Bioinformatics Analysis And Mechanism Study Of Anti-colorectal Cancer Targets Of Aspirin

BackgroundColorectal cancer(CRC)is a common malignant tumor,with the third highest incidence and the second highest mortality among malignant tumors in the world.At present,aspirin is one of the most promising chemoprevention drugs for CRC.Although the preventive effect of aspirin on CRC has been recognized to a great extent,the molecular mechanism of aspirin in the prevention and treatment of CRC has not been reached.Bioinformatics analysis shows its unique advantages such as high efficiency,economy,flexibility and so on.A variety of public gene expression profile databases and Drug database provide a broad platform for us to predict key genes and pathways related to diseases,and to find potential biomarkers and new drug targets.ObjectiveThis paper aims to predict and analyze the candidate genes of aspirin in the prevention and treatment of CRC through a variety of public databases,to explore the potential targets of aspirin against CRC,and to further verify the biological function of aspirin on HT-29 cells and its effects on potential targets.Methods1.Identification of targets of aspirin:DrugBank 5.1.7 was searched to identify direct protein targets(DPTs)of aspirin.The protein-protein interaction(PPI)network of DPTs was constructed and signaling pathways involved were analyzed.2.Screening of differentially expressed genes related to CRC:CRC-associated gene expression datasets were downloaded from GEO database to analyze the differentially expressed genes in colorectal cancer tissues.The common differentially expressed genes were obtained by intersection of which the top twenty differentially expressed genes with the highest degree were screened as Hub genes.3.Predication and analysis of targets of aspirin in the treatment of CRC:Common genes between the genes associated with the DPTs and the Hub genes of CRC were the potential targets of aspirin against CRC.Examine the potential targets in TCGA-COAD samples.GO functional enrichment analysis and KEGG signaling pathway of the potential targets were performed.4.Effect of aspirin on biological function of colon cancer cells:CCK8 assay,immunofluorescence assay,scratch test,Transwell invasion assay and flow cytometry were used to detect different concentrations of aspirin on cell viability,migration,invasion,cell cycle and apoptosis of HT-29 cells.5.Effect of aspirin on the expression of the potential targets:The potential targets of aspirin were verified by reverse transcription-polymerase chain reaction(RT-PCR)and western blot(WB).Results1.11 DPTs of aspirin were identified in DrugBank 5.1.7.KEGG signaling pathway showed that 6 genes(EDNRA,IKBKB,NFKB2,NFKBIA,PTGS2 and TP53)were associated with the occurrence and development of cancer.By intersecting 20 Hub genes of CRC with genes associated with the DPTs of aspirin,it was found that 5 genes(CDK1,AURKA,CCNB1,MAD2L1 and TPX2)may be the potential targets of aspirin against CRC.2.CDK1,AURKA,CCNB1,MAD2L1 and TPX2 were all up-regulated in TCGA-COAD samples,and the gene functions were mainly enriched in regulation of cell cycle phase transition,microtuble cytoskeleton organization and so on.3.The results of CCK8 assay and immunofluorescence assay showed that the cell viability and proliferation ability of HT-29 cells decreased significantly with the increase of aspirin concentrations.The results of scratch test showed that the migration ability of HT-29 cells decreased significantly with the increase of aspirin concentration.The results of Transwell invasion assay showed that the invasive ability of HT-29 cells decreased significantly with the increase of aspirin concentration.The results of flow cytometry showed that aspirin could block HT-29 cells in G0/G1 phase,and the apoptosis rate of HT-29 cells increased with the increase of aspirin concentration.4.The results of RT-PCR and WB showed that aspirin could down-regulate the expression levels of mRNA and protein of CDK1,AURKA,CCNB1,MAD2L1 and TPX2 in HT-29 cells in a concentration-dependent manner.ConclusionAspirin can significantly inhibit the proliferation,migration and invasion of colon cancer cells,block the cell cycle and promote apoptosis.CDK1,AURKA,CCNB1,MAD2L1 and TPX2 may be potential targets of aspirin in the treatment of CRC.