| Objective: Tumor epidemiological statistics show that morbidity and mortality of breast cancer both rank first among female cancers,and there are nearly 1.7 million new breast cancer cases every year worldwide.The morbidity of breast cancer is increasing year by year,and the age of onset is younger.Despite the continuous improvement in the comprehensive treatment of breast cancer,the prognosis of breast cancer continues to improve,but breast cancer is still an important cause of death in women.Previous studies have shown that the main function of VASP is to regulate cytoskeletal rearrangement and play an important role in tumor cell metastasis.Recent studies have suggested a link between VASP and Wnt/β-catenin signaling pathways,however,the mechanism of action remains unclear.The purpose of this study was to investigate the role of VASP and Wnt/β-catenin signaling pathways in the development of breast cancer.Methods: 1.To explore the mechanism of Wnt/β-catenin signaling pathway activating VASP in breast cancer cells: bioinformatics analysis of TCF7L2 protein(TCF4)enrichment in VASP promoter region;the interaction between β-catenin and TCF4 and VASP promoter was detected by Ch IP assay and dual luciferase reporter gene assay.The regulation mechanism of Wnt/β-catenin signaling pathway on VASP expression was detected by RT-q PCR,western blot and immunofluorescence assay.2.The relationship between the expression level of VASP and the development of breast cancer and Wnt/β-catenin signaling pathway: the correlation of VASP with the development of breast cancer was detected by bioinformatics analysis,RT-q PCR,immunohistochemistry;the correlation between VASP and Wnt/β-catenin signaling pathway was observed by transcriptome sequencing,RT-q PCR and western blot in breast cancer cells.3.The mechanism of VASP activating Wnt/β-catenin signaling pathway in breast cancer cells: Western blot,TOP/FOP flash,cellular immunofluorescence assay and FRAP assay were used to observe the effect of VASP on the nuclear import of β-catenin and DVL3 proteins.Protein profiling,Co-IP,pulldown assay,Ch IP assay were used to explore the formation of protein complexes between VASP and DVL3,β-catenin and TCF4,by which regulating Wnt/β-catenin signaling pathway.4.VASP and Wnt/β-catenin signaling pathways form a positive feedback loop to regulate the proliferation and migration of breast cancer cells: In breast cancer cells,the effect of Wnt/β-catenin/VASP feedback loops on cell proliferation and migration is observed by CCK-8 and Transwell assays.Results: 1.In breast cancer cells,β-catenin and TCF4 proteins can interact with the promoter of VASP to increase the activity of VASP promoter.Activation of Wnt/β-catenin signaling pathway can up-regulate the expression level of VASP,suggesting that VASP is target gene of the Wnt/β-catenin signaling pathway.2.The expression level of VASP in breast cancer tissues is significantly increased,which is closely related to pathological differentiation,clinical stage,lymph node metastasis,recurrence and prognosis of breast cancer patients.3.In breast cancer cells,transcriptome sequencing shows that VASP is closely related to Wnt/β-catenin signaling pathway,and VASP can activate Wnt/β-catenin signaling pathway.4.In breast cancer cells,VASP can promote the import of β-catenin and DVL3 proteins into the nucleus and activate the Wnt/β-catenin signaling pathway.5.The expression level of VASP in the nucleus of breast cancer cells is increased,and its nuclear import is related to the EVH1 and EVH2 domains.6.In the nucleus,VASP forms a protein complex with DVL3,β-catenin and TCF4 through the EVH1 and EVH2 domains,activating the expression of Wnt/β-catenin signaling pathway target genes,and promoting proliferation and migration of breast cancer cells.Conclusion: This study demonstrates that the Wnt/β-catenin/VASP malignant positive feedback loop promotes proliferation and migration of breast cancer cells,and breaking this positive feedback loop may provide a new strategy for breast cancer treatment. |
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