Preliminary Study On The Effects And Mechanisms Of Ubiquitin-specific Peptidase 11 On Radiation Induced Lung Injury

Objective : In order to study the biological effects and regulatory mechanisms of Ubiquitin-specific peptidase 11(USP11)on the development and progression of radiation-induced lung injury.Methods :(1)Usp11 gene knockout mice were constructed,reproduced,and identificated.The growth and development of gene knockout mice were recorded as well.(2)The variations of leukocytes and lymphocytes of mice with different Usp11 gene status were observed after irradiated with 9 Gy of total body irradiation(TBI).(3)and the overall survival were examined after exposed to 7.5Gy TBI.(4)The radiation-induced lung injury mouse model was established by exposing the right lungs to 30 Gy X-ray using a precision irradiation X-ray apparatus.(5)At different time intervals after irradiation,pulmonary iconography,histopathology,serum TGF-?1,and lung tissue ?-SMA expression were determined,using living imaging,HE and Masson staining,ELISA,and Western blot assay,respectively.(6)The primary pulmonary cells from mice with different Usp11 gene status were isolated and cultured.The DNA double strand breaks were observed by immunofluorescence staining of phosphorylated ?-H2 AX in the nuclei after irradiation.The variations of cells cycle distribution in primary cells were analyzed by flow cytometry assay.TUNEL assay was used to detect the apoptosis of primary cells after irradiation.(7)High-throughput proteomics analysis was performed using lung tissues extracted from radiation-induced lung injury mice.The differentially expressed proteins were screened out between different genotypes with/without irradiation exposure,meanwhile the differential proteins were undergone bioinformatics analysis to understand the cellular localization as well as the potential biological processes and related signaling pathways.Results :(1)Usp11 gene knockout has no obvious effect on mouse growth and development.(2)When acute radiation injury occurred,leukocytes in Usp11-/-mice declined slower than Usp11+/+ mice,while the recovery rate of lymphocytes is faster than that in USP11 wild-type mice.(3)The overall survival rate of Usp11-/-mice was significantly longer after 7.5 Gy TBI,compared to USP11 wild-type mice.(4)USP11 gene knockout alleviated radiation-induced lung injury,represented as the obviously relieved iconography images of “cloudy” shadows in irradiated lungs,less collagens around the pulmonary blood vessels in MASSON staining,lower level of serum TGF-?1,and suppressed expression of ?-SMA in lung tissues as compared with USP11 wild-type mice.(5)The primary lung cells from Usp11-/-mice formed less ?-H2 AX foci after irradiation than that from USP11 wild-type mice.Six hours after irradiation,primary cells from Usp11-/-mice manifested typical G2/M phase arrest,whereas the most obvious change in wild-type mouse primary cells was the significant increase in S phase.The difference between these different cell cycles returned to normal 12 h after irradiation.The apoptotic rate of primary cells from Usp11-/-lungs was lower than that in the wild-type mouse primary cells at 24 h after irradiation;48h later,there were no significantly different between the two types cells in the number of apoptotic cells.(6)Using high-throughput proteomics analysis,180 differential proteins were screened in the lung tissues between two genotype mice,22 were up-regulated and 158 were down-regulated.Bioinformatics analysis revealed that these differentially expressed proteins were mainly enriched in the extracellular region part,extracellular exosomes,extracellular membrane-bounded organelle,extracellular vesicle and extracellular organelles.The functional annotation found they were involved in several biological processes,for example,the negative regulation of catalytic activity,organ regeneration,the negative regulation of hydrolase activity,regulation of developmental process and the cellular response to nitric oxide and so on.These proteins enriched in signal pathways that related to tight junctions,leukocyte transendothelial migration,focal adhesion and carbon metabolism.In addition,GSEA enrichment analysis revealed that Usp11 knockout also resulted changes in TGF-? signaling pathways,transcriptional misregulation pathways in tumors,vascular endothelial growth factor(VEGF)signaling pathways,and P13K-AKT signaling pathways.Conclusions:Usp11 gene knockout to some extend mitigated the radiation-induced lung injury.The mechanisms involved may be related to its regulation of TGF-? signaling pathway,transcriptional misregulation in tumors,vascular endothelial growth factor(VEGF)signaling pathway,and P13K-AKT signaling pathway.Further studies are extremely necessary to verify the exact mechanism of Usp11 gene in regulating radiation-induced lung injury.