Molecular Target And Preliminary Verification Of Coptisine In The Treatment Of Colon Cancer Based On Network Pharmacology

Objective: Coptisine(COP),a natural isoquinoline alkaloid extracted from Coptidis rhizoma,has been reported to exhibit anticancer activity in colon cancer.However,the molecular mechanism of coptisine in the treatment of colon cancer is still unclear,and the molecular targets of its effect on colon cancer remain to be elucidated.This study aims to reveal the potential molecular mechanism of coptisine in the treatment of colon cancer using network pharmacology,explore the molecular targets of coptisine in the treatment of colon cancer and conduct preliminary verification.Methods: Firstly,we studied the effect of coptisine on the biological behavior of colon cancer by in vitro validation method;then predicted coptisine targets and colon cancer targets by network pharmacology,took the intersection of coptisine and colon cancer targets and screened out coptisine Targets for alkali therapy in colon cancer.The obtained targets were then imported into the string database,and a protein-protein interaction(PPI)network was constructed.Cytoscape software was used to analyze the PPI network,and the core targets of coptisine in the treatment of colon cancer were extracted,and the core target network was constructed.We then used the MCODE module to perform cluster analysis on the PPI network and performed Gene Ontology Functional Annotation(GO)and Kyoto Encyclopedia of Gene and Genomes Pathway Analysis(KEGG)enrichment pathway analysis on the targets involved in the cluster analysis to reveal the potential molecular mechanism of coptisine in the treatment of colon cancer.Next,we analyzed the differential expression of the obtained core targets in colon cancer through the GEPIA2 website,and further screened out the specific targets of coptisine in the treatment of colon cancer according to the results of the differential analysis.Then,these targets are used as receptors,and coptisine is used as a ligand for molecular docking to verify the binding ability between coptisine and the target.Finally,the target is preliminarily verified by Q-PCR analysis.Results:1.Coptisine has inhibitory effects on the viability,migration and proliferation of human colon cancer cell lines DLD1 and HCT-116.2.Target screening of coptisine in the treatment of colon cancer and construction of PPI network: 295 coptisine targets were screened through pharm Mapper website and Swiss Target Prediction website.16054 colon cancer targets were obtained from Gene Cards,OMIM,Drug Bank websites.The intersection of coptisine and colon cancer targets was obtained,and 91 targets of coptisine in the treatment of colon cancer were obtained.We constructed a PPI network with these 91 targets.3.Gene enrichment analysis: Cytoscape software was used to perform cluster analysis on the PPI network,2 clusters were obtained,and all clustered targets were subjected to GO and KEGG enrichment analysis.GO enrichment was mainly related to reproductive system development,regulation of intracellular estrogen receptor signaling pathway,positive regulation of DNA replication,and drug binding and steroid-binding.KEGG enrichment analysis suggested that these targets were mainly related to cancer in pathway,fcγR-mediated phagocytosis,prostate cancer,progesterone-mediated oocyte maturation,estrogen signaling pathway,proteoglycan in cancer,human papillomavirus infection and PI3K-Akt signaling pathway.4.Core target screening and preliminary validation: We used the Cyto Hubba plugin in Cytoscape software to screen out the top 10 hub genes in the PPI network:ESR1,AR,ALB,CDK2,PARP1,HSP90AB1,IGF1 R,CCNE1,CHEK2 and CDC42.Then,the differential expression analysis of the hub gene was carried out on the GEPIA2 website.The results showed that CCNE1,CDK2,HSP90AB1 and CHEK2 were up-regulated in colon cancer samples.Molecular docking showed that coptisine has a good binding ability to the above four targets.The results of Q-PCR showed that the expressions of CCNE1 and HSP90AB1 were significantly down-regulated after coptisine-treated colon cancer DLD1 cells,while CDK2 and CHEK2 had no significant difference in expression.Conclusion: Coptisine may be a candidate drug for the treatment of colon cancer.CCNE1,CDK2,CHEK2 and HSP90AB1 may be the potential targets of coptisine in the treatment of colon cancer,and further research is needed to verify it.