Expression Of SphK1/S1P Signaling Pathway In Keloids And Related Research

Background:Keloid is fibroproliferative disease that arises from deep skin lesions caused by trauma,burns,or surgery.Whether it is in appearance,or in functions(such as contracture)or in subjective feelings(including pruritus),it affects the life quality of patients.There are many related studies,but its formation mechanism has not yet been established,prevention and treatment strategies are still unsatisfactory.Sphingosine-1-phosphate(SIP)and its upstream key enzyme responsible for its production,sphingosine kinase 1(SphK1),are widely studied in a variety of human diseases,including various solid cancers,muscular contracture,inflammatory disease,Neurological Diseases,Age-Related Macular Degeneration(AMD),and Fibrosis Diseases,but as a tumor-like disease,and who has fibrotic feature,keloid has not been reported in almost literature so far.This experiment was designed to explore the relationship between keloid,SphK1/SlP pathways and fibrosis,with the results of oncological research,to provide a basis for new therapeutic targets for keloids.Objective:To analyze the relationship between SphK1/SlP pathway and keloid,by examining the differential expression of SphK1 and S1P in histology and cytology;to investigate how will the gene silencing impact on the proliferation and migration of keloid fibroblasts and how will the fibrosis-related protecins express by inducing the SIP gene silencing.To provide evidence and practical value to reveal targets for keloid treatment further.Methods:1.Keloid surgical specimens and normal skin specimens from surgical patients were selected from May 2016 to December 2017 in Yanbian University Affiliated Hospital,and those were used to prepare paraffin specimens and conduct primary cell culture.2.The paraffin specimens were prepared for HE staining,and immuno histochemistry(IHC).The expression differences of SphK1 and SlP in tissues were detected by IHC.3.3-10 generation of fibroblasts were selceted to cytological experiments,using Western blot assays to detect the expression differences on SphK1 and SlP between primary cultured keloid fibroblasts cell and normal fibroblasts cell.4.RNAi technology was used to transfect specific S1PsiRNA into keloid fibroblast cell,and the silencing effect was detected by Western blotting assays.The gene sequence with the most silence efficiency was screened.5.Western blot assays were performed to detect the expression of fibrosis-related proteins ?-SMA and Collagen type III.6.The cell scratch experiment was used to detect the effect of S1P gene silencing on the inhibition of migration of keloid fibroblast cell.Results:1.Successfully cultured primary cells and wax block specimens were selected for the experiment.2.Immuno histochemistry showed that in the organization the expression of SphK1 and SlP were up-regulated in keloid group compared with normal skin group.3.There was no significant difference in the expression of SphK1 between keloid fibroblasts cell and normal fibroblasts cell by Western blotting assays.The expression of SlP in keloid fibroblasts cell was significantly higher than that of nomal fibroblasts cell,and the difference was statistically significant(P<0.01).4.The cell gene silencing effect was clearly observed in the 3 strands,and the S1Psi-RNA1,S1Psi-RNA2,S1Psi-RNA3 transfection groups protein were decreased by 34.7%,20.1%,43.8%,(P<0.05),and the difference between the experimental group and the control group was greatest at the concentration of 100 nmol and the transfection time was 24 hours.5.After gene silencing,the expression levels of fibrosis-related proteins?-SMA and Collagen type III were down-regulated(P<0.05).6.Scratch test showed that the cell migration distance gradually decreased with time(P<0.05).The difference in migration distance between the experimental group and the control group was the largest at 24 h(P<0.05).Conclusions:1.The expression of SphK1/SlP axis is increased in keloids,and SlP is related to fibrosis and promote cell migration.2.The SphK1/SlP pathway has an intervention effect on the control of keloid fibrosis,and we conclude that the SphK1/SlP pathway may be used as a new potential target for the treatment of keloid patients(KD)in the future and should be further studied.