Study On The Molecular Mechanism Of Icariside Ⅱ In Regulating EMT In Triple-negative Breast Cancer Through Wnt7b/β-catenin Signaling Pathway

Objective:Triple-negative breast cancer（TNBC）is the most challenging subtype of breast cancer with high invasive power,high metastasis rate and poor prognosis,clinical treatments are limited.Therefore,exploring effective drug has become a hot topic of research.IcarisideⅡ（ICS-Ⅱ）is a monomeric compound extracted from the traditional Chinese medicine Epimedium,and is the major effective ingredients responsible for the pharmacological effects of Epimedium.Studies have shown that ICS-Ⅱhas anti-tumor activity,but its mechanism of action on TNBC cells is still unclear.The aim of this study was to investigate the pharmacological effects and mechanism of ICS-Ⅱon TNBC cells.Methods:CCK-8 assay was used to detect the effect of ICS-Ⅱon the viability of TNBC cells.Bioinformatics methods were used to analyze the difference of Wnt7b expression between breast cancer tissues and normal tissues,and exploring the effect of Wnt7b expression on the overall survival in breast cancer patients.Western blotting assay was used to detect the changes of ICS-Ⅱon the expression levels of Wnt7b/β-catenin signaling pathway and EMT-related proteins in TNBC cells.The Ed U incorporation assay was used to assessment the effect of ICS-Ⅱon the proliferation ability of TNBC cells.Transwell assay was used to evaluate the effect of ICS-Ⅱon the migration and invasion ability of TNBC cells.To clarify the correlation between the interventional effect of ICS-Ⅱon TNBC cells and the Wnt7b/β-catenin signaling pathway,a rescue experiment was performed using the Wnt/β-catenin signaling pathway activator SB216763.Results:The results of CCK-8 assay showed that ICS-Ⅱinhibited the cell viability of two TNBC cell lines（MDA-MB-231 and MDA-MB-468）in a dose-dependent manner,with an IC₅₀of 19.57μM for ICS-Ⅱintervention in MDA-MB-231 cells and its IC₅₀of 32.75μM for MDA-MB-468 cells.According to the ICS-Ⅱconcentration,the study was further divided into control group（ICS-Ⅱ0μM）,low dose group（ICS-Ⅱ10μM）,medium dose group（ICS-Ⅱ20μM）and high dose group（ICS-Ⅱ40μM）.Bioinformatics analysis showed that the expression of Wnt7b in breast cancer tissues was significantly higher than in normal tissues（P<0.05）,and the overall survival of breast cancer patients with high expression of Wnt7b was lower than that of patients with low expression of Wnt7b（P<0.05）.Western blotting results showed that the medium and high dose groups were also able to significantly inhibit the protein expression levels of Wnt7b,β-catenin and p-GSK3βin MDA-MB-231 and MDA-MB-468 cells（P<0.05）.Meanwhile,the medium and high dose groups also inhibited the Epithelial-mesenchymal transition（EMT）of MDA-MB-231and MDA-MB-468 cells by promoting the expression of the epithelial marker E-cadherin and inhibiting the expression of the mesenchymal marker Vimentin in MDA-MB-231 and MDA-MB-468 cells（P<0.05）.The results of Ed U incorporation assay showed that the low,medium and high dose groups significantly inhibited the proliferation of MDA-MB-231and MDA-MB-468 cells（P<0.05）and the medium and high dose groups suppressed the expression of proliferation-related proteins PCNA and CCND1（P<0.05）.Transwell assay results showed that the medium and high dose groups were able to significantly inhibit the migration and invasion of MDA-MB-231 and MDA-MB-468 cells（P<0.05）,as well as inhibit the expression of MMP2 and MMP9 at the protein level（P<0.05）.SB216763,an activator of Wnt/β-catenin signaling pathway,significantly attenuated the inhibitory effect of ICS-Ⅱon EMT,proliferation,migration and invasion in MDA-MB-231 and MDA-MB-468 cells and reversed the inhibitory effect of ICS-Ⅱon expression level of Wnt7b/β-catenin signaling pathway and its downstream related proteins.Conclusion:ICS-Ⅱcan inhibit the proliferation,migration and invasion of MDA-MB-231 and MDA-MB-468 cells by regulating the EMT process mediated by Wnt7b/β-catenin signaling pathway,which has anti-TNBC effect,providing the basis of theoretical basis and in terms of experimental for the treatment of TNBC by Epimedium.