Research Of Generation And Characterization Of EIF3a CKO Mice And Their Lewis Lung Cancer Model

Background:Transcription and translation are two crucial stages of gene regulation.Transcription is the process of transforming geneticinformation from DNA to RNA,and translation is the process of converting mRNA to protein.Protein translation includes initiation,extension,termination,and ribosome recycling.Translation initiation is the rate-limiting stage of protein synthesis,and at least 12 translation initiation factors(eukaryotic initiation factors,eIFs)involve in this process.Among the translation initiation factors,eIF3 is the largest and most complex one.It consists of 13(eIF3a-m)subunits,of which eIF3a is the largest.It participates in protein synthesis and plays an essential role in cell proliferation,differentiation,and apoptosis.It affects follicle development,fibrotic diseases,carcinogenesis and tumor development,and sensitivity to chemotherapy and radiation therapy.eIF3a is widely expressed in various tissues,and the expression level alters in different tissues.The expression level also fluctuates at different developmental stages in the same tissue.It suggests that the proteins profilings under the regulation of eIF3a may be tissue specific.It was reported that eIF3a regulated about 15-20%of protein synthesis in vitro.But there is still no study in vivo nor eIF3a knockout model mouse available around the world.The research of eIF3a in oncology focuses on the tumor cells,but whether eIF3a plays a role in the tumor microenvironment to affect the occurrence and development of tumors has not been reported.Objective:Generate eIF3a conditional knockout mice,and explore its phenotypic characteristics and protein expression profile characteristics to unveil new functions of eIF3a.Build a Lewis lung cancer model in eIF3a conditional knockout mice to reveal its gene expression profile characteristics and its role in the tumor microenvironment.Methods:Cre-LoxP system was used to generate eIF3a floxed mice in C57BL/6 mice by inserting Floxp between exons 2 and 3.UBC-Cre-ERT2 transgenic mice(from Jackson lab)were bred with eIF3aflox/flox mice to remove the loxP-flanked exons in all tissues following tamoxifen induction.Knockout efficiency was validated in DNA,mRNA,and protein levels.For the mature knockout mice,common vital signs were recorded,and the effect of eIF3a on embryonic development was also observed.T cell-specific eIF3a knockout mice model was generated.Tandem mass tagging(TMT)coupled with liquid chromatography-tandem mass spectrometry(LC/MS/MS)was used to research the proteomics of fat,lung,skin,and spleen tissues from eIF3a knockout and control mice.The role of eIF3a in reactive oxygen species generation was verified by the DCF method in embryonic fibroblasts.Illumina platform was used to research transcriptomics of eIF3a knockout mice-bearing Lewis lung cancer.Primary tumor cells were extracted from LLC orthotopic tumors of eIF3a knockout and control mice and then reinoculated into BALB/c-nu immunodeficient mice and C57BL/6 wild-type mice.Immune cells in the tumor cells of C57BL/6 wild mice were detected by flow cytometry.Lewis lung cancer model was generated in T cell-specific knockout eIF3a mice.Tumor-infiltrating T cells were analyzed by flow cytometry.Results:1.Phenotype and proteomics of eIF3a conditional knockout miceThe eIF3a conditional knockout mice were successfully constructed,and the knockout of eIF3a in the embryonic and mature stages resulted in the death of the mice.After knocking out eIF3a in mice,their body weight and body temperature decreased,hair loss and diarrhea symptoms occurred,and they died quickly.The spleen and thymus atrophied significantly.HE staining showed histological changes in fat,lung,skin,and immune system.T cell-specific knockout eIF3a mice had no noticeable phenotype changes.The protein expression profiles of fat,lung,spleen,and skin tissues of eIF3a knockout mice were significantly changed when compared with the controls,and the differentially expressed proteins(DEPs)were 588,237,944,and 324,respectively.DEPs functions in fat were enriched in cellular response to cytokine stimulus,cellular response to organic cyclic compound,regulation of sterol transport,positive regulation of cytokine production,regulation of lipid metabolic process,lipid-related metabolism,and endocytosis.KEGG pathways were enriched in leishmaniasis,peroxisome,Fc gamma R-mediated phagocytosis,complement and coagulation cascades,PPAR signaling pathway,DNA replication,B cell receptor signaling pathway,leukocyte transendothelial migration,asthma,and metabolism-related pathway.DEPs functions in the lung were enriched in neutrophil-mediated immunity,regulated exocytosis,regulation of immune response,inflammatory response,and defense response,regulation of cell,neutrophil,and leukocyte activation.TOP KEGG pathways were staphylococcus aureus infection,leishmaniasis,phagosome,rheumatoid arthritis,tuberculosis,natural killer cell-mediated cytotoxicity,and transcriptional misregulation in cancer.DEPs functions in the skin were enriched in the regulation of peptidase and hydrolase activity,platelet degranulation,monocarboxylic acid metabolic process,and regulation of proteolysis.KEGG pathways were enriched in complement and coagulation cascades,PPAR signaling pathway,fatty acid metabolism,staphylococcus aureus infection,phagosome,and ferroptosis.DEPs functions in the spleen were enriched in response to steroid hormones,DNA replication and repair,aminoglycan catabolic process,acute inflammatory response,and G1/S transition of the mitotic cell cycle.TOP KEGG pathways were DNA replication,mismatch repair,lysosome,intestinal immune network for IgA production,systemic lupus erythematosus,ECM-receptor interaction,focal adhesion,and hematopoietic cell lineage.There were only 8 proteins sheared in the four tissues from the Venn diagram for the DEPs in each tissue.The tissue-special DEPs were 129 in the lungs,738 in the spleen,177 in the skin,and 369 in the fat.2.Establishment and characterization of Lewis lung cancer model in eIF3a conditional knockout miceWhen compared with control,984 differentially expressed genes(DEGs)were identified in eIF3a knockout mice-bearing tumors.GO enrichment analysis found DEGs enriched in metabolism and reaction of phosphoric acid&phosphate,stress and inflammatory response,protein phosphorylation,stress protein modification,response to stimulus and chemicals,and cell communication.KEGG pathway enriched in IL-17 signaling pathway,malaria,cytokine interaction,TNF signaling pathway,viral protein interaction with cytokine and cytokine receptor,cytokine-cytokine receptor interaction,legionella,African trypanosomiasis,rheumatoid arthritis.Knockout of eIF3a in the tumor microenvironment inhibits tumor growth.The flow cytometry results of tumor-bearing C57BL/6 mice showed that the infiltration of lymphocytes,T lymphocytes,and CD4-positive T lymphocytes increased,and M2-type macrophages decreased.The growth of LLC tumors was accelerated in T cell-specific eIF3a-deficient mice,and the infiltration of CD4-positive T cells was reduced.Conclusion1.eIF3a plays a vital role in sustaining life,and the downstream proteins and functions regulated by eIF3a are tissue specificity.2.The expression profile of Lewis tumor-bearing genes in eIF3a knockout mice was significantly changed,and eIF3a in the tumor microenvironment played an essential role in maintaining the tumor phenotype.