| Background and objective:Keloids are benign fibroproliferative tumors caused by dysregulation of the wound healing process.It is characterized by excessive proliferation of fibroblasts,reduced apoptosis and excessive deposition of extracellular matrix.The clinical symptoms of keloids affecting the appearance and itching cause great pain to patients.Because the growth characteristics of keloids are similar to that of tumors that continue to exceed the wound boundary and the high recurrence,it brings great difficulty to clinical treatment.The pathogenesis of keloids has not been clarified.There is no specific and completely cured treatment method.It is the focus of researchers to clarify the mechanism and improve the therapy.Fibronectin,as one of the extracellular matrix increased in keloids,participates in various physiological and pathological processes.Among various isoforms of fibronectin,fibronectin extra domain B(EDB)that highly expressed in various tumors and fibrotic diseases is involved in the fibroblast morphology,matrix assembly and synthesis process.But EDB has not been studied in keloids.The present study aimed to investigate the expression and the influence of EDB on keloid and elucidate the putative signaling pathway.Methods:1.Collect keloid tissue and foreskin tissue removed from 10 clinical patients.Hematoxylin and eosin staining was performed on keloids and surrounding normal tissues to observe the pathological morphology of keloids.Immunohistochemical staining was used to detect EDB protein in keloid tissue and normal skin tissue.Tissue protein was extracted for Western blot experiment to detect the expression of EDB protein in the tissue.The keloid and normal foreskin fibroblasts were cultured in vitro using digestion and tissue block methods.Western blot was used to identify relevant proteins based on the characteristics of these two fibroblasts and detect the expression of EDB in cells and cell culture medium.2.After knockdown EDB in keloid fibroblasts.Western blot was used to check the expression of EDB in cells and culture medium,and to estimated the expression of EDA and FN to confirm the specificity of EDB knockdown.MTT method and flow cytometry were used to verify the effect of EDB knockdown on keloid fibroblast cell proliferation and apoptosis.The scratch experiment tested the effect of EDB knockdown on cell migration,and examined the expression levels of proteins MMP-2 and MMP-9 that affected migration.The changes of collagen and α-SMA in cells and culture medium was detect by Western blot after EDB knockdown.Finally,in order to explore the potential mechanism through which the above functional changes may be passed,we investigated the expression of TGF-β1/Smad,MAPK.PI3K/AKT pathway-related proteins.3.Treat keloid fibroblasts with 10ng/mL TGF-β1 recombinant protein to simulate the internal environment of keloids.MTT assayed cell proliferation changes with or without TGFβ1 treatment after EDB knockdown.Western blot and qRT-PCR were used to test collagen secretion.We also examined the expression of TGF-β receptor after TGF-β1 treatment and knockdown of EDB protein and re-examined the expression of TGF-β1/Smad.MAPK,PI3K/AKT pathway related proteins.Results:1.HE staining showed that there was a lot of collagen fiber accumulation in the keloid tissue,fibroblasts increased,and the epithelial feet became dull and disappear.EDB immunohistochemistry showed that compared with normal skin tissue,the positive epidermal and dermal layers of keloids showed obvious EDB positive expression,mainly expressed in cytoplasm and extracellular matrix.Western blot results support the results of immunohistochemistry.The expression of EDB in the protein extracted from keloid tissue was significantly increased.The morphology of fibroblasts cultured in vitro is classic fusiform,and compared with normal fibroblasts,keloid fibroblasts highly express FN,pro-Col Ⅰ,Col-Ⅲ,αSMA,which is consistent with the characteristics of keloid fibroblasts.EDB.EDA and FN are highly expressed in KFs cells and in cell culture medium,the difference was statistically significant(P<0.05).2.EDB knockdown efficiency reached about 70%.EDB Knockdown is specific and has no significant effect on other isoforms.EDB knockdown inhibited keloid fibroblast proliferation,but had no effect on KFs apoptosis.In the scratch test results,it can be observed that the migration rate of KFs becomes slower,and the expression levels of MMP-2 and MMP9 proteins decrease.The expression level of cells and extracellular collagen suppressed by EDB knockdown,but the expression level of α-SMA protein was not affected.In the pathways of TGF-β1/Smad,MAPK and PI3K/AKT,the expression of phosphorylated Smad2,Smad3,ERK and AKT was suppressed,but p-P38 and p-JNK had no effect,the difference was statistically significant(P<0.05).3.After treatment with TGF-β1 recombinant protein,the cell proliferation rate increases,but this cell proliferation of KFs effect was suppressed by EDB knockdown.TGF-β1 can induce KFs to produce EDB.It also can obviously promote the intracellular and extracellular collagen deposition of KFs,EDB siRNA has an inhibitory effect on these promotions.EDB knockdown can also reduce the increase in TGF receptors on the cell surface caused by TGF-β1.During the detection of the pathway.it was found that TGF-β1 stimulated the activation of TGF-β1/Smad.MAPK,PI3 K/AKT pathway.while the inhibitory effect of EDB knockdown was only reduced in Smad2,Smad3.ERK.AKT,the difference was statistically significant(P<0.05).Conclusions:1.It was the first time to proved that EDB is highly expressed in keloid tissue and KFs.2.Knockdown of EDB has a inhibitory effect on the proliferation,collagen deposition and migration of keloid fibroblasts,but it has no significant effect on apoptosis and α-SMA protein expression.The above biological functions were mediated by TGF-β1/Smad.ERK/AKT signaling pathway.3.EDB Knockdown can suppress the proliferation of KFs induced by TGF-β1,reduce the expression of TGF-β receptors on the cell surface and collagen synthesis.The underlying mechanism was verified again by the inhibition of TGF-β1/Smad,ERK/AKT signaling pathway.The results show that EDB cooperates with TGF-β1 in the formation of keloids,which can regulate cell proliferation and collagen expression.EDB is a new target for the treatment of keloids. |
