Study On The Expression And Role Of CCNA2 In Lung Adenocarcinoma

Objective:Lung cancer is one of the most common malignant tumors in the world.It is divided into non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC).NSCLC accounts for about 83%of lung cancers.Lung adenocarcinoma(LUAD)is the most common pathologic type of NSCLC.The process of LUAD is very complicated.With the development of molecular biology technology,it is possible to distinguish benign and malignant tumors according to the abnormal expression of molecules.The study of abnormal molecular expression in LUAD will provide a new guidance for molecular pathological diagnosis,clinical prognosis and treatment of LUAD.Cyclin A2(CCNA2)is an important cell cycle regulator.The purpose of this study was to explore the level of CCNA2 expression between LUAD and normal tissues,detect the correlation between CCNA2 and clinical characteristics of patients and search the relationship between CCNA2 and tumor immunity.We aimed to assess the value of CCNA2 in the diagnosis and prognosis of LUAD.Methods:1.We detected the expressed difference of CCNA2 in LUAD and normal tissues by GEPIA and HPA database.2.Survival Analysis and Clinical Relevance Analysis:Survival analysis was applied in LUAD samples which were downloaded from TCGA database and GEO database.The Kaplan-Meier(KM)survival curve was pictured by "survival R" package in R software.Patients’ gender,age,cancer stages,tumor size and nodal metastasis status were selected to perform the stratified analysis.3.GO function analysis and KEGG pathway analysis were used to predict related functions of CCNA2-related differentially expressed mRNAs.4.TIMER database was applied to analyze the relationship between CCNA2 expression and tumor immune in LUAD.Results:1.Detection of CCNA2 expression in LUAD:CCNA2 expressed highly in LUAD tissues compared with normal tissues.2.Survival analysis and clinical relevance analysis:The Kaplan-Meier(KM)survival curve illustrated that LUAD patients with high CCNA2 expression usually had lower overall survival.The expression level of CCNA2 was significantly correlated with gender,clinical stage,T classification and N classification.3.KEGG and GO enrichment analysis:The functional analysis revealed that the function of CCNA2 was mainly involved in the DNA replication,cell cycle,nucleocytoplasmic transport,cellular senescence,apoptosis and p53 signaling pathway.4.Tumor immune:CCNA2 had a negative correlation with the levels of CD4+T cells and B cells,while had the positive correlation with the expression level of CD8+T cells,Neutrophil and Macrophage.CCNA2 had a positive correlation with the expression of CTLA-4,PD1(PDCD1),TIM3(HAVCR2),LAG3 and TIGIT.Conclusion:1.CCNA2 expressed highly in LUAD tissues and had relationship with patients’ overall survival and patients’ clinical features.2.CCNA2 mainly participated in the regulation of cell cycle.3.CCNA2 was significantly correlated with immune cell infiltration and immune checkpoint molecules.Objective:In first part of the study,we confirmed that CCNA2 expressed higher in lung adenocarcinoma tissues compared with normal tissues.To further inquire the effects of CCNA2 in lung adenocarcinoma cells,we conducted in vitro experiments to explore the effects of CCNA2 on the proliferation,invasion,migration and cell cycle of lung adenocarcinoma cells.Methods:1.The expression of CCNA2 in normal bronchial epithelial cell(16HBE)and human LUAD cells(H1975,H1299 and A549 cell)lines was detected by RT-qPCR and Western Blot.2.We establish the cells with si-CCNA2 and over expression of CCNA2.Western blot and RT-qPCR were used to identify the expression of CCNA2.3.Cell Function Test:EdU cell proliferation test was used to detect the proliferation effect of CCNA2 in LUAD cells.Transwell cell method was applied to detect the migration and invasion level.We use flow cytometry to test the cell cycle progression of H1975 cell.Results:1.RT-qPCR and Western Blot showed that the expression of CCNA2 was up-regulated in LUAD cells compared with normal bronchial epithelial cell.2.Knockdown of CCNA2 suppressed the proliferation,migration and invasion of H1975 cell and H1299cell.Over expression of CCNA2 can promote the proliferation,migration and invasion of A549 cell.When CCNA2 was knocked down,there were more cells were arrested in G2 phase.3.Explore the Mechanism of CCNA2 in LUAD Cells:After knocking down CCNA2 in H1975 cells,P53 has an upward trend,which may suggest that the function of human CCNA2 in LUAD cells plays a role through P53.Conclusion:1.CCNA2 knockdown inhibits the proliferation,migration and invasion of LUAD cells,while over-expression of CCNA2 promotes the proliferation,migration and invasion of LUAD cell.2.CCNA2 affects the cell cycle of LUAD cell by arresting in G2 phase.3.The function of CCNA2 in LUAD cells plays a role through P53.