Role And Mechanism Of Benzo(α)pyrene In Chronic Obstructive Pulmonary Disease Transformation Into Lung Adenocarcinoma

Objective: The experiment is designed to explore the expression changes of key genes and the biological processes involved in the transformation of Chronic obstructive pulmonary disease(COPD)into Lung adenocarcinoma(LAC).Meanwhile,the role and mechanism of Benzo[a]pyrene(Ba P)in the progression of COPD to LAC will be clarified.Methods: 1.By using the microarray data of COPD and LAC included in the Gene expression omnibus(GEO),the differentially expressed genes between the two diseases and their respective control groups are screened and classified.2.Routine bioinformatics analysis such as functional analysis,expression verification,immune infiltration analysis,protein interaction analysis and pathway enrichment are performed for the screened genes.3.The in vitro COPD model is established using Cigarette smoke extract(CSE)and Lipopolysaccharides(LPS).4.The proliferation,invasion and migration abilities of the cells are detected by CCK-8,transwell and scratch tests,and the expression of related indicators of the proliferation,apoptosis and EMT are observed by q PCR and Western Blot experiments.Combined with the experiment,we elaborate the effect of Ba P on tumor characteristics of COPD model in vitro.As distinct genes with opposite expression trends in both diseases,we observe the changes of NQO1 before and after exposure to Ba P in COPD models.5.The experimental method of adding NQO1 agonist(t BHQ)and inhibitor(DIC)is to make clear the effects of NQO1 on A549 cell proliferation,apoptosis,migration,invasiveness and EMT.6.The experimental method of treating cells with Ba P after knocking down the NQO1 gene is used to determine whether Ba P exerted effects on cell proliferation,apoptosis,migration and EMT through NQO1.Results: 1.The datasets of GSE151052,GSE118370 and GSE140797 were obtained after screening.GSE151052 included 77 COPD samples and 40 control samples,GSE140797 included 7 pairs of LAC tissue and control tissue samples,and GSE118370 included 6 pairs of LAC tissue and control tissue samples.Compared with the respective control groups,the differential expression results were as follows:52 up-regulated genes and 44 down-regulated genes of GSE151052.245 up-regulated genes and 152 down-regulated gene of GSE118370.1291 up-regulated genes and1483 down-regulated genes of GSE140797.In the classification analysis,COPD up-regulated and the LAC down-regulated genes were RTKN2,SLC6A4,and HBB.COPD down-regulated and the LAC up-regulated gene was NQO1.Genes upregulated in both COPD and LAC without screening conditions.Both COPD and LAC down-regulated gene were FPR1,LYVE1,and PKHD1L1.2.The main enrichment pathways were cell cycle,EMT,PI3K/AKT,and apoptosis.In data included by GEPIA,PKHD1L1,FPR1,LYVE1,RTKN2,HBB,and SLC6A4 were significantly down-regulated(P<0.05)and NQO1 was up-regulated(P<0.05)in LAC relative to controls.Besides,the target genes were all highly correlated with the expression of B cells,CD8+T cells,CD4+T cells,macrophages,neutrophils and dendritic cells(P<0.05).There were 46 proteins in the target interaction,whose enrichment functions were hydrogen peroxide catabolism,oxygen transport,antibiotic catabolism,etc..3.When A549 cells were stimulated with 100 ng/m L LPS+10% CSE,COX-2 expression indicated that the in vitro COPD model was successfully established.4.Compared with the control group,the number of cell proliferation and invasive cell in the Ba P group was significantly increased(P<0.01),the scratch distance of COPD and Ba P groups was significantly reduced(P<0.05).Compared with the COPD group,the COPD+Ba P group showed increased cell proliferation,shortened migration distance,and more significant increase in the number of invasive cells(P<0.01).It was further found that the expression trend of NQO1 in the COPD model was not consistent with that under the stimulation of Ba P.Compared with the control group,the expression of NQO1 in the COPD model was decreased(P<0.01),and that of Ba P group was increased(P<0.01).Compared with the COPD model,the expression of NQO1 in the COPD+Ba P group was significantly increased(P<0.01).Secondly,compared with the control group,the expressions of PCNA,Vimentin and Snail in COPD group and Ba P group were significantly increased(P<0.01),besides Caspase-3 and E-cadherin was significantly decreased(P<0.01).Compared with the COPD group,the expression of PCNA,Vimentin and Snail were increased(P<0.01),and the expression of Caspase-3 was decreased significantly in the COPD+Ba P group(P<0.01).5.Compared with the control group,t BHQ-stimulated cells exhibited increased proliferative and invasive activities(P<0.01),and shorter migration distances(P<0.01).Compared with the t BHQ group,the proliferation and invasion abilities were decreased(P<0.01),and the migration distance was increased of the t BHQ+DIC group(P<0.05).Compared with the control group,the expressions of NQO1,PCNA,Vimentin and Snail were significantly increased(P<0.01),and the expressions of Caspase-3 and E-cadherin were significantly decreased in the t BHQ group(P<0.01).Compared with the t BHQ group,the expressions of NQO1,PCNA,Vimentin and Snail were significantly decreased(P<0.01),and the expressions of Caspase-3 and E-cadherin were significantly increased in the t BHQ+DIC group(P<0.01).6.Compared with the sh Control group,the proliferation,migration and invasion abilities of the sh Control+Ba P group were increased(P<0.01).At the same time,the expression levels of NQO1,PCNA,Vimentin and Snail were significantly increased(P<0.01),while Caspase-3 and E-cadherin were decreased(P<0.01).Compared with the sh Control+Ba P group,the proliferation,migration and invasion abilities of cells in the sh NQO1+Ba P group were decreased(P<0.01),the expressions of NQO1,PCNA,Vimentin and Snail were decreased(P<0.01),and the expressions of Caspase-3 and E-cadherin were significantly increased(P<0.01).Conclusion: The expression changes of PKHD1L1,FPR1,LYVE1,RTKN2,HBB,SLC6A4 and NQO1 may play an important role in the progression of COPD to LAC.NQO1 may improve proliferation and migration of lung adenocarcinoma A549 cells,Ba P may promote the state of cellular EMT by increasing the expression of NQO1 in COPD models,which in turn exposes COPD models to tumor features.