(*P<0.05,**P<0.01).2.SDC-1 siRNA 3 was selected for the downstream studies(**P<0.01).Intriguingly,the SDC-1 siRNA-treated group showed lower levels of cell proliferation than the NC siRNA-treated group in KFs(*P<0.05).Knockdown of SDC-1 could stagnate the cell cycle G1/S transition and suppress the expression of cell cycle-related target gene Cyclin D1 in KFs(*P<0.05).However,the knockdown of SDC-1 did not affect the level of cell apoptosis of KFs(P>0.05).SDC-1 siRNA-treated group showed lower ECM proteins expression in cell lysate as compared to the NC siRNA-treated group(*P<0.05,**P<0.01).In addition,the knockdown of SDC-1 suppressed the ECM protein levels in the culture media(*P<0.05,**P<0.01).Additionally,SDC-1 knockdown inhibited the level of TGF-β1/Smad,p-ERK,p-AKT and p-P38 signaling pathways related proteins in KFs(*P<0.05,**P<0.01).3.Treatment of KFs with different concentrations of TGF-β1 significantly increased the expression of ECM proteins compared with the control group(*P<0.05,**P<0.01).TGF-β1+SDC-1 siRNA group could inhibit the expression of TGF-β1-induced cell proliferation compared with the TGF-β1 group in KFs(#P<0.05).Knockdown of SDC-1 could inhibit the expression of TGF-β1-induced ECM proteins,TIMP1 and MMP-2 in KFs(#P<0.05,##P<0.01).Moreover,knockdown of SDC-1 suppressed TGF-β1-induced TGF-β receptor I,TGF-β1/Smad,p-ERK,p-AKT and p-P38 signaling pathways related proteins expression(#P<0.05,##P<0.01).Conclusions:1.The level of SDC-1 was markedly higher in the KFs and keloid tissues.2.Knockdown of SDC-1 could suppress cell proliferation in KFs.However,there was no significant effect on cell apoptosis.3.SDC-1 regulated ECM expression in keloid fibroblasts via TGF-β1/Smad,p-ERK,p-AKT and p-P38 signaling pathways.4.Knockdown of SDC-1 possibly inhibited TGF-β1-induced ECM proteins,TIMP-1 and MMP-2 expression via TGF-β1/Smad,p-ERK,p-AKT and p-P38 signaling pathways,thereby promoting ECM degradation and inhibiting KFs migration.