The Role Of NFE2L1 And NFE2L2 In The Development Of Urethane-Induced Lung Adenocarcinoma In Mice

Objective:Cancer is a majoy threat to human life all over the world.Lung cancer is on the top of cancer list because of its high morbidity,mortality and poor prognosis.The molecular mechanism of lung cancer development and progression is very complicated.Oxidative stress is one of the most recognized mechanisms in the development of lung cancer.NFE2L1 and NFE2L2 are transcription factors in the CNC-bZIP family,which play important roles in resisting oxidative stress and maintaining redox homeostasis.It is known that NFE2L2 can prevent diseases related to oxidative stress and inhibit tumorigenesis.However,cancer cells take advantage of NFE2L2 to promote tumor growth and metastasis.Therefore,NFE2L2 has a dual role in tumorigenesis and development.NFE2L1 exhibits stronger physiological and pathological effects than NFE2L2.Therefore,NFE2L1 may be a more important target in prevention and therapeutics of lung cancer.In this study,we used alveolar type ? epithelial?AT??cell-specific knockout Nfe2l1 and Nfe2l2?Nfe2l1?AT??-KO and Nfe2l2?AT??-KO?mice to investigate the role of NFE2L1 and NFE2L1 in the development of mouse lung adenocarcinoma induced by urethane.Potentied underlying mechanisms were also explored.Methods:1.The construction of Nfe2l1?AT??-KO and Nfe2l2?AT??-KO mice:Nfe2l1-KI/Sftpc-rtTA^+/-/Teto-Cre^+/-and Nfe2l2-KI/Sftpc-rtTA^+/-/Teto-Cre^+/-triple transgenic mice were constructed using C57BL/6 genetic background mice.According to the results of genotyping,Nfe2l1-KI,Nfe2l1-KI/Sftpc-rtTA^+/-,Nfe2l1-KI/Teto-Cre^+/-,Nfe2l1-KI/Sftpc-rtTA^+/-/Teto-Cre^+/-;Nfe2l2-KI,Nfe2l2-KI/Sftpc-rtTA^+/-,Nfe2l2-KI/Teto-Cre^+/-,Nfe2l2-KI/Sftpc-rtTA^+/-/Teto-Cre^+/-and Sftpc-rtTA^+/-/Teto-Cre^+/-mice were given drinking water containing 2 mg/ml doxycycline?DOX?,5%sucrose from 6-week old to 8-week old.2.Isolation and identification of mouse primary AT? cells:Preparation of mouse lung tissue single cell suspension by Dispase ?.Cells of Lin^-/Ep-CAM⁺were selected from the lungs of mice by immunomagnetic beads.After 48-h culture the morphology of adherent cells was observed by an inverted microscope.The expression of SP-C,AT? cell-specific marker,was detected by immunofluorescence.3.Verification of AT? cell-specific Nfe2l1 and Nfe2l2 knockout:AT? primary cells of Nfe2l1?AT??-KO,Nfe2l2?AT??-KO and corresponding KI control mice were isolated.The mRNA levels of Nfe2l1 and Nfe2l2 in AT? cells were detected by qPCR.The mRNA levels of Nfe2l1 and Nfe2l2 were detected in Ep-CAM^-cells and used as negative control.4.Urethane-induced lung adenocarcinoma in mice:Wild-type mice with C57BL/6genetic background were injected intraperitoneally with urethane at the dose of 1 g/kg once a week for 10 weeks.Mice were sacrificed and examined 10 weeks after the last injection.AT?-cell specific gene knockout mice were randomly assigned to the experimental group injected with urethane?1 g/kg?and the control group injected with physiological saline according to the genotype.Intraperitoneal injections were given once a week for 10 weeks.Mice were sacrificed and examined 17 weeks after the last injection.5.Evaluation and identification of urethane-induced lung adenocarcinoma in mice:Tumor burden was evaluated by the number,diameter and the volume of tumors on the surface of the lungs as well as the lung weight.HE staining was evaluated of lung tumor pathology,and SP-C was detected by immunohistochemistry to identify the source of lung adenoma cells.PCNA and TTF1 were detected by immunohistochemistry to identify lung adenocarcinoma.6.Nfe2l1 silencing and identification:MLE-12 mouse AT? cells were transfected with a lentivirus carrying Nfe2l1 shRNA targeting and a non-target negative control?Scramble?,and stably infected cells were screened with 0.5?g/m L puromycin.The silencing effect was identified by detecting the mRNA and protein levels of NFE2L1 in MLE-12 cells.7.RNA-Sequencing?RNA-Seq?and analysis:AT? cells from Nfe2l1?AT??-KO mice and Nfe2l1-KI mice,as well as Nfe2l1-KD and Scramble MLE-12 cells were dissolved in Trizol.RNA-seq was performed by Genedenovo Biotechnology?Guangzhou,China?.Differential gene expression and function analysis were performed by the Kyoto Encyclopedia of Genes and Genomes?KEGG?.The mRNA levels of target RNA obtained by RNA-Seq were detected by qPCR.8.Statistical analysis:All data were analyzed using GraphPad Prism 5.Results:1.Successful construction of Nfe2l1?AT??-KO and Nfe2l2?AT??-KO mice:The isolated mouse AT? primary cells were round in shape and grew in pave stone-like morphology,and positive in expression of SP-C,an AT? cell-specific marker in immunostaining.The expression of Nfe2l1 in AT? cells of Nfe2l1?AT??-KO group was significantly lower than Nfe2l1-KI group?P<0.05?.The expression of Nfe2l1 in negative control cells was unchanged.The expression of Nfe2l2 in AT? cell of Nfe2l2?AT??-KO group was significantly lower than Nfe2l2-KI group?P<0.05?.The expression of Nfe2l2in negative control cells was unchanged.2.Urethane-induced lung adenocarcinoma in wild-type C57BL/6 mouse:Milky tumors were observed on the surface of the lung.Epithelioid hyperplasia,adenomatous hyperplasia,and several typical lung adenocarcinoma lesions of adenoma were observed after HE staining.3.Nfe2l2?AT??-KO did not affect susceptibility to urethane-induced lung adenocarcinoma in mice:There was no significant difference in the lung mass index between Nfe2l2-KI mice and Nfe2l2?AT??-KO mice at the end of the observation.There were also no significant difference in tumor burden assessment such as the number of lung surface nodules,nodule burden,average tumor diameters.4.Nfe2l1?AT??-KO mice were more susceptible to urethane-induced lung adenocarcinoma:In both male or female mice,Nfe2l1?AT??-KO group showed more severe lung tumors than Cre and Nfe2l1-KI group.Tumor burden assessment found that the weight of lung tissue of Nfe2l1?AT??-KO mice in urethane injection group was significantly heavier than that of Nfe2l1-KI mice in the same group?P<0.05?.The number of lung surface tumors in Nfe2l1?AT??-KO mice was significantly higher than that in the same group of Nfe2l1-KI mice?P<0.05?.The lung surface tumor volume of Nfe2l1?AT??-KO mice was significantly larger than that of the same group of Nfe2l1-KI mice?P<0.05?.However,the mean diameter of the lung surface tumor of Nfe2l1?AT??-KO mice was not significantly different from that of Nfe2l1-KI mice with the same treatment.Furthermore,HE staining revealed that the tumor area of Nfe2l1?AT??-KO mice was significantly larger than Nfe2l1-KI mice according to quantitative analysis?P<0.05?.Immunohistochemical staining of lung tissue sections revealed that SP-C,PCNA and TTF1 were expressed in tumor tissues.Quantitative analysis showed that the area of SP-C,PCNA and TTF1positively stained cells in Nfe2l1?AT??-KO group was significantly higher than that of Nfe2l1-KI group?P<0.05?.5.The effect of Nfe2l1 knockdown or silencing on RNA expression in AT? cells:According to the RNA-Seq,there were up-regulated 669 genes and down-regulated 406genes in AT? cells of Nfe2l1?AT??-KO group compared with Nfe2l1-KI group.There were up-regulated 657 genes and down-regulated 220 genes in MLE-12 of Nfe2l1-KD group than Scr group.After Nfe2l1 inhibition,primary AT? and MLE-12 cells had similar trend in changes of cytokine-cytokine receptor interaction,focal adhesion,protein digestion and absorption,cell cycle,ECM-receptor interaction,and PI3K-Akt signaling pathways.Representative genes were verified by qPCR.Conclusion:1.Nfe2l1?AT??-KO and Nfe2l2?AT??-KO mice were successfully constructed.2.NFE2L1 in alveolar type ? epithelial cells plays a tumor suppressive role in the development of lung adenocarcinoma induced by urethane.