Application Of Dioscorea Bulbifera L. And Its Main Ingredient Diosbulbin B To Triple-negative Breast Cancer

Objective: Explore the main active compounds,potential targets and mechanism of Dioscorea bulbifera L.against triple-negative breast cancer,and provide new scientific methods and basis for the application of Dioscorea bulbifera L.in triple-negative breast cancer.Methods: 1.Obtain the genes of the intersection of Dioscorea bulbifera L.active ingredient target genes and triple-negative breast cancer targets through TCMSP,Gene Cards,GEO and TCGA databases,and use Kaplan-Meie,TCGA,and GSEA databases to conduct survival analysis and pathway enrichment analysis and biorelock analysis in breast cancer.2.Through the network pharmacology of Chinese medicine were analysised the main active components of Dioscorea bulbifera L.anti-triple-negative breast cancer and the main anti-tumor components,and the main compounds of Dioscorea bulbifera L.and the main anti-tumor compound Diosbulbin B were selected and screened for in vitro verification experiments.CCK-8 and scratch experiments were used to study the effect of Diosbulbin B on the proliferation and migration of MDA-MB-231 breast cancer cells;Hoechst staining and flow cytometry were used to detect the effect of Diosbulbin B on MDA-MB-231 breast cancer cell apoptosis;Real-time fluorescent quantitative PCR(q RT-PCR)verifies that Diosbulbin B has an effect on MDA-MB-231 breast cancer cell TP63 gene expression;Western-blot method(Western-blot)verifies that Diosbulbin B has an effect on MDA-MB-231 breast cancer cell P63 protein and its Apoptosis-related proteins Bax,Bcl-2 and Caspase 3 are expressed.Results: 1.A total of 14 active ingredients were screened,and the targets of the active ingredients were combined with the triple-negative breast cancer-related targets in the Gene Cards database,TCGA and GEO databases,and three genes(CCNB1,PGR,TP63)and Dioscorea bulbifera L.were found Related to anti-triple-negative breast cancer.Kaplan-Meie survival analysis showed that only TP63(P<0.05)was related to the survival rate of breast cancer.According to the analysis of TCGA clinical database,the differential expression of TP63 gene in triple-negative breast cancer was the most significant,while GSEA pathway enrichment analysis showed that TP63 Genes are related to the pathway of apoptosis in breast cancer(P<0.01).2.Through the network pharmacological analysis of Chinese medicine Dioscorea bulbifera L.The main active ingredients of anti-breast cancer and the main anti-tumor ingredients Diosbulbin B was selected for in vitro verification experiments.The results showed that Diosbulbin B is main anti-tumor compound in Dioscorea bulbifera L.,can inhibit the proliferation and migration of MDA-MB-231 breast cancer cells in a dose-dependent manner.Observed by Hoechst staining method,the higher the concentration of Diosbulbin B,the stronger the transparency of the blue cells undergoing apoptosis.The results of flow cytometry showed that the number of apoptotic cells also increased with the increase of Diosbulbin B concentration.The results of q RT-PCR showed that the higher the concentration of Diosbulbin B,the higher the expression of TP63 gene in MDA-MB-231 breast cancer cells.Western blotting results showed that P63,Bax,and Caspase3 proteins would be up-regulated with the increase of Diosbulbin B concentration,Bcl-2 protein would be down-regulated,and the Bcl-2/Bax ratio would gradually decrease.Therefore,it was verified that Diosbulbin B the main anti-tumor compound of Dioscorea bulbifera L.,may inhibit the proliferation and migration of MDA-MB-231 breast cancer cells and induce cell apoptosis by regulating the expression of TP63 gene.Conclusions: 1.Successfully screened Dioscorea bulbifera L.as the main component of anti-breast cancer and the main target for the treatment of triple-negative breast cancer.2.It was verified that Diosbulbin B the main anti-tumor component in Dioscorea bulbifera L.can inhibit the proliferation and migration of MDA-MB-231 breast cancer cells as the concentration increases.3.It was verified that Diosbulbin B the main anti-tumor component in Dioscorea bulbifera L,can promote the expression of TP63 gene with increasing concentration,and promote the expression of apoptosis-related P63,Bax,and Caspase 3 proteins,and make Bcl-2 protein gradually Decrease,the ratio of Bcl-2/Bax gradually decreases,indicating that Diosbulbin B may induce apoptosis of MDA-MB-231 breast cancer cells by regulating the expression of TP63 gene.