| Background:Colon cancer is one of the most common malignant tumors,and colonic adenocarcinoma is the most common in patients with colon cancer.Surgery combined with chemotherapy is the primary treatment for colon adenocarcinoma.However,the high cost of surgery and the risk of chemotherapy resistance has become a significant obstacle in treatment,and the search for new drugs is imminent.Many research found that berberine can inhibit the growth of colorectal adenocarcinoma cells,but the mechanism of action is not completely clear.Methods:The action genes related to berberine were obtained from the Chinese Traditional Pharmacology System and Swiss Target Prediction database,the data of GSE44076,a gene expression microarray for colon adenocarcinoma,were obtained from the comprehensive gene expression database,and the differentially expressed genes for colon adenocarcinoma were obtained after analysis by R language,and the genes related to colon adenocarcinoma were obtained from the Gene Cards database.After taking the intersection with the acquired berberine-related genes,the shared genes of berberine and colon adenocarcinoma were visualized by Cytoscape software,and the shared genes were analyzed by Gene Ontology and Kyoto Gene and Genome Encyclopedia,then,the STRING database was used to obtain the core genes in the shared genes,and the core network of the core genes was constructed,and the GEPIA database tool was used to analyze the differential expression of the core genes in normal colon tissues and colon adenocarcinoma tissues,to screen out genes with statistical significance on survival prognosis,to obtain their immunohistochemical staining maps in normal and tumor tissues from the Human Protein Atlas database,to obtain the 3D structures of their proteins from the Protein Institution database,and to obtain the 3D designs of berberine from the Pubchem database,and simulated molecular docking was performed using Autodock Vina.Configure the medium required for SW480 cells,fully dissolve berberine into DMSO solution,configure the necessary concentration of drug solution,add complete medium according to the principle of ready-to-use,configure the required attention of the drug-containing medium,remove the frozen cells from liquid nitrogen,quickly put them into a 37℃ water bath that has been preheated,shake continuously,and after the liquid in the freezing tube melts,use a pipette gun to transfer to a centrifuge tube.After blowing and centrifuging,discard the supernatant,add 10 ml of complete culture medium,blow repeatedly,mix thoroughly,and after using the cell counting plate technique,dispense the cell suspension that meets the cell counting requirements into the culture dish,shake gently from left to right and mix well,put it into the incubator,and change the liquid after 24 hours.The cell activity assay was performed using colon adenocarcinoma cell line SW480 cells,which were inoculated into 96-well plates at a density of 3000 cells/(100μL-well).After the cells were plastered,the medium in the vessels was aspirated and treated with appropriate concentrations of berberine for 48 hours,and the IC50 of berberine was calculated by reading the absorbance at 450 nm by an enzymatic standard.SW480 cells were treated with an IC50 concentration of berberine,and an equal volume of DMSO solution without berberine was added to the control group.After 48 hours,the complete medium containing berberine and DMSO in the culture dish was poured off,washed with PBS,lysis solution was added,left for a moment to allow cells to fully lyse,transferred to a centrifuge tube,operated according to the instructions of the kit,and total RNA was extracted.The RNA concentration was measured,reverse transcribed to cDNA,and a real-time polymerase chain reaction was performed.The experimental group was incubated with berberine at a concentration of IC50,and the control group was set with an equal volume of DMSO solution for 48 h.The medium in the culture dish was aspirated using a pipette gun,and the cells were washed twice by adding pre-cooled PBS at 4℃.The cell lysate containing PMSF was added and placed on ice for 30 min to allow the cells to lyse fully,and the lysate containing the cells was transferred to a centrifuge tube using a pipette gun.After centrifugation,the lysate was placed at -80℃ and set aside.The relative concentrations of the extracted total proteins were determined using Kemas Brilliant Blue solution,and the protein expression of the experimental and control groups was obtained by SDS-PAGE electrophoresis experiments.Results:A total of 118 prediction target genes of berberine were obtained from Chinese traditional pharmacology System and Swiss Target Prediction database,1598 differential genes of colorectal adenocarcinoma were obtained through analysis of colorectal adenocarcinoma gene expression chip,and 8644 colorectal adenocarcinoma related genes were obtained from Gene Cards database.A total of 17 genes were obtained after intersection.In the gene ontology analysis,we found that the G2/M cycle-related biological processes were at the top of the q-value list.The Kyoto Gene and Genome Encyclopedia analysis found that the cell cycle pathway qvalue ranked third.In the protein interaction network analysis,7 core genes were screened out,and the prognosis analysis found that among the 7 core genes,only AURKA and CCNB1 had statistically significant survival curves.In the protein immunohistochemical analysis,the expression levels of AURKA and CCNB1 were increased in the colorectal adenocarcinoma tissue.Molecular docking results showed that berberine had good binding ability with the two targets.In the cell viability assay experiment,the IC50 of berberine was calculated to be 28.28μM,and the Q-pcr results showed that berberine could down-regulate the m RNA expression of AURKA and CCNB1.Western blotting showed that AURKA and CCNB1 protein expression in berberine group decreased significantly compared with control group.Conclusion:Berberine can inhibit the growth of colon adenocarcinoma cells by downregulating AURKA and CCNB1,with the most likely pathway of action being the cell cycle signalling pathway.. |
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