Identification Of Potential Hub Genes Via Bioinformatics Analysis Combined With Experimental Verification In Colorectal Cancer

Colorectal cancer(CRC)is a malignant tumor with high morbidity and mortality,which is a serious threat to human health.The purpose of this study is to find the potential hub genes related to colorectal cancer through bioinformatics analysis and experimental verification.First of all,the gene expression profile data of colorectal cancer patients were downloaded from Gene Expression Omnibus database(GEO),and the Differentially Expressed Genes(DEGs)were detected in colorectal cancer tissues and paracancerous tissues respectively.Among them,there are 1131 DEGs in microarray GSE81558,1837 DEGs in GSE21510 microarray and 1507 DEGs in GSE32323 microarray.A total of 484 DEGs are shared by the three microarrays.The 484 genes were selected for Gene Ontology(GO)analysis,and then the pathways of significant enrichment of DEGs were analyzed by using Kyoto Encyclopedia of Genes and Genomes(KEGG).The results showed that the DEGs were significantly enriched in the biological processes such as flavonoid glucuronidation,cell division,cell cycle phase transition and monocarboxylic acid metabolism,the changes of cell components mainly included the changes of cytoplasmic spindle and cytoplasmic perinuclear region,and the molecular functions such as oxidoreductase activity were significantly enriched in DEGs.KEGG analysis showed that DEGs play an important role in pentose and glucuronic acid transformation,glucuronic acid pathway and drug metabolism.To further analyze the interactions between DEGs,Metascape database was used to explore the enrichment of protein-protein interactions.And use MCODE algorithm to identify tightly linked network components.Eight core modules were mainly enriched from 484 proteins.We select the genes in the most important module MCODE1 for further study.To verify the effectiveness of module gene acquisition,we use a variety of databases based on TCGA patient samples to further verify and analyze the core module genes.First of all,the effects of module genes on patients' viability were pre-screened by the optimal cut-off value.It was found that the abnormal expression of RRM2,CCNB1,MAD2L1,AHCY,BUB1 B,ERCC6L,KIF18 A and DSN1 has a significant effect on the overall survival rate of patients with colorectal cancer.Furtherverify the expression of these genes in patient samples.The results showed that these genes were significantly up-regulated in colorectal cancer patients.We further analyzed the genetic changes of these genes in colorectal cancer patients.Among these core genes,DSN1 and AHCY have the highest frequency of gene change,and DSN1 has a high frequency in colorectal adenocarcinoma,rectal adenocarcinoma and mucinous adenocarcinoma.We selected DSN1,AHCY and ERCC6 L genes to verify their expression in colorectal cancer cell lines.The results showed that the mRNA expression of DSN1,AHCY and ERCC6 L in colorectal cancer cells was significantly higher than that in normal colorectal cells,and the protein level of DSN1 in SW480 cell line was significantly increased.It is suggested that DSN1 may play a significant role in the occurrence and development of colorectal cancer.We selected DSN1 gene for gene function analysis.si RNA is used to knock down the expression of DSN1.To analyze its effects on the proliferation,migration and colony formation of colorectal cancer cells.The results showed that the decreased expression of DSN1 significantly inhibited the cell migration and colony formation ability of colorectal cancer cells caco-2 and SW480,but did not significantly inhibit the growth rate of the two kinds of cells.Then,the pathway of significant enrichment of DSN1 gene in colorectal cancer samples with high expression of DSN1 gene was further analyzed by(Gene Set Enrichment Analysis,GSEA)method.It was found that pyrimidine metabolism and nucleotide excision and repair pathway related genes were significantly enriched in colorectal cancer patients with high DSN1 expression.Finally,STRING database and GEPIA database were used to explore the possible mechanism of the role of DSN1 in colorectal cancer.It is found that there is a close relationship between DSN1 and BUB1 B,a member of BUB1 family.Therefore,we think that the interaction between DSN1 and BUB1 B may be the potential mechanism of DSN1 affecting the migration and colony formation ability of CRC cells.In short,this study shows that the combination of bioinformatics analysis and experimental method verification can provide more potentially important targets for the prevention and treatment of colorectal cancer.