Study Of The Role Of NFE2L1/NRF1 Deletion In Exacerbating Kras Mutations Causing Lung Adenocarcinoma

Objective:Lung cancer is one of the most common malignancies,the leading cause of cancer deaths,and a public health problem that needs to be addressed urgently.As the smoking population has declined in recent years,lung adenocarcinoma has become the main histological subtype of lung malignancy in humans.The factors affecting the occurrence and development of lung cancer include environmental factors and genetic factors.Kirsten rat sarcoma virtal oncogene is one of the most common mutant oncogenic drivers in non-small cell lung cancer,playing an important role in regulating cell growth and proliferation,and about 30%of lung adenocarcinomas have KRAS mutations.KRAS mutations induce significant inflammatory cell infiltration and lipid metabolism.Although research on KRAS has been going on for decades,it is still a challenging treatment point.Nuclear factor erythroid-derived 2-like 1（NFE2L1/NRF1）is a transcription factor with basic lysine zipper structure belonging to the Cap’n’Collar basic leucine zipper（CNC-b ZIP）family,which plays a regulatory role in redox homeostasis,proteasome homeostasis,and cell differentiation.In addition to the above effects,NFE2L1 also has a regulatory effect on inflammatory response and lipid metabolism in a variety of disease processes,but there are few studies on NFE2L1 and the mechanism of action is not clear.In this study,the role of NFE2L1 in lung adenocarcinoma caused by Kras mutation was analyzed from the perspective of inflammatory cell infiltration characteristics and lipid droplet accumulation using a mouse model of lung-specific Kras activation and Kras activation combined with Nfe2l1 deletion and NFE2L1 silent human non-small cell lung cancer cell A549 cell line.Methods:1.Construction of lung-specific Kras activation and Kras activation combined with Nfe2l1 Knockout（KO）mouse lung adenocarcinoma model:Using Kras^G12D mice with C57BL/6 genetic background,Nfe2l1 Knockin（KI）mice were crossed with them,and mice with genotype Nfe2l1-KI/Kras were obtained,and AAV-CMV-Cre virus and its control AAV-Empty virus bronchial perfusion were given at 8weeks of age along with Kras^G12D mice.2.Identification and comparison of lung adenocarcinoma in mice:the approximate distribution and size of in vivo tumors were observed by Micro-CT;The collected lung tissue is weighed to calculate the lung tissue organ coefficient;HE staining was performed on lung tissue,and the tumor tissue in the staining results was quantitatively analyzed to assess tumor burden,and the pathological type,cell source,proliferation ability and malignancy of lung tumor were identified by immunohistochemistry.3.Silencing and identification of human non-small cell lung cancer cell A549 gene:A549 cells were transfected with lentivirus（NFE2L1-KD）containing NFE2L1 sh RNA and non-target negative control lentivirus（Scramble）,stable transfected cells were screened with 2μg/m L puromycin,and the m RNA level of NFE2L1 was detected by q PCR.Western blot detects NFE2L1 protein expression levels.4.Detection of malignant phenotypic indexes of A549 cell line:A549cells using Scramble and NFE2L1-KD were evaluated by soft agar clonal formation experiment,cell scratch healing experiment and Transwell experiment to evaluate the degree of malignancy between the two groups.5.Evaluation the levels of lipid droplet accumulation in A549 cells:Scramble and NFE2L1-KD A549 cells were plated onto 6-well plates,treated with oleic acid and then stained with Oil Red O to evaluate the level of lipid droplet accumulation in both groups of cells.6.Statistical analysis:All data from this study were analyzed using Graph Pad Prism 5 software.Results:1.Lung-specific Kras activation and Kras activation combined with Nfe2l1deletion mouse lung cancer models were successfully constructed.2.The lung tissues of the collected mice were stained with HE and the tumor tissues of the staining results were quantitatively analyzed,and the tumor burden of Nfe2l1（P）-KO/Kras group mice was heavier than that of Kras group mice.3.F4/80 and Ly6G immunohistochemical staining of lung tissue sections showed that the degree of impotence of Ly6G in lung tissue of the two groups was lighter and there was no difference.Compared with the control group of Kras group mice,Nfe2l1（P）-KO/Kras group mice had a mild degree of positive F4/80 staining.4.CD86 and NOS2 immunohistochemical staining of mouse lung tissue sections showed no obvious positive staining cells in the lung tissue tumor sites of CD86 and NOS2 in the lung tissue tumor of the mice in both groups.CD163and CD206 immunohistochemical staining of mouse lung tissue sections showed that CD163 showed no obvious impotence cells in the lung tissue tumor site of the two groups of mice,while CD206 had a higher number of positive cells in the lung tissue tumor site of the two groups,and compared with the Kras group,the number of positive cells in Nfe2l1（P）-KO/Kras group mice was less.5.The number of lipid droplets was counted on the HE staining results,and the Nfe2l1（P）-KO/Kras group mice had a heavier degree of lipid droplet accumulation in lung tissue compared with the control group of Kras group mice.6.NFE2L1 silent A549 cell lines were obtained by lentiviral stable transfection,and RNA and protein samples were collected to verify their silencing effect.The experimental results showed that the cell line was successfully constructed.7.Through soft agar cloning experiments,it was found that the NFE2L1-KD group had stronger cell clonal formation ability than the Scramble group.Through cell scratch healing experiments,it was found that after 48 hours,the scratch area of NFE2L1-KD cells was smaller than that of cells in the Scramble group.Transwell experiments showed that after 24 h,the NFE2L1-KD group had a higher number of cells passing through the pores of matrigel and polycarbonate membrane.8.A549 cells were treated with sodium oleate and stained with Oil red O after 24 h.Compared to control Scramble cells,lipid droplets were more abundant in the cytoplasm of the NFE2L1-KD group.Intracellular lipid droplet-colored Oil red washed down for quantitative analysis using isopropanol,the NFE2L1-KD group was higher than the Scramble group.Conclusion:1.Nfe2l1 deletion promotes the progression of Kras mutation-induced lung adenocarcinoma in mice and changes the proportion of M2a-type macrophages in their tumor tissues.2.Nfe2l1 deletion may promote the progression of Kras mutation-induced lung adenocarcinoma in mice by altering lipid metabolism in cancer cells.