Quantitative Proteomic Analysis Of Keloids And The Mechanisms Of HIF-1α Regulating Hypoxia-induced Function Of Human Skin Fibroblasts

BackgroundKeloids are pathological fibrous proliferative disorders of the skin with complex and unclear molecular pathology,and new biomarkers and associated mechanisms are needed to improve current therapeutic approaches.The behaviour of skin fibroblasts will have a direct impact on wound healing and scar growth,and the effect of hypoxia on the development of keloid scarring and the mechanisms involved deserve further investigation.ObjectivesTo identify novel biomarkers and underlying pathological mechanisms of keloid scarring,to clarify the role of hypoxia in initiating the development of keloid scarring and to explore the effect of hypoxia-inducible factor 1α(HIF-1α)on hypoxia-induced fibroblast function.MethodsSix pairs of keloid tissues and corresponding normal skin tissues were first quantified by high-resolution label-free liquid chromatography tandem mass spectrometry(LC-MS/MS).The different protein expression data were further analysed by a comprehensive bioinformatics approach to identify new biomarkers and mechanistic pathways of keloid formation.The candidate biomarkers were also validated by immunoprotein blotting(Western blot,WB)and quantitative real-time PCR(qPCR)methods.Fibroblasts from normal human skin cells were extracted by enzymatic digestion and cultured in vitro using 1%O2 to simulate a hypoxic environment.The changes in proliferation,apoptosis and migration functions in response to hypoxic stress were observed using live cell proliferation curve analysis,flow analysis and scratch assay.The difference of HIF-1α expression in normal skin and keloid tissue was detected by WB analysis.Then,the expression levels of HIF-1α and Spliced X-box binding protein 1(XBP1)in hypoxic-cultured human normal skin fibroblasts were investigated by WB,qPCR and immunofluorescence staining.The effects of HIF-1α and XBP1 on the function of hypoxic-induced fibroblasts were studied by means of inhibitor and living cell proliferation curve analysis,flow analysis and scratch test.ResultsA total of 1359 proteins were identified and quantified by proteomic analysis of keloid and adjacent normal skin tissue samples.Of these,206 proteins were significantly different in expression between keloids and normal skin tissues,including Secreted protein acidic and rich in cysteine,SPARC),Reticulocalbin 3(RCN3),Reticulocalbin 1(RCN1),Calumenin,Reticulocalbin 3,Reticulocalbin 1,RCN1 Platelet-derived growth factor like(PDGFRL)and Platelet derived growth factor like(PDGFRL),which may play an important role in keloid formation.Pathway analysis indicated that the XBP1-mediated Unfolded protein response(UPR)pathway plays a role in it unfolded.In addition,a gene co-expression network centered on PDGFRL was constructed to illustrate its function in skin healing.In addition,the extracted primary human skin fibroblasts showed a long spindle shape,with typical fibroblast morphological characteristics,and expressed specific Vimentin,but did not express KRT10.The 1%O2 hypoxia treatment for 24 h promoted the migration and apoptosis of human skin fibroblasts,and inhibited the proliferation of human skin fibroblasts,suggesting that hypoxia environmental stress affects the biological function of human skin fibroblasts.HIF-1α was highly expressed in keloid tissue.The expression of HIF-1α and XBP1 in human skin fibroblasts was promoted by 1%O2 hypoxia for 24 h.After the intervention of HIF-1α inhibitor LW6,the protein level of HIF-1α in human skin fibroblasts induced by hypoxia was reduced,and the apoptosis and migration of fibroblasts under hypoxia were inhibited,without significant effect on cell proliferation.XBP1 inhibitor 4μ8c also decreased the expression of HIF-1α in hypoxic-induced human skin fibroblasts,which had no effect on cell proliferation,but inhibited cell apoptosis and migration under hypoxia.ConclusionsIn this study,five new biomarkers for keloid were proposed and a gene co-expression network centered on PDGFRL was constructed to elucidate its function in the skin.The role of XBP1-mediated UPR pathway in keloid pathology is emphasized.It provides new biological insights that could help in the development of new keloid treatment strategies.Primary human skin fibroblasts were extracted and hypoxic culture conditions were set in vitro to simulate the hypoxic internal environment of keloid.It was found that hypoxia promoted the migration and apoptosis of human skin fibroblasts and inhibited cell proliferation.HIF-1α and XBP1 were highly expressed in keloid tissue and hypoxic human skin fibroblasts.After the intervention of HIF-1α inhibitor LW6 and XBP1 inhibitor 4μ8c,it was found that both of them could reduce the expression level of HIF-1α in hypoxic-induced human skin fibroblasts,inhibit the apoptosis and migration of hypoxic-induced human skin fibroblasts,and had no effect on cell proliferation.