Study On The Effect And Mechanism Of USP47 On Radiation-induced Skin Injury

Objective The ubiquitination of proteins is involved in the post-translational modification of proteins in important cell processes.Ubiquitin specific proteases 47(USP47)is a cysteine protease with the function of deubiquitinating substrates,belong to the deubiquitinases family.This study mainly explored the role of USP47 in radiation-induced skin injury and its mechanism.Methods(Ⅰ)Exploring the effect of USP47 on the progression of radiation-induced skin injury at the animal level:1.The knockout mice were identified and bred,and the body weight during the growth period was tracked to evaluate the effect of the knockout USP47 gene on growth,development and reproduction in mice.2.USP47 knockout mice were used to establish a radiation-induced skin injury model.The wound score statistics,taking wound photos and tissue hematoxylin staining were used to assess the skin injury of mice.(Ⅱ)Exploring the effect of USP47 on the progression of radiation-induced skin injury at the cellular level:1.The expression and localization of USP47 after skin cells irradiated were detected by immunofluorescence and Western blot.2.USP47 knockout lentivirus was used to infect skin cells,and its knockout effect were verified by Western blot and fluorescent quantitative PCR detection;3.CCK-8 and EdU experiments were used to explore the effect of USP47 gene knockout on the proliferation of irradiated skin cells;4.Flow cytometry was used to explore the effect of USP47 gene knockout on the apoptosis rate of irradiated cells.5.Scratch test was used to explore the effect of USP47 gene knockout on skin cell migration ability.6.Flow cytometry method was used to detect the effect of USP47 gene knockout on the cell cycle distribution of irradiated skin cells.(Ⅲ)High-throughput proteomics was used to analyze the skin tissues of mice with radiation-induced skin damage,to study the differences in proteins between USP47 knockout mice and wild mice after irradiation,and to explore the changes in protein functions that USP47 affected skin tissues.Results(1)There was no significant difference in growth and development between USP47 knockout mice and wild-type mice,but USP47 knockout mice have lower reproductive rate.(2)In the radiation damage model established by USP47 knockout mice,USP47 knockout mice compared with wild mice had severe skin damage and slow healing.(3)Compared with wild-type mice,USP47 knockout mice showed no significant difference in skin appendages and epidermal thickness after exposure.(4)Immunofluorescence and Western blot results showed that USP47 was located in the nucleus and the expression level of protein increased with dose and changed with time.(5)The proliferation ability and migration ability of skin cells with USP47 gene knockout decreased after exposure.(6)The apoptosis rate of skin cells with USP47 gene knockout increased after exposure,and the cell cycle of skin cells with USP47 gene knockout was arrested.(7)Through proteomics,3,251 differentially expressed proteins were quantified,of which 63 were upregulated 1.5 times and 170 were down-regulated 1.5 times.(8)Bioinformatics analysis revealed that the differentially expressed proteins were mainly concentrated in extracellular regions,the nucleus and cytoplasm of cells,and mainly involved in several biological processes including post-translational modification,protein renewal,molecular chaperone and defense mechanism.The major signaling pathways involved in the study were complement and coagulation cascade signaling,prion disease signaling,retinol metabolism signaling and iron death signaling.Conclusion Ubiquitin-specific protease 47(USP47)is involved in the progression of radiation-induced skin injury.The absence of USP47 will cause more severe damage to the skin of mice after irradiation,USP47 silenced skin cells will also have varying degrees of negative effects after irradiation.USP47 is expected to be a new target for the prevention and treatment of radiation-induced skin injury.