The Effect And Mechanism Of IFI6 On The Progression Of Radiation-induced Skin Cells Damage

Objective:Skin that composed of epidermis,derma and subcutaneous tissue is the largest organ of human body.Radiation-induced skin injury caused by radiotherapy and nuclear accidents is difficult to cure and often stay for a long time,which seriously affect the survival and prognosis of patients,and there is no specific treatment for that.Exploring the mechanism of radiation-induced skin injury and finding effective methods to prevent and treat this disease becomes very important.Methods:Human epidermal keratinocyte cell line HaCaT and human dermal fibroblast cell line WS1 were utilized for this study.Affymetrix HTA2.0 expression microarray analysis was performed to identify the differentially expressed genes in irradiated skin keratinocytes(HaCaT);Immunohistochemical experiment was performed to detect the expression of IFI6 between irradiated and non-irradiated skin tissues;HaCaT and WS1 cells were infected with IFI6 overexpression and silencing lentivirus and the protein expression level of IFI6 was verified by western blot assay;The functional changes of irradiated and nonirradiated skin cells were detected by flow cytometry,ROS probe and Edu apollo567 cell kit;Immunofluorescence experiment was utilized to figure out the distribution of relative proteins in skin cells,and immunoprecipitation(IP)combined with mass spectrometry was carried out to detect the interacting proteins of IFI6 in skin cells after irradiation.The global gene changes in IFI6-overexpressed skin cells after irradiation were detected by RNA sequencing.Results:Ionizing radiation modulated the expression of 63 genes(50 upregulated genes and 13 downregulated genes)in human keratinocyte HaCaT cells,which were implicated in multiple pathways including cytokine-and type ? interferon mediated signaling pathways and immune response.Among the dysregulated genes,interferon a inducible protein 6(IFI6)was upregulated by 7.55-fold.Immunohistochemical experiment indicated IFI6 was highly expressed in irradiated skin tissues.In IFI6-silencing human skin cells,the intracellular ROS level and apoptosis rate were significantly increased and cell proliferation was inhibited,and conversely,IFI6-overexpression decreased the radio-sensitivity of skin cells.IFI6 could translocate from cytoplasm to nucleus and was figured out 35 interacting proteins in irradiated human skin cells.IFI6 interacting protein single stranded DNA binding protein 1(SSBP1)was also found translocated to nucleus after irradiation.Nuclear translocation of SSBP1 was realized by its binding to heat shock regulator 1(HSF1).Luciferase-reporter based assay showed that ionizing radiation promoted the transcriptional activity of HSF1,and the transcriptional activity of HSF1 was positively correlated with the expression of IFI6.IFI6 overexpression combined with radiation affected more than twenty thousand genes expression in skin cells and 500 genes(409 upregulated genes and 91 downregulated genes)were changed by two or more folds.Among the upregulated 409 genes,176 genes have HSF1-binding sites which indicating the radio-sensitivity of skin cells regulated by IFI6 was associated with heat shock response(HSR)Conclusions:In conclusion,IFI6 is highly expressed in irradiated skin cells and skin tissues and decrease the radio-sensitivity of skin cells through affecting its anti-apoptotic and proliferative abilities.IFI6 was found translocated into nucleus after radiation in skin cells and could regulate the transcriptional activity of HSF1;IFI6 overexpression combined with radiation upregulated 176 genes which have HSF1-binding sites in skin cells.These results suggesting that the radio-sensitivity of skin cells regulated by IFI6 is relative to HSR.