The Effect Of SPP1 In Lung Adenocarcinoma And Its Prognostic Correlation Analysis

Objective:Lung adenocarcinoma(LUAD)is one of the most common malignant diseases in the world,and its morbidity and mortality have been high these years.It is necessary to improve the understanding of the molecular mechanism of LUAD in order to predict the prognosis and develop new treatment strategies for target genes.This study used bioinformatics methods to screen out differentially expressed genes related to LUAD prognosis with WGCNA in GEO and TCGA databases,and verified the expression and function of this gene in lung adenocarcinoma cell lines through cell experiments.Furthermore,we analyzed the possible molecular mechanism and function of this gene in the development and prognosis of LUAD by bioinformatics.Methods:1.Screening of key genes:Screen the genes differentially expressed in LUAD tissues and its paired adjacent tissues through GEO database and TCGA database,and then through the limma software package of R software and the weighted gene co-expression network(WCGNA)software package identifies differentially expressed genes and key co-expression modules.And the expression of differential genes in LUAD were verified through the Human Protein Atlas Database(HPA).2.Verifying the expression of key genes in lung adenocarcinoma cell lines:qRT-PCR and Western Blot methods were used to detect differences in the expression of key genes in lung adenocarcinoma cell lines A549 and H1299 and normal bronchial epithelial cells 16HBE.3.Cell function experiments:siRNA sequence for specific gene were designed to transfect LUAD cell line A549 and H1299 cells to knock down the expression of target gene,using qRT-PCR method to detect cell interference efficiency,and after transfection of siRNA,CCK8 cell proliferation experiment was performed to detect the effect of the target gene on the proliferation of LUAD cells.4.Predicting the function and mechanism of the target gene in LUAD:PPI protein interaction network was constructed through Cytoscape software and STRING database,GO function analysis and KEGG function analysis were performed through DAVID database.And through Kaplan-Meier univariate survival analysis and GAPIA2 tool,the relationship between disease-free survival(DFS)and key genes in LUAD patients were explored.At the same time,the gene expression levels of patients in different clinical stages were analyzed to explore the relationship between target genes and clinical stages.Results:1.By analyzing the chip data in GEO and TCGA by WCGNA software package,a total of 327 differentially co-expressed genes were identified(|logFC|≥ 1.0,P<0.05,).10 key genes were selected through the PPI network using Cytoscape.Among these key genes,SPP1 was up-regulated in LUAD tissues compared with normal tissues.The HPA database has also verified this.2.The expression of SPP1 gene in lung adenocarcinoma cell lines A549 and H1299 and normal human bronchial epithelial cells 16HBE was verified by qRT-PCR method,and it was confirmed that SPP1 gene expression was up-regulated in lung adenocarcinoma cell lines(P<0.01).Western Blot experiment results also confirmed that SSP1 was up-regulated in lung adenocarcinoma cell lines.3.Survival analysis showed that up-regulation of SPP1 in LUAD patients was associated with poor overall survival(OS).4.Cell function experiments showed that after interfering with SPP1,the proliferation of A549 and H1299 cells was significantly inhibited,indicating that SPP1 could promote the proliferation of LUAD cells.5.Bioinformatics analysis predicted the protein interaction network of key genes,GO and KEGG pathway analysis showed that SPP1 may be involved in the occurrence and development of LUAD.Conclusion:1.SPP1 is differentially expressed in lung adenocarcinoma cell lines A549 and H1299 and human normal bronchial epithelial cells 16HBE,and has the potential as a LUAD biomarker.2.The high expression of SPP1 can promote the proliferation of LUAD cells.3.Bioinformatics analysis found that the high expression of SPP1 is related to the poor prognosis of LUAD patients,and may be involved in the occurrence and development of LUAD.