Ginger GEO Through DNA Methylation And PTEN-PI3K-AKT Pathway Inhibiting The Proliferation Of Triple Negative Breast Cancer Mechanism Research

Objective:To explore the pathway of therapeutic effect of ginger volatile oil(GEO)on triple negative breast cancer.Through network pharmacology,we collected data about ginger in the treatment of triple negative breast cancer on various platforms,predicted its possible mechanism,and the prediction results of data mining are verified by in vivo and in vitro experiments.Part Ⅰ Data mining of Ginger treating triple negative breast cancerMethods:The effective components and target proteins of Ginger were predicted and screened through multiple drug databases and literature records.Collect disease targets of TNBC through multiple disease databases.The core genes(Hub genes)in the interaction network of ginger and triple negative breast cancer target protein interaction(PPI)were screened out after the collected PPI.Metascape data platform was used to conduct pathway enrichment analysis to obtain the mechanism pathway of ginger in the treatment of triple negative breast cancer.Ultimately,the binding activity between the triple negative breast cancer target and the effective components of ginger was obtained by molecular docking.Results:According to TCMSP and other databases and literature searches,there are 265 known components in Ginger.After downloading the 3D structure diagram of the components on PUBCHEM and uploading it to Swiss ADME database for pharmacokinetic(ADME)prediction and screening,10 potential effective components of Ginger are obtained.Finally,after target prediction and merging by Pharmamapper,53 target points of ginger are obtained by deleting duplicate targets.Through the disease database,1729 TNBC related targets were obtained after deleting duplicate targets by taking triple negative breast cancer as the key word.The Hub gene in the interaction network of ginger and triple negative breast cancer target protein interaction(PPI)was screened out after the collection of obtained PPI,which includes ESR1,ALB,EGFR,CASP3,ANXA5,MAPK1,PGR,MAPK14,AR,MAPK8.KEGG results of ginger in treating triple negative breast cancer were obtained by using Metascape data platform to conduct pathway enrichment analysis,which include TNF,IL-17,FoxO,MAPK,intestinal flora,PI3K/AKT and other pathways.The molecular docking of 10-gingerol,geraniol,citral,dihydrocapsaicin,L-linalool,6-gingerol with core targets ALB,ESR1,EGFR,CASP3,ANXA5,MAPK1 shows that the active components have good and stable binding with the target and high binding activity.Conclusion:Ginger treats triple negative breast cancer through TNF,IL-17,FoxO,MAPK,intestinal flora,PI3K/AKT and other pathways.The active ingredients of ginger have high affinity with the triple negative breast cancer target,and the combination is good.Part Ⅱ GEO Regulates the Diversity of Intestinal Flora and Affects the Expression of DNA Demethylation Genes to Inhibit the Proliferation of Triple Negative breast cancerMethods:A nude mouse model of triple negative breast cancer was built.Rectal feces and tumor tissue after GEO administration intervention were collected,and fecal genomic DNA was extracted and amplified,and intestinal microbes of nude mice with triple negative breast cancer were sequencing analysed by NovaSeq6000.The effect of GEO on the relative expression of DNA demethylation gene mRNA in nude mice with triple negative breast cancer was detected by RT-qPCR.Results:After the nude mice bearing MDA-MB-231 breast cancer tumor were given low,medium and high doses of GEO,the tumor growth of mice was inhibited to a certain extent,and the average tumor weight of high dose mice was significantly lower than that of the control group.The intestinal species composition of nude mice bearing MDA-MB-231 breast cancer tumor was analyzed.The number of OTU species was 490 in the blank control group,597 in the GEO low dose group,665 in the GEO medium dose group,and 507 in the GEO low dose group.Via analyzing the effect of GEO on the relative abundance of intestinal microbial species in nude mice bearing MDA-MB231 breast cancer tumor,five phylum level species were detected,of which Bacteroidota was the dominant flora in GEO group and control group.A total of 17 genus level(Genus)species were detected and one other species was not studied and named.Among them,Bacteroides accounted for the same proportion in the four groups.Muribaculaceae was the dominant flora in the high dose group of ginger oil(HGEO),and Alloprevotella was the dominant flora in the control group.The alpha diversity of intestinal microbial species samples of nude mice bearing MDA-MB-231 breast cancer tumor caused by GEO was analyzed and alpha diversity indicators Chao,Ace,Shannon and Simpson were statistical analyzed.The Chao index and Ace index of the GEO group were higher than those of the control group.The Shannon index of the GEO group was higher than that of the control group,while the Simpson index was lower than that of the control group.After tumor inoculation,the abundance of the intestinal microbial species in nude mice decreased,and the abundance of the community increased after GEO treatment.The relative expression of DNA demethylase TET1 mRNA in nude mice bearing MDA-MB-231 breast cancer tumor increased after GEO treatment,and there was a significant difference in the high dose GEO group,it was positively correlated with Enterobacter,Acinetobacter,and Enterococcus.The relative expression of mRNA of DNA demethylases TET2 and TET3 tended to increase,but there was no significant difference,and TET2 and Lactobacillus showed a significant positive correlation,and Bacteroides and Prevotellaceae UCG-001 showed a very significant negative correlation.TET3 was positively correlated with Oscillospiraceae,Candidatus Saccharimonas,norank f Peptococcaceae,Lactococcus,and Mucispirillum.Conclusion:GEO inhibits the proliferation of triple-negative breast cancer by increasing the diversity of intestinal flora,the abundance of microbial species and the alpha diversity of microbial species.GEO may affect the expression of DNA demethylation genes TET1,TET2 and TET3 by affecting the richness of intestinal flora,thereby inhibiting the proliferation of triple-negative breast cancer.Part Ⅲ GEO Regulates PTEN-PI3K-AKT Signal Pathway/DNA Methylation to Inhibit the Proliferation of Triple negative breast cancer CellsMethods:Effect of GEO on the activity of MDA-MB-231 cells in triple negative breast cancer was determined by MTT.Effect of GEO on the migration ability of MDA-MB231 cells was determined by cell scratch recovery test.The effect of GEO on apoptosis/cycle of MDA-MB-231 cells was determined by flow cytometry.The effect of GEO on the relative expression of DNA methylation gene and PTEN-PI3K-AKT channel gene in MDA-MB-231 cells were determined by RT-qPCR.The effect of GEO on the relative expression of PTEN-PI3K-AKT channel protein and DNA methylation protein in MDA-MB-231 cells were determined by Western Blot experiment.Results:The IC50 of GEO was determined by MTT to be 130 mg/L.When the concentration of GEO was 50 mg/L,there was a significant difference in the inhibitory effect of drug intervention on cell proliferation for 48 hours.When the concentration of GEO was 100,150,200,250 mg/L,there was a significant difference in the drug intervention for 24 or 48 hours,and there was a certain dose-effect relationship.The cell cross section test showed that GEO could significantly inhibit the horizontal migration of MDA-MB-231 cells when the concentration of GEO was 50,100,and 150 mg/L 24 h after administration.Cell apoptosis and cell cycle were measured by flow cytometry.The experimental results showed that the proportion of early apoptosis and late apoptosis in GEO group was significantly higher than that in the control group,and the proportion of total apoptosis showed a certain concentration-dependent trend.The total apoptotic ratio of the control group was 9.13 ± 1.12%,while the total apoptotic ratio of the GEO group with the concentration of 100,150,200mg/L increased to 123.19± 1.90%,26.43 ± 1.32%and 36.70 ± 0.88%,respectively.After treating MDA-MB-231 cells with 200 mg/L GEO,the percentage of G0/G1 phase cells decreased significantly,and the proportion of S phase cells increased significantly,indicating that GEO blocked the S phase of MDA-MB-231 cells.RT-qPCR results showed that GEO could reduce the expression of DNA methyltransferase gene DNMT1,and increase the expression of PTEN,CASP3,CASP9 and DNA demethylation gene TET1.Western blot set up four groups,namely PTEN-PI3K-AKT pathway inhibitors wortmannin,Control,200mg/L GEO,and DNA methyltransferase inhibitor 5-Aza.The results showed that GEO decreased the protein expression of p-PI3K/PI3K,p-Akt/Akt,DNMT1 and increased the protein expression of PTEN and CASP9.Conclusion:The cell phenotype experiment showed that GEO could inhibit the proliferation activity,migration movement,cycle arrest and induce apoptosis of MDA-MB-231 cells.GEO may play the role of methylation or demethylation to regulate the expression of key genes/proteins in PTEN-PI3K-AKT signal pathway,further activate the downstream signal of PTEN-PI3K-AKT,and affect the proliferation,cell survival and cell cycle of MDA-MB-231 cells.