| Background:Breast phyllodes tumors are rare breast fibroadenoma with epithelial and mesenchymal biphasic malignant transformation[1],which is an ideal carrier for studying epithelial-mesenchymal interaction and epithelial-mesenchymal transition.Breast phyllodes tumors have the characteristics of local recurrence and distant metastasis,which seriously affect the prognosis and health of patients.EZH2 is involved in tumor development,invasion and migration[2,3],Wnt/?-catenin signaling pathway affects cell polarity,cell adhesion,apoptosis and tumorigenesis[4,5],both in tumor stemness play an important role in self-renewal and epithelial-mesenchymal transition.In immunohistochemical studies,the researchers found that activation of the epithelial Wnt/?-catenin signaling pathway in breast phyllodes tumors can promote mesenchymal hyperplasia of breast phyllodes tumors[6,7].At the same time,it was found that EZH2 was overexpressed in malignant breast phyllodes tumor tissues,and together with the high expression of subcutaneous ALDH1in stromal cells,the hypothesis that breast phyllodes tumors may be derived from tumor stem cells[8].Studies have confirmed that EZH2 is involved in the regulation of the Wnt/?-catenin pathway[9]and promotes the process of epithelial-mesenchymal transition.By activating the signaling of?-catenin,EZH2 can enhance the self-renewal and proliferation of breast cancer stem cells.However,there is still a lack of experiments to confirm whether EZH2 can affect breast phyllodes tumor cells through Wnt/?-catenin signaling pathway,and thus affect the biological behavior of breast phyllodes tumors.There is a lack of effective treatment for breast phyllodes tumors,and it is especially necessary to demonstrate effective targets to resolve recurrence and chemoradiotherapy resistance.EZH2 may be a potential important target for the treatment of phyllodes tumors of the breast.It may promote the occurrence,development,recurrence and chemoradiotherapy of breast phyllodes tumors by promoting the stem cells of breast phyllodes tumor cells,which needs further confirmation.Objective:In this study,human malignant breast phyllodes tumor cell line?MCP-1?was used as the research object to investigate the effects of EZH2 on proliferation,migration,invasion,apoptosis and EMT on human breast phyllodes tumor cells;The influence of Wnt/?-catenin signaling pathway on breast phyllodes tumor cells and related mechanisms provide a theoretical basis for new treatment options for breast phyllodes tumors.Methods:Breast phyllodes tumor cell lines?MCP-1?were divided into two groups:unrelated sequence transfection group?NC group?and EZH2 shRNA transfection group?EZH2 shRNA group?.?1?CCK-8 assay and colony formation assay were used to detect the change of the proliferation after downregulated EZH2 in MCP-1 cells,and Western Blot was used to detect the expression of proliferation-related protein PCNA protein.?2?Wound healing assay,Transwell chamber migration and Transwell matrigel invasion assay measured the change of the migration and invasion after downregulated EZH2 in MCP-1 cells,and Western Blot was used to detect invasion-related proteins MMP-2,MMP-9 change in protein expression level.?3?Tunel assay and Hoechst 33258 assay were used to measure the change of the apoptosis after downregulated of EZH2 in MCP-1 cells.Western Blot was used to detect the expression change of apoptosis-related proteins Bax,Bcl2,cleaved PARP and cleaved Caspase3.?4?Immunofluorescence stain assay was used to detect the change of the EMT after down-regulating EZH2 in MCP-1 cells.Western Blot to detect changes of EMT markers E-cadherin,N-cadherin,Snail1 in protein expression levels.?5?Sphere formation assay was used to detect the change of the stem cell proliferation after down-regulating EZH2 in MCP-1 cells.Western Blot was used to detect the expression change in protein level of cancer stem cell markers ALDH1,Sox2and Oct4.?6?RT-PCR was used to detect the RNA expression change of?-catenin and GSK-3?in the wnt/?-catenin pathway in MCP-1 cells of EZH2,Western Blot to detectthe?-catenin and GSK-3?,p-GSK-3?protein expression changes in the wnt/?-catenin pathway.Results:?1?The effects of EZH2 down-regulation on proliferation of MCP-1 in breast phyllodes tumor cellsCompared with the NC group,the results of CCK-8 showed that the absorbance of the EZH2-shRNA group significantly decreased,the results of the colony formation assay showed that the colony shape of the EZH2-shRNA group significantly smaller and the number of cloned cells significantly decreased,the results of Western Blot showed the protein expression of PCNA in the shRNA group were significantly decreased.It is indicated that that down-regulation of EZH2 significantly decreased the proliferation of MCP-1 cells.?2?The effects of down-regulation of EZH2 on migration and invasion of MCP-1in breast phyllodes tumor cellsCompared with the NC group,wound healing assay results showed that the mobility of the EZH2-shRNA group significantly decreased,the transwell chamber migration results showed that the number of cells transdermally transduced by the EZH2-shRNA group significantly decreased.The transwell matrigel invasion assay showed that the number of EZH2-shRNA group cells penetrating the matrigel gel significantly reduced.The results of Western Blot showed that the protein expression of MMP-2 and MMP-9 in EZH2-shRNA group were significantly decreased.It is indicated that down-regulation of EZH2 significantly decreased the migration and invasion of MCP-1 cells.?3?The effects of EZH2 down-regulation on the apoptosis ability of MCP-1 in breast phyllodes tumor cellsCompared with NC group,the results of the Tunel apoptosis experiment showed that the proportion of Tunel-labeled positive fluorescentcells in the EZH2-shRNA group was higher.The apoptosis of Hoechst 33258 showed that the EZH2-shRNA group showed a significant increase in the proportion of labeled positive fluorescent cells,the results of Western Blot showed that the protein expression of Bcl2 was significantly decreased?the protein expression of Bax,cleaved PARP and cleaved Caspase3 were significantly increased.It is indicated that down-regulation of EZH2 significantly increased the apoptosis ability of MCP-1 cells.?4?The effects of EZH2 down-regulation on EMT of MCP-1 in breast phyllodes tumor cellsCompared with the NC group,the results of immunofluorescence assay showed that the fluorescence intensity of the E-cadherin group was significantly increased in the EZH2-shRNA group and the fluorescence intensity of the N-cadherin and Snail1 groups significantly decreased,the results of Western Blot showed that the EZH2-shRNA group,the protein E-cadherin expression was significantly increased and the protein expression of N-cadherin?Snail1 were significantly decreased.It is indicated that down-regulation of EZH2 significantly inhibited EMT in MCP-1 cells.?5?The effects of EZH2 down-regulation on stemness of MCP-1 in breast phyllodes tumor cellsCompared with the NC group,the results of sphere formation assay showed that the germination ability of EZH2-shRNA group significantly decreased,the results of Western Blot showed that the protein expression ALDH1,SOX2 and OCT4 in EZH2-shRNA group were significantly decreased.It is indicated that EZH2 was down-regulated significantly could decrease the stemness effect of MCP-1 cells.?6?The effects of EZH2 down-regulation in Wnt/?-catenin signaling pathway of MCP-1 in breast phyllodes tumor cellsCompared with the NC group,RT-PCR results showed that the RNA expression of?-catenin in the EZH2-shRNA group significantly decreased,and the RNA expression of GSK-3?significantly increased,results of Western Blot showed that EZH2-shRNA group the expression of?-catenin and p-GSK-3?protein were significantly decreased,and the protein expression of GSK-3?was significantly increased.It is indicated that down-regulation of EZH2 significantly inhibited Wnt/?-catenin pathway in MCP-1cells.Conclusions:?1?Down-regulation of EZH2 in breast phyllodes tumor cells?MCP-1?can inhibit tumour cell proliferation,migration,invasion and tumour stemness,EMT,and promote tumour cell apoptosis;?2?Down-regulation of EZH2 in breast phyllodes tumor cells can inhibit Wnt/?-catenin signaling pathway.So inhibition of EZH2 expression has multiple Potential clinical implications for the treatment of breast phyllodes tumors. |
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