The Role Of NFE2L1 In The Development Of Lung Adenocarcinoma And Its Effect On The Malignant Degree Of Lung Cancer Cells

Object:Lung cancer is a common malignant tumor,the incidence and death number increase year by year,but its survival rate is low,the prognosis is poor.The morbidity and mortality rate of lung cancer are high in China,which is a public health problem that cannot be ignored.Lung adenocarcinoma accounts for a large proportion of lung cancer cases.Due to its disease characteristics and detection methods,it is usually detected in the middle and late stages.Nuclear factor-erythroid 2 related factor 1 is a member of the CNC-bZIP transcription factor family,and plays an important role in embryonic development,tissue and organ growth,maintenance of redox homeostasis,proteasome homeostasis,and anti-inflammatory.In the present study,it is suggested that NFE2L1 is involved in the occurrence and development of cancer,and it has been confirmed that NFE2L1 can inhibit the occurrence of lung cancer,but the role and mechanism of NFE2L1 in the development stage of lung cancer have not been clearly studied.In this study,the role of Nfe2l1 in the development of lung adenocarcinoma was investigated using NFE2L1 specific knockout mice of alveolar type II epithelial cells established earlier in our group and lentivirus statically transfected NFE2L1 silenced lung adenocarcinoma cells,providing a theoretical basis for clarifying the role of NFE2L1 in the development of lung adenocarcinoma.Methods:1.To establisha mouse lung adenocarcinoma model:In this experiment,8-week-old male mice were used,and the genotypes were Nfe2l1-KI、Nfe2l1-KI/Sftpc-rt TA^+/-、Nfe2l1-KI/Teto-Cre^+/-and Nfe2l1-KI/Sftpc-rt TA^+/-/Teto-Cre^+/-.Mice were randomly assigned to the modeling group and the control group.The urethane modeling group was intraperitoneally injected with a dose of 1g/kg once a week for 10 weeks,while the control group was injected with normal saline.2.Establishment of Nfe2l1 specific knockout mice of alveolar Type II epithelial cells in the development stage of lung adenocarcinoma:Mice were intraperitoneally injected with urethane for 10 weeks,and were observed for another 10 weeks.After lung tumors appeared,mice were given drinking water containing 2 mg/ml doxycycline and 5%sucrose for 2 weeks.Nfe2l1 specific knockout mice of alveolar type II epithelial cells（Nfe2l1（ATII）-KO）and Nfe2l1 unknockout mice（Nfe2l1-KI）were obtained.3.Tumor evaluation:The number of tumors on the lung surface of mice was counted and the tumor diameter was measured to evaluate the tumor load of mice.Immunohistochemical staining was used to detect PCNA and TTF-1 and identify tumor growth and malignancy.4.NFE2L1 gene knockdown:A549 cells were transfected with a lentivirus carrying NFE2L1 shRNA targeting（NFE2L1-KD）and a non-target negative control lentivirus（Scramble,Scr）.Purinamycin was screened and the effect of gene silencing was determined by the expression of NFE2L1 protein in A549 cells5.Detection of indicators related to the malignancy degree of A549 cells:cell morphological changes were observed,cell proliferation,migration and invasion ability were detected by clonal formation assay,scratch assay and Transwell invasion assay,and changes of proteins related to migration and invasion were detected.6.Statistical analysis:Statistical analysis of data by Graph Pad Prism 5.0 software.Results:1.Mouse lung tumor model was successfully established by continuous injection of urethane:Lung cancer was modeled in mice by continuous intraperitoneally injected with urethane for 10 weeks.White tumors were observed on the lung surface of mice after continuous observation for 10 weeks after the injection.2.Nfe2l1 specific knockout of alveolar type II epithelial cells promotes the development of lung adenocarcinoma in the development stage of lung adenocarcinoma:Lung cancer modeling was performed in mice by continuous intrabitoneal injection of urethane,and Nfe2l1 specific knockout of alveolar type II epithelial cells was performed 10 weeks after the injection.At the end of,it was observed that the Nfe2l1（ATII）-KO group had more tumors than the Nfe2l1-KI group,but there was no significant difference in the mean tumor diameter between the two groups of mice.3.Nfe2l1 specific knockout of alveolar type II epithelial cells in the development stage of lung adenocarcinoma increases the malignancy of lung tumors:Immunohistochemical staining of lung tissue sections showed that there was infiltration of macrophages in tumor sites,but there was little infiltration of neutrophils.Quantitative analysis showed that macrophages in Nfe2l1（ATⅡ）-KO group had no significant difference compared with Nfe2l1-KI group.It was also found that TTF-1 and PCNA were expressed at the tumor site,quantitative analysis found that compared with Nfe2l1-KI group,Nfe2l1（ATⅡ）-KO group had fewer TTF-1 positive staining cells,while PCNA had more positive staining cells.4.NFE2L1 knockdown cells were established successfully:The protein level of NFE2L1was decreased in NFE2L1-KD cells.5.NFE2L1 knockdown promoted the malignant phenotype of A549 cells:cell morphology changed,with Scr cells exhibiting normal epithelioid cell morphology and NFE2L1-KD cells exhibiting long spindle and spindle morphology.The clones of NFE2L1-KD cells were larger than those of Scr cells,and the number of clones larger than 0.1 mm in diameter was more.Scratch healing effect of NFE2L1-KD cells at 24 h and 48 h was more obvious than that of Scr cells.The number of NFE2L1-KD cells passing through the stroma-coated compartments was more than that of Scr cells.The expression of matrix metalloproteinase 9（MMP9）protein was increased.Conclusion:NFE2L1 plays a tumor suppressor role in the development stage of lung adenocarcinoma in mice,and inhibits cell proliferation,migration,and invasion in lung adenocarcinoma cells.