Bioinformatics-based Mining Of Key Genes In Colorectal Cancer Liver Metastasis And Exploring Their Functions

Objective:To screen out the key genes involved in the occurrence and development of colorectal cancer liver metastasis(CRLM),and study their functions through cell experiments by bioinformatics methods.In order to explore the molecular mechanism of CRLM,provide theoretical basis for clinical research,basic research and targeted therapy of CRLM.Methods:3 gene chips(GSE103340、GSE49355、GSE51244)of colorectal cancer(CRC)primary tumor tissue and CRLM tissue were selected and downloaded from the gene expression database(GEO),the GEO2R online tools of GEO was used to screen the differentially expressed genes(DEGs),and the Venn diagrams was used to pick up the co-expressed DEGs.Then we performed gene ontology(GO)function and KEGG pathway enrichment analysis on the DAVID online website and drew the protein-protein interaction network diagram(PPI)on the STRING online website.Used Cytoscape software and MCODE plug-in to analyze and get the core DEGs in PPI,GEPIA online website was used to analyze the relationship between core DEGs and the prognosis of CRC,and get the genes related to the prognosis of CRC.Collected clinical CRC primary tumor tissues and CRLM histopathological specimens,and used immunohistochemical staining techniques to verify the expression of related genes.DLD-1 cells were transfected with APOE-siRNA and APOE plasmids to construct APOE low and high expression cell lines,and the expression of mRNA in each group of cells after transfection was verified using Real-time PCR,and then studied by Sphere-forming assay,Colony formation assay,CCK-8 assay and Transwell assay respectively to investigate the effects of APOE knockdown and high expression on the self-renewal,in vitro tumourigenic,proliferative and invasive capacities of DLD-1 cells.Results:435 DEGs were selected from the 3 gene chips,including 354 up-regulated DEGs and 81 down-regulated DEGs,and then 56 co-expressed DEGs were picked out by Venn diagram.After carrying out KEGG pathway enrichment analysis,GO function analysis,PPI mapping and PPI core network screening,a total of 41 core DEGs were obtained.3 genes were determined after performing subsistence analysis on the GEPIA online website,including 3 up-regulated DEGs,Apolipoprotein E(APOE),Cytochrome P450 Family 2 Subfamily E Member 1(CYP2E1)and secreted phosphoprotein 1(SPP1).The immunohistochemical results of clinicopathological specimens showed that the expression of APOE gene in CRLM tissue was significantly higher than that in CRC primary tissue(P<0.05).After transfection,the mRNA expression of APOE high expression group was significantly higher than that of control group(P<0.05),and the mRNA expression of APOE low expression group was significantly lower than that of control group(P<0.05).APOE was shown to promote self-renewal,in vitro tumour formation,proliferation and invasion ability of DLD-1 cells by Sphere-forming assay,Colony formation assay,CCK-8 assay and Transwell assay.Conclusions:APOE,CYP2E1 and SPP1 were screened out by bioinformatics methods may be the key genes in the development of CRLM.APOE is highly expressed in CRLM tissues,and its expression level affects the self-renewal capacity,in vitro tumourigenic capacity,proliferative capacity and invasive capacity of DLD-1 cells and promotes CRLM.