| Objective: 1.To explore and analyze the existing genetic data in the Gene Expression Omnibus(GEO)database to find the key dysregulated genes in the human Dukes’ B stage colorectal cancer(CRC).2.To predict the target and molecular mechanisms of Ebracteolatain A(EA)and human Dukes’ B CRC based on the network pharmacology and bioinformatics.3.To explore the hub genes and their effects of EA on human Dukes’ B CRC and then verify them by molecular biological methods.Methods: 1.Screened the dysregulated genes in Dukes’ B CRC: Collected,processed and integrated the existing chip data of human Dukes’ B CRC in the GEO database,and used the online analysis tool GEO2R(GEO to R)to analyze and normalize the selected chip data for the hub genes which are related to the human Dukes’ B stage CRC;used the R language program to analyze the gene expression between CRC and normal colorectal tissue and the Volcano Plot to visualize the results of the difference analysis;2.Predicted the mechanism of EA’s effect on human Dukes’ B CRC: The possible targets of EA and the target information were collected from the Swisstarget Prediction database and Target NET database,combined with the results of the screened differential expressed genes,further screened and analyzed the targets of EA on human Dukes’ B CRC;used STRING database and Cytoscape＿v3.8.2 to visualize the data and draw the protein-protein interaction(PPI)network diagram;based on the R language program package,the predicted targets were enriched and analyzed to obtain the key hub genes;3.Verified the effect of EA on the key hub genes of human Dukes’ B CRC cells: detected the toxicity of EA to human normal colorectal mucosal cells NCM-460,and determined the median inhibition concentration(IC50)of EA on human Dukes’ B CRC cells SW480.SW480 was treated with EA at IC50,real-time fluorescent quantitative PCR and Western Blot were used to detect the m RNA and protein expression of key hub genes in SW480.Results: 1.The key hub genes in human Dukes’ B CRC: The gene chip GSE37364 was selected and the control and experimental groups had 38 and 14 samples respectively(Affymetrix Human Genome U133 Plus 2.0 Array);we obtained 5411 differential expression genes and there were 3469 genes could be clearly annotated.From the 3469 genes we found that 1474 were up-regulated genes and 1995 were down-regulated genes.2.The molecular mechanism of EA on Dukes’ B CRC: 217 possible drug targets were obtained and 57 targets can match disease targets and drug targets simultaneously(19 up-regulated genes and 38 down-regulated genes).Using STRING database and Cytoscape＿v3.8.2,co-action targets were formed into a PPI network,and 46 targets can match into the network to obtain 2 clusters and 4 key hub genes such as PTGS2,ESR1,MMP9,SERPINE1;Gene Ontology(GO)analysis found that these 46 gene targets are mainly involved into 757 biological processes(BP),37 cellular components(CC),and 63 molecular functions(MF);Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis found that the 46 predictive targets are involved into 38 signaling pathways,and among them,enrichment in the Nitrogen metabolism pathway has the most significant correlation,which contains 4 predicted targets such as carbonic anhydrase 2(CA2),CA4,CA7 and CA9,and the 7 signal pathways such as Pathways in cancer,IL-17 signaling pathway,TNF signaling pathway,Micro RNAs in cancer,Endocrine resistance,Estrogen signaling pathway and Proteoglycans in cancer.3.The effect and mechanism of EA on human Dukes’ B CRC cells: CCK-8 assay showed that EA has no obvious toxicity to normal colorectal cells NCM-460 at low doses,and only 25-35% inhibition rate at high doses;The IC50 of SW480 treated with EA for 24 h,48h and 72 h were 41.24μg/m L,30.20μg/m L and 29.65μg/m L,respectively;RT-q PCR and Western Blot results showed that the m RNA and protein expression of PTGS2,MMP9 and SERPINE1 genes in CRC cell SW480 decreased after EA intervention,while the ESR1 gene increased,which are consistent with the results of network pharmacology.Conclusions: The key dysregulated genes for human Dukes’ B CRC were screened out;the possible key targets of EA’s effect on human Dukes’ B CRC and the signal pathways involved are predicted,including the up-regulated genes PTGS2,MMP9,SERPINE1 and down-regulated gene ESR1,etc.;biological studies have found that EA has low toxicity to normal cells,but can inhibit the expression of the aforementioned up-regulated genes and promote down-regulated gene expression in CRC,and significantly inhibit Dukes’ B CRC cells SW480. |
