Study On The Role Of Splicing Factor SRSF2 In Keloid Fibroblasts

BackgroundKeloids are benign fibroproliferative tumors that can occur following skin trauma,characterized by the proliferation of dermal fibroblasts and the increase of extracellular matrix secretion.Keloids are inclined to recur after resection,and there is no effective treatment for now.The pathogenesis of keloids has not been clear,and it is believed that fibroblasts play a major role in the formation of keloids.The study of the biological characteristics of fibroblast proliferation,apoptosis and migration,as well as its extracellular matrix and cytokines secretion function,is of great significance to the study of the pathogenesis of keloids.The post-transcription regulation of genes can affect the biological characteristics of cells without causing changes in DNA.The post-transcription regulation of keloids fibroblasts is seldom studied at present.The splicing factor SRSF2(serine and arginine rich splicing factor 2,SRSF2)is also known as SC35,which regulates the expression of target genes by affecting the splicing and mRNA stability of the precursor mRNA.Studies have shown that SRSF2 can affect the apoptosis,proliferation and other biological characteristics of a variety of cells,but the role in the adult fibroblasts has not been related to the study.Therefore,the study of the effect of SRSF2 on the biological characteristics and function of fibroblasts may provide a new insight into keloids pathogenesis.Objective1.To clarify the expression of SRSF2 in normal skin and keloids.2.To clarify the effect of SRSF2 on the biological characteristics of normal skin and keloids fibroblasts.3.To clarify the effect of SRSF2 on the expression of extracellular matrix and the secretion of cytokines in normal skin and keloids fibroblasts.Methods and results1.The expression of SRSF2 in normal skin and keloidsMethods:The expressions of SRSF2 were assessed by immunohistochemistry staining in normal skin and keloid tissues.Results:Compared to normal skin,the expression of SRSF2 was upregulated in keloids,and SRSF2 was located in the nucleus of fibroblasts.2.Bioinformatics analysis of the possible role of SRSF2 in keloidsMethods:Bioinformatics analysis was carried out to analyze the SRSF2 target genes aquired from online database.Gene expression profiles of normal skin and keloids were obtained from GEO datasets,and the differential genes were screened for bioinformatics analysis.Also,bioinformatics analysis was carried out by the intersection of SRSF2 target gene and the selected differential genes.Results:SRSF2 target genes were mainly enriched in transcription coregulator actitity,extracellular exosome,intracellular signal transduction,cell cycle and so on.The difference genes of normal skin and keloid tissue were mainly enriched in extracellur matrix structural constituent,collagen binding,skeletal system development,cell adhesion and so on.The SRSF2 targeted differential genes are mainly enriched in extracellur matrix structural constituent,collagen binding,growth factor binding and so on.3.The effect of SRSF2 on the biological characteristics of normal skin and keloids fibroblasts.Methods:Lentivirus carrying SRSF2-overexpression plasmids and lentiviral sh-RNA-expression vector targeting SRSF2 were constructed to change the expression of SRSF2 in fibroblasts.Annexin V17-ADD staining and flow cytometry were used to analyze the apoptosis of fibroblasts.PI staining and flow cytometry were performed to analyze the cell cycle.The cell proliferation curves were plotted by Cell Counting Kit-8(CCK-8)assay,and migration abilities were assessed by scratch wound healing assay and transwell assay.Results:SRSF2 was related to the apoptosis,proliferation and migration of fibroblasts.Normal skin fibroblasts with excessive expression of SRSF2 had enhanced proliferation and impaired migration.Downregulation of SRSF2 in normal skin and keloids fibroblasts decreased the proliferation and increased the apoptosis of cells.4.The effect of SRSF2 on the function of normal skin and keloids fibroblasts.Methods:Upregulate the expression of SRSF2 in normal skin fibroblasts,or decrease the expression of SRSF2 in normal skin and keloids fibroblasts.Then detect the expression of extracellular matrix genes and the secretion of some cytokines.Furthermore,SRSF2 would be detected after the stimulation of TGF-?1 in fibroblasts.Results:After the knock-down of SRSF2,the expressions of COL3A1,FN1,TGF-?1 and CXCL12 were declined in both normal skin and keloids fibroblasts.In addition,SRSF2 was up-regulated after TGF-?1 stimulation.Conclusions1.The expression of SRSF2 increased in keloid,and located in the nucleus of fibroblasts.2.SRSF2 was involved in the apoptosis,proliferation and migration of fibroblasts.Normal skin fibroblasts with excessive expression of SRSF2 had enhanced proliferation and impaired migration.Downregulation of SRSF2 in normal skin and keloids fibroblasts decreased the proliferation and increased the apoptosis of cells.3.SRSF2 was up-regulated by TGF-?1 in fibroblasts.The inhibition of SRSF2 in fibroblasts restricted the expression of extracellular matrix COL3A1?FN1 and cytokines TGF-?1?CXCL12.