Analysis Of The Correlation Between The Expression Of CENP-N And Autophagy In Lung Adenocarcinoma

ObjectiveIn the world,despite the significant effects of lung cancer screening,targeted drug therapy,internal medicine chemotherapy,immunotherapy,and surgical treatment,the most common cause of death in the occurrence and development of cancer is still lung cancer.Among primary lung tumors,the most common histological subtype is lung adenocarcinoma,which accounts for more than 40%of the total incidence rate of lung cancer cases,and its relative frequency is even increasing year by year.In the Centromere protein(CENP)family,CENP-N is the core member of the family.CENP-N has been found to be overexpressed in various tumors and affect tumor progression and prognosis,possibly significantly related to cell cycle and p53 signaling pathways,but its molecular mechanisms have not been systematically reported.Especially,the expression in lung adenocarcinoma tissue and how to further regulate the development of lung adenocarcinoma are still unknown and require in-depth research.This topic is to explore the clinicopathologic factors,prognostic value,apoptosis and autophagy function related to the total survival rate of lung adenocarcinoma patients through the application of bioinformatics analysis and molecular biological experiment verification of CENP-N to lung adenocarcinoma,so as to provide reference for CENP-N as a possible marker of poor prognosis of lung adenocarcinoma,a new target for clinical screening and treatment.MethodsWe used a variety of databases,including Gene Expression Profiling Interactive Analysis(GEPIA),Gene Expression Omnibus(GEO)database,The Cancer Genome Atlas(TCGA)and Genotype Tissue Expression database,to evaluate the differential expression,survival prognosis.Immunohistochemistry was used to investigate the expression of CENP-N in lung adenocarcinoma and its pathological factors.The relationship between CENP-N and autophagy in lung adenocarcinoma was investigated by transmission electron microscopy,immunofluorescence and Western blot.ResultsFirst,at the mRNA level,we found that CENP-N was more expressed in up to 21 tumors,including Adrenocortical carcinoma ACC,Bladder urothelial carcinoma BLCA,Breast invasive carcinoma BRCA,Lung adenocarcinoma LUAD,pancreatic cancer PAD,and Glioblastoma multiform GBM,The GEO database data set on lung adenocarcinoma showed that CENP-N was highly expressed in lung adenocarcinoma compared with precancer tissues.Second,ACC,Kidney Chromophobe KICH,BRCA,Kidney Renal Clear Cell Carcinoma KIRC,Skin Melanoma SKCM,Kidney Renal Papillary Cell Carcinoma KIRP,Liver Hepatocellular Carcinoma LIHC There is a correlation between the expression of CENP-N and clinical stage in nine types of ovarian serous cystadenocarcinoma(OV)and LUAD.In lung adenocarcinoma,the expression of CENP-N was also significantly correlated with age(P=0.041),clinical stage(P=0.007),survival status(P=6.554e-04),gen-der(P=0.026),T stage(P=0.004),and N stage(P=0.011),but not with M stage(P<0.095).Third,the high expression of CENP-N was significantly positively correlated with the overall survival OS in 7 tumors including ACC,BRCA,LUAD,PAAD,LGG,Mesothelioma MESO and uveal Melanoma UVM.The high expression of CENP-N affects the disease-specific survival rate(DSS)of eight cancers,including ACC,BRCA,LUAD,PAAD,LGG,KIRC,KIRP and MESO.In lung adenocarcinoma,high expression of CENP-N was positively correlated with OS and progression-free survival(p<0.05).The risk ratio(HR)in a single factor Cox model was 1.077;95%confidence interval:1.009-1.150,P=0.024;In multivariate COX model analysis,the HR was 1.372(95%CI:1.050-1.792,P=0.020),suggesting that high expression of CENP-N is an independent risk factor for overall survival in patients with LUAD.Fourth,Nomogram analysis shows that CENP-N has high diagnostic value in lung adenocarcinoma;The area under curve AUC results showed 0.852(95%CI:0.8270.877),indicating that CENP-N not only has the potential to distinguish between tumor tissue and normal tissue,but also can be used as a predictor of survival in patients with lung adenocarcinoma.Fifth,we performed immunohistochemical analysis on 114 cases of lung adenocarcinoma and precancerous specimens.The results showed that CENP-N protein was highly expressed in cytoplasm and nucleus;The positive expression rate of CENPN in 114 cases of lung adenocarcinoma was 57%(65/114),and the high expression of CENP-N in lung adenocarcinoma was significantly correlated with poor tumor differentiation(P=0.001),clinical stage(P=0.021),late T stage(P=0.028),and lymph node metastasis(P=0.022).Sixthly,Bioinformatics analysis of CENP-N expression and autophagy related proteins showed that BCL2(R=-0.25,P=2.6e-13),LC3(R=-0.52,P=1.4e-58),ATG5(R=0.45,P=0),ATG7(R=0.18,P=2.6e-07),P62(SQSTM1)(R=0.074,P=0.033),and Beclinl(R=0.31,P=1.5e-19),suggesting that autophagy and CENP-N expression have varying degrees of correlation in lung adenocarcinoma,especially in LC3 autophagy proteins.Seventh,transmission electron microscopy showed that the number of autophagosome and auto phagolysosome increased significantly after CENP-N knockdown in A549 cells compared with the control group.Eighth,the results of immunofluorescence detection showed that CENP-N and LC3 were co-localized in the nucleus,and the expression of BCL2 decreased and Beclinl and LC3 increased after CENP-N interference.Ninth,the results of Western blot experiment clarify:After CENP-N interference,the expression of pro-apoptotic protein BAX increased,while the expression of antiapoptotic protein BCL2 decreased,and the expression of LC3 and Beclinl increased.Conclusions1.CENP-N is highly expressed in pan-cancer tissues,especially in lung adenocarcinoma,and its high expression is actively correlated with clinical prognosis,low differentiation,T stage and N stage.2.High expression of CENP-N can affect tumor prognosis,and CENP-N can be a valuable prognostic biomarker for lung adenocarcinoma.3.CENP-N may regulate the development of lung adenocarcinoma by inhibiting autophagy.