Screening And Mechanism Of Prognostic Markers For Lung Adenocarcinoma Based On Bioinformatics

Objective: Lung adenocarcinoma(LUAD)is the most common pathological type of lung cancer,with high incidence and mortality.To improve the curative effect and prolong the survival of patients,it is necessary to find new biomarkers to accurately predict the prognosis of patients and explore new strategy to treat high-risk LUAD.Methods: 1.Use the retrospective database of LUAD patient data from the Cancer Genome Atlas(TCGA)and Gene Expression Synthesis(GEO)datasets,including GSE31210,GSE18842,GSE19188,GSE40791 and GSE50081,to compare the gene expression of "ANGPT(L)s" family and "5 gene prognostic signature" and its relationship with the clinical characteristics of lung cancer patients.2.Using Cox proportional hazard model and Kaplan-Meier survival analysis method,?~2 test analysis to test the relationship between the "ANGPT(L)s" family and "5-gene prognostic characteristics" and the clinicopathological characteristics and prognosis of patients with lung adenocarcinoma;3.Using genes Gene Set Enrichment Analysis(GESA)method predicts related pathways in lung adenocarcinoma that may be regulated by the "ANGPT(L)s" family and "5 gene prognostic characteristics".4.The Connectivity map(Cmap)is used to predict potential drugs for the treatment of LUAD.5.Use Auto dock Vina v.1.1.2 molecular docking software to analyze the binding between the drug and the target.6.Cell viability experiments,colony formation experiments,Transwell experiments,Western blot(WB)experiments,and flow cytometry are used to explore the potential biological functions and mechanisms of the "ANGPT(L)s" family and Gliclazide in lung adenocarcinoma cells.8.Statistical analysis: Use SPSS 17.0 and Graph Pad statistical software for statistical analysis.All experimental results were repeated 3 times and expressed as mean ± SD.P<0.05 is considered statistically significant.Results: 1.The mRNA level of ANGPT1 in lung cancer tissues was lower than that in normal tissues;its expression level was negatively correlated with T stage(P=0.013).2.The prognosis of lung adenocarcinoma patients with low ANGPT1 expression was significantly worse than that of patients with high expression(P=0.006).3.ANGPT1 is an independent protective factor for lung adenocarcinoma(P=0.008).3.GSEA results show that ANGPT1 may affect the occurrence and development of lung adenocarcinoma by regulating G2 M checkpoints,DNA damage repair,apoptosis,and cell proliferation-related pathways such as mTOR signal,MYC signal,and mitotic spindle.4.ANGPTL4 gene is highly expressed in lung adenocarcinoma tissue;it is positively correlated with later age and N stage(P<0.05).5.The prognosis of lung adenocarcinoma patients with high ANGPTL4 expression is significantly worse than that of patients with low expression(P<0.05).6.ANGPTL4 is an independent protective factor affecting the prognosis of lung adenocarcinoma(P<0.05).6.The results of GSEA show that ANGPTL4 may affect the occurrence and development of lung adenocarcinoma by regulating glycolysis,hypoxia,mismatch repair,MTORC1 and other related pathways.7.Q-RT-PCR and WB fully verified the knock-down efficiency of silencing A549 and H1299 by si-RNA;after silencing ANGPTL4,the results of Transwell experiment indicated that the migration and invasion ability of A549 and H1299 cells was weakened.8.After ANGPTL4 transfection,WB plot found that LDHA,PKM1,PFKFB3,PFKFB4 decreased to varying degrees,while PKM2 did not change significantly;in addition,E-CA and ZO1 increased to varying degrees,while the expression of Vimentin has been downregulated.9.A risk score(RS)based on 5 gene prognostic characteristics(KIF20A,KLF4,KRT6 A,LIFR and RGS13)is determined to be significantly related to overall survival(OS),and it is obtained in both the training set TCGA and the validation set GSE50081 verification.10.Constructed a Nomogram combining RS and clinicopathological parameters can effectively predict the prognosis of LUAD patients.11.Use the Cmap website to further screen Gliclazide as a drug candidate for the treatment of high RS LUAD.12.In vitro experiments show that Gliclazide may induce cell cycle arrest and apoptosis of LUAD cells by targeting CCNB1,CCNB2,CDK1 and AURKA.Conclusion: 1.ANGPT1 and ANGPTL4 are independent prognostic protective factors for lung adenocarcinoma,and may become potential prognostic markers and therapeutic targets for lung adenocarcinoma.2.This study identified and verified 5 gene markers that can predict the prognosis of LUAD patients,and its reliability and effectiveness are worthy of recognition.3.Gliclazide can be used as a potential drug for the treatment of lung adenocarcinoma.It provides a new direction for the prognosis and treatment of lung adenocarcinoma.