| Background: Keloid is a kind of benign fibrous tumor of the skin caused by excessive proliferation of fibroblasts and excessive deposition of extracellular matrix in the dermis,as well as an abnormal wound healing response caused by skin injury and irritation.Because of the unclear specific pathogenesis of keloid,clinical treatment is limited.It is still a huge challenge to surgeons due to the high recurrence rate.Early growth response factor 1(Egr1)is an important transcription factor in a variety of physiological and pathological pathways,and its abnormal expression is related to a variety of human disease processes,including cancer,inflammation,atherosclerosis and some fibrotic diseases.Abnormal Egr1 expression may affect the pathogenesis of keloid by regulating downstream target genes related to inflammation,immune response types,cell proliferation and apoptosis and EMT,etc.Numerous studies have confirmed the abnormal high activation of the TGF-?1/smad signaling pathway in keloids.In addition,there are many studies in scleroderma,pulmonary fibrosis,and animal models of colitis have confirmed that TGF-?1 can up-regulate the expression of Egr1 through the TGF-?1/smad signaling pathway.However,the relationship between TGF-?1/smad signaling pathway and Egr1 in keloids has not been clarified yet.Objective:We aim to investigate the correlation between the expression of Egr1 and TGF-?1/smad signaling pathway in human keloids.It may help to elucidate the molecular mechanism of the interaction between Egr1 and TGF-?1/smad signaling pathway,so as to provide a new theoretical basis and target for keloid treatment.Experimental method:In this study,keloids are used as the research object by collecting keloid samples.Screening target genes(TGF-?1/smad signal pathway and Egr1)based on keloid gene expression profiles and Meta analysis results.Immunohistochemistry,western blot,and q-PCR are used to detect mRNA and protein expression changes of Egr1,?-SMA,Col1,Col3 and key target genes in TGF-?1/smad pathway in keloids.Secondly,construct human fibroblast fibrosis modelby adding TGF-?1 to human skin fibroblasts(HSF).Western blot and q-PCR are used to detect mRNA and protein expression changes of Egr1,?-SMA,Col1,Col3,and key target genes in TGF-?1/smad pathways in HSF fibrosis models induced by TGF-?1.Results:1.Results of mRNA expression profiles of human keloids:Compared with normal skin,777 genes are up-regulated and 568 genes are down-regulated in the skin of the keloid central area.Compared with skin larger than 3 mm from the keloid margin,the skin of the keloid central area has 962 genes up-regulated and 464 genes down-regulated.Compared with normal skin,37 genes are up-regulated and 173 genes are down-regulated in areas larger than 3 mm from the keloid margin.According to the differential gene enrichment analysis,we find that the differential genes are mainly included in the extracellular matrix formation and degradation,collagen metabolism and other signal pathways,and there are significant changes in genes related to the TGF-?signal pathway.2.Changes in mRNA expression of ?-SMA,Col1,Col3,key target genes in TGF-?1/smad pathway and Egr1 in human keloids:(1)Changes in mRNA expression of ?-SMA,Col1,Col3,and Egr1 in the three groups of samples are consistent:the mRNA expression in the skin of the keloid central area is higher than the skin larger than 3mm from the keloid margin and normal skins,the skin larger than 3mm from the keloid margin group is higher than normal skin.(2)Changes in TGF-?1,Smad3,and Smad7 mRNA expressions are inconsistent with the above trends:The trend of mRNA expression of TGF-?1 is that the skin larger than3 mm from the keloid margin is significantly higher than normal skin,and the skin in the keloid central area is higher than the normal skin and skin larger than 3mm from the keloid margin,but there is no statistical significance.The expression trend of Smad3 mRNA is that the skin larger than 3mm from the keloid margin is significantly higher than the skin of keloid central area and normal skin,and the skin area of the keloid central area is significantly higher than normal skin.The trend of mRNA expression of Smad7 is there is no significant difference in the skin of the keloid central area,the skin larger than 3 mm from the keloid margin and normal skin.3.Changes in protein expression of ?-SMA,collagen,key target genes inTGF-?1/smad pathway and Egr1 in human keloids(1)Changes of protein expression of ?-SMA and key target genes in TGF-?1/smad pathways in human keloids:The results of immunohistochemistry show that:the expressions of ?-SMA,TGF-?1,Smad3,Smad2,and Smad7 in the three groups of skin samples are mainly concentrated in the epidermis and dermis.Among them,the dermis of normal skin is relatively loose and scattered with a little of cells.Compared with the skin larger than 3mm from the keloid margin and the normal skin,the skin of keloid central area's stratum corneum and dermis are thicker and the fibrous tissue is denser.The results of western blot show that the expression of ?-SMA,TGF-?1,Col1,Smad3,and Smad2 in the skin larger than 3 mm from the keloid margin group are higher than those in the normal skin,but the differences between the skin of the keloid central area and skin larger than 3 mm from the keloid margin,skin of the keloid central area and normal skin are different.(2)Changes of collagen expression in human keloids:The results of masson staining show that compared with the skin of the keloid central area,the expression of collagen in the areas larger than 3mm from the keloid margin and the normal skin is smaller.It mainly concentrate in the fibrous tissue of the dermis,and the collagen arrangement is loose and regular,and mostly parallel to the dermis.Compared with the areas larger than3 mm from the keloid margin and normal skin,skin of the keloid central area has denser collagen,and the collagen is arranged in disorder,some are even perpendicular to the dermis.(3)Changes in protein expression of Egr1 in human keloids:Immunohistochemical results show that Egr1 expression is mainly concentrated in the basal of the epidermis and dermal cells.The expression of Egr1 in the skin of the keloid central area is higher than that in the normal skin and areas larger than 3mm from the keloid margin.There is no difference between the skin larger than 3mm from the keloid margin and normal skin.The results of western blot show that the expression of Egr1 protein in skin of the keloid central area is lower than that in skin larger than 3mm from the keloid margin,but higher than the normal skin.3.Changes in mRNA and protein expressions of ?-SMA,Col1,key target genes inTGF-?1/smad pathway and Egr1 in HSF fibrosis model induced by TGF-?1(1)Dose-effect relationship between TGF-?1 and?-SMA,Col1,key target genes in TGF-?1/smad pathway and Egr1 in HSF fibrosis model:The results show that with the increase of TGF-?1 concentration,mRNA expression of ?-SMA,Col1,Smad3,Smad2,Smad7 and Egr1 show an upward trend before 2ng/ml,and a downward trend after2ng/ml.The mRNA expression of these factors reached the highest when the TGF-?1concentration is 2ng/ml.(2)Time-effect relationship between TGF-?1 and ?-SMA,Col1,key target genes in TGF-?1/smad pathway and Egr1 in HSF fibrosis model:The results show that over time,the mRNA expression of ?-SMA,Col1,Smad3,Smad2,and Smad7 show an upward trend before 6h,and a downward trend after 6h.At 6h,the mRNA expressions are highest and all are higher than those of the control group(Time is 0h).Conclusion:1.The expression profile of human keloid mRNA is significantly changed compared with normal skin and skin more than 3 mm from the keloid margin.These differential genes are mainly included in the extracellular matrix formation and degradation,and collagen metabolism signal pathways.And the expression of genes related to TGF-? signaling pathway has changed significantly.2.The extracellular matrix in human keloids changes,the expression of fibrosis markers and Egr1 changes significantly,and the TGF-?1/smad signaling pathway is activated.3.Exogenous TGF-?1 can activate the expression of TGF-?1/smad signaling pathway and fibrosis markers in the cell model.The activation of TGF-?1/smad signaling pathway is positively correlated with the expression of Egr1.Safely and effectively blocking the TGF-?1/smad signaling pathway and inhibiting the expression of Egr1 are expected to become a new method for the clinical treatment of keloids. |
PDF Full Text Request