| Background: Primary lung cancer is the malignant tumor with the highest incidence and mortality worldwide,and it is one of the critical killers that threaten human health.The National Cancer Data Annual Report jointly issued by the Centers for Disease Control and Prevention and the American Cancer Society and other institutions pointed out that in 2020 the number of new cases of lung cancer in both men and women in the United States ranked second only to prostate cancer and breast cancer,respectively.But the mortality was the highest,accounting for almost a quarter of the total mortality of malignant tumors.According to the global lung cancer statistics published by the World Health Organization in 2020,the mortality of new cases in lung cancer was approximately 2.1 million,and the deaths was approximately 1.8 million.China is also a high incidence area in lung cancer,where the lung cancer ranks the first place among all malignant tumors.In terms of mortality,lung cancer also ranks first in China.With the augmentation of public health awareness,the extensive development of lung cancer screening,the continuous improvements for lung cancer treatments are widely launched and applied,such as molecular targeted therapy,immunotherapy and so forth.However,the mortality of lung cancer is showing a downward trend,but the 5-year overall survival is still awfully low,the clinical benefits are still limited,recurrence after surgery,relapse and drug resistance after treatments are still the important issues that bother the clinicians.KEAP1,encoded by the KEAP1 gene,was discovered in 1999 in Japan.A twohybrid screening assay identified it as a protein repressing NRF2 transcriptional activity,sensitive to electrophiles.It is also an intracellular sensor of oxidants.The NRF2/KEAP1 axis has been recognised as a central node for a cross-talk of cellular defence,survival pathways and the crucial modulator of cell homeostasis.Nowadays,some researches have focused on the mutation of KEAP1 in different cancers,such as breast cancer,lung cancer,gallbladder cancer,liver cancer and endometrial cancer,while most of these studies have mainly emphasized on the impact of KEAP1/NRF2 axis on cancer,which exerted the carcinogenic effect of cancer.But,the focus on KEAP1 rarely goes beyond the association of NRF2,little has been known about the role of KEAP1 plays in the biological processes of lung cancer,and there is also no clear conclusion.Cancer has been widely regarded as a metabolic disease,and metabolic reprogramming is an important characteristic of cancer.Cancer metabolism is partly regulated by signalings and transcription networks activated by mutations of oncogenes and tumor suppressor genes,resulting in a heterogeneous cell-autonomous phenotype in genetical diversity of cancer cells.Emerging evidences have uncovered that specific metabolic activities can disturb the carcinogenic activity of key enzymes in the oncogenic signaling pathways leads to metabolic reprogramming of cancer cells and directly participates in the biological processes resulting in supporting the tumor growth.In the early stage of this study,bioinformatics technology was used to find that the level of KEAP1 expression is closely related to clinical prognosis of lung cancer.In the pre-experimental stage,it was found that the expression of KEAP1 in lung cancer tissue specimens was significantly lower than that in non-cancerous adjacent tissues(NATs).It was preliminarily judged that KEAP1 seemed to be a tumor suppressor gene.However,the changes of KEAP1/NRF2 in tissue specimens are not consistent with the expression in cells.NRF2 can cause metabolic reprogramming in cancer,but there is no clear research that KEAP1 can directly affect the metabolic pathways of lung cancer,and the regular changes of KEAP1 in lung cancer tissues are not accidental.Therefore,the main purpose of this study is to explore the effect of KEAP1 on the proliferation of non-small cell lung cancer(NSCLC)and to analyze the molecular mechanism of influence of KEAP1 on the metabolism of NSCLC by metabonomics to clarify the application value of KEAP1 in clinical research.Methods:1.The expression of KEAP1 gene in lung cancer and NATs were analyzed from the transcription level and protein level by bioinformatics,and the statistical analysis of clinical prognostic survivorship curve were performed.The metabolic pathways closely related to lung adenocarcinoma and lung squamous cell carcinoma were predicted by enrichment pathway analysis.All tissue specimens of 34 patients with lung cancer who underwent surgical treatment were collected from the department of Thoracic Surgery of Liaoning Cancer Hospital & Institute.None of the patients were received preoperative antitumor combination therapy.Western blot was used to verify the expression difference of KEAP1 in cancer tissues and NATs,and the results of bioinformatics analysis were further compared to verify mutually.2.Human non-small cell lung cancer cell lines A549,H1299,H460 and H2030 were cultured.The expression of KEAP1 gene was knocked down by lentivirus-mediated RNA interference,and the effects of KEAP1 on the proliferation of non-small cell lung cancer cell lines were detected by MTT,crystal violet staining and CCK8 assay.The effect on cell migration was detected by scratch assay.3.Thirty-four pairs specimens provided by Liaoning Cancer Hospital & Institute were metabonomics analyzed by CE-MS and LC-MS platform.Meanwhile,the H1299 cell line with KEAP1 knockdown was analyzed by CE-MS platform.Comparing metabolites of tissue specimens and cells to find common metabolites,analysis of metabolic pathways and possible associated oncogenic signaling pathways.4.According to the results of metabonomics,Western blot and q PCR techniques were used to verify relevant proteins such as S6,m TOR and Rag D of m TOR signaling pathway affecting amino acids,so as to clarify the correlation between KEAP1 and m TOR signaling pathway.Results:1.KEAP1 gene expression profile was analyzed at the protein level by CPTAC database,and the results showed that KEAP1 gene expression level in lung adenocarcinoma tissues was lower than that in NATs,with significantly statistical difference(p<0.05).Transcriptome level analysis was conducted on the correlation between KEAP1 and the clinical prognosis of NSCLC by GEO database and R language extraction data.It was found that the survival of KEAP1 group with high expression was significantly higher than that of the low expression group(p=0.029).KEGG pathway enrichment analysis showed that KEAP1 was significantly correlated with metabolization-related signaling pathways in both LUAD and LUSC.2.Western blotting experiments were performed on 34 pairs of non-small cell lung cancer(NSCLC)and NATs.Results showed that KEAP1 expression in lung cancer tissues was lower than that in NATs,with significant statistical difference(p<0.05);In H460 and H1299 cell lines,KEAP1 expression interference did not change the proliferation ability of lung cancer cells significantly.The proliferation of KEAP1 overexpressed in lung cancer cells was not significantly inhibited,but KEAP1 interference did not significantly affect the migration of lung cancer cells.3.A total of 253 metabolites were detected in the tissues of NSCLC,PLS-DA analysis was performed to show the difference in metabolic characteristics between samples.Using Me V software for heat map drawing,it is found that when some metabolites of NATs are down-regulated,the corresponding metabolites of cancer tissues are up-regulated,and when other metabolites of NATs are up-regulated,the corresponding metabolites of cancer tissues are down-regulated.There are significant differences and trends,the changes in the part of metabolites are related to the pathological type.Aggregation of metabolites were collected and analyzed on the Metabo Analyst website(http://www.metaboanalyst.ca)for differential metabolite pathway enrichment analysis.Results were that the changes of metabolites induced by KEAP1 deletion in 34 pairs of lung cancer tissue specimens were correlated with glycine,serine,glutamic acid,methionine and other amino acid metabolic pathways,and were also closely related to Warburg effect,urea cycle and other metabolic pathways.4.A total of 108 metabolites were detected in KEAP1 knockdown H1299 cell line.The Me V software was also used to process the metabolic datas and draw the heat map.The results showed that,compared with the control group,some metabolites in the KEAP1 knockdown had significantly changed.In KEAP1 knockdown group,amino acids such as serine,glutamine,leucine,isoleucine and tyrosine were significantly down-regulated,while metabolites such as NADH,FAD,Co A,GSH and taurine were significantly up-regulated.Aggregation of metabolites were collected and analyzed on the Metabo Analyst website(http://www.metaboanalyst.ca)for differential metabolite pathway enrichment analysis.Results were that the changes of metabolites induced by KEAP1 knockdown were correlated with biosynthesis of amino acid,central carbon metabolism in cancer,alanine,aspartate acid,glutamate metabolism,arginine metabolism and synthesis,proline metabolism,protein digestion and absorption.5.The commom metabolites between NSCLC tissues and cell lines were amino acids,which were down-regulated.KEAP1 knockdown H1299 cell line was used to detect the m TOR signaling pathway affected by amino acids.Western blot detection showed that the phosphorylation levels of m TOR and S6 decreased significantly after KEAP1 interference.q PCR was used to detect the RNA level of Rag D,the activator of m TOR,and the RNA level of Rag D was down-regulated after KEAP1 interference.Conclusion:1.In NSCLC,KEAP1 expression is closely related to clinical prognosis,and the survival in high KEAP1 expression is significantly better than that in low KEAP1 expression.2.KEAP1 knockdown induces metabolic reprogramming in NSCLC and is independent of NRF2.The amino acid is significantly down-regulated.3.Amino acid metabolic reprogramming induced by KEAP1 knockdown is closely related to the m TOR signaling pathway,which may be regulated by Rag D. |
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