EYA4-targeted SIX1 Regulates Breast Cancer Cell Stemness And Related Pathological Features Through Wnt Signaling Pathway

Background of the study:Breast cancer is one of the most common malignant tumors worldwide and the leading cause of cancer death.The incidence of breast cancer is increasing year by year and still poses a great threat to women’s health.Therefore,it is essential to explore the molecular mechanisms involved in the development of breast cancer and to explore early diagnostic markers and effective therapeutic targets.EYA4(eye deletion homolog 4)is one of the members of the EYA gene family.A growing body of literature elaborates that EYA4 plays a crucial role in the development of many cancers.However,the specific role of EYA4 in breast cancer is unclear.Sine oculis homology frame homolog 1(SIX1)is a homologous protein that controls cell proliferation,migration,invasion and epithelial mesenchyme(EMT),producing transformations in the development of many organs.It has been reported in the literature that during development,SIX1 can interact with EYA family members to form transcription factors that are essential for organ development.However,the interaction between SIX1 and EYA4 has not been reported in breast cancer.Through relevant literature and databases we found that the Wnt pathway may play an important role as a regulatory mechanism of EYA4 in the complete process of tumor formation.Objectives:(1)To investigate the effects of EYA4 on proliferation,regulation,migration and invasion of breast cancer cells(2)To investigate the effect of EYA4 on EMT and stemness of breast cancer cells(3)To investigate the interaction between EYA4 and SIX1 on breast cancer development(4)To explore the effect of EYA4 on Wnt signaling pathwayMethods:(1)The expression levels of EYA4 in breast normal epithelial cells MCF-10A,breast cancer MDA-MB-231 and MCF-7 cell lines were detected by RT-qPCR and Western blot assay.(2)Lipofectamine 2000 was used to transfect si-EYA4 group and NC group into breast cancer cells MDA-MB-231 and MCF-7,and the transfection efficiency was detected by RT-qPCR technique using SYBR Green dye method.(3)Cell colony formation assay and CCK-8 assay were used to detect the proliferation ability of breast cancer cells in different subgroups;Hoechst 33258 staining assay to detect apoptosis of breast cancer cells;scratch assay to detect the migration ability of breast cancer cells in each group;Transwell assay(with or without Matrigel)to detect the migration and invasion ability of breast cancer cells in different subgroups The Transwell assay(with or without Matrigel)was performed to detect the migration and invasion of different groups of breast cancer cells.In addition,changes in the expression of proliferation,apoptosis,invasion-related proteins,tumor stem cell markers,andβ-catenin and ID2 in the Wnt signaling pathway were analyzed by Western blot.(4)The relationship between EYA4 and SIX1 in breast cancer cells was detected by immunoprecipitation and cellular immunofluorescence assays.Results:(1)EYA4 expression was elevated in MCF-7 and MDA-MB-231 of breast cancer cells compared with MCF-10A of normal breast epithelial cells.(2)Compared with the experimental results obtained from different groups of cells,down-regulation of EYA4 expression level decreased the proliferation level,scratch healing,invasive metastatic ability,and microcellular sphere-forming ability of MDA-MB-231 and MCF-7 cells,respectively,however,the number of apoptotic cells was significantly increased.(3)Immunoprecipitation results showed that EYA4 and SIX1 interacted in MDA-MB-231 and MCF-7 cells.(4)The expression levels of both β-catenin and ID2,related molecules in the Wnt signaling pathway,were reduced after successful si-EYA4 transfection.Conclusions:(1)EYA4 showed abnormally high expression in both cancer cells in this experiment,and EYA4 could inhibit the proliferation of breast cancer cells and promote apoptosis.(2)Down-regulation of EYA4 could inhibit the EMT process in breast cancer cells,and could inhibit the sphere-forming ability and stemness of breast cancer cells.(3)EYA4 interacted with SIX1 and upregulated its expression.the expression levels ofβ-catenin and ID2,which are related to Wnt signaling pathway,were decreased,and downregulation of EYA4 in breast cancer could inhibit the activity of Wnt signaling pathway.