Mechanistic Study Of The Role Of The SWI/SNF Complex Subunit BAF170 In Regulating Breast Cancer Cell Proliferation

Background: SWI/SNF is an ATP-dependent chromatin remodeling complex,which could utilize the energy released by ATP hydrolysis to change the binding position of nucleosomes to DNA,such as gene promoters and enhancers,thereby changing the chromatin structure and regulating gene transcription.Scientific studies have found that genes encoding the SWI / SNF complex subunit are mutated in cancers which contain multiple cancer types.And most mutations lead to the function loss of subunit protein,suggesting that the SWI / SNF subunits may act as tumor suppressors.However,with the further study of SWI / SNF subunits,the abnormal expression of certain subunits in tumor cells was found to promote the proliferation and migration ability of tumor cells.Therefore,the regulation of SWI/SNF complex subunits has subunit specificity and tissue specificity in tumor cells.Objectives: 1.To clarify the expression of SWI/SNF complex subunit BAF170 in breast cancer cell lines;2.To clarify the effect of SWI/SNF complex subunit BAF170 on proliferation,cell cycle and apoptosis of breast cancer cells;3.To clarify the effect of SWI/SNF complex subunit BAF170 on cancer stem-like cell phenotype in breast cancer cells;4.To clarify the regulatory mechanism of SWI/SNF complex subunit BAF170 on cell proliferation in breast cancer cells;Methods: 1.Western Blot was used to detect the expression of BAF170 at the protein level in normal breast cells and eight different breast cancer cells.2.Lentivirus infect MCF-7,T-47 D cells,express sh RNA against BAF170 gene,knockdown the expression of BAF170 in breast cancer cells.3.Western Blot was used to detect the expression of BAF170 in MCF-7 and T-47 D cells,and to verify the knockdown efficiency of lentivirus.4.MTT assay was used to detect the effect of BAF170 knockdown on the proliferation of breast cancer cells MCF-7 and T-47 D.5.Colony formation assay was used to detect the effect of BAF170 knockdown on the proliferation of breast cancer cells MCF-7 and T-47 D.6.Ed U assay was used to detect the effect of BAF170 knockdown on the proportion of S phase cells in breast cancer cells MCF-7 and T-47 D.7.Flow cytometry was used to detect the effect of BAF170 knockdown on the cell cycle of breast cancer cells MCF-7 and T-47 D.8.Annexin V-647/PI double staining assay was used to detect the apoptosis of breast cancer cells MCF-7 and T-47 D after BAF170 knockdown.9.Mouse xenograft model was established to explore the regulation of BAF170 on breast cancer cell proliferation in vivo.10.Flow cytometry was used to detect the surface antibody of breast cancer cells,and to detect the effect of BAF170 knockdown on the expression of cancer stem cellassociated proteins in breast cancer cells.11.Mammosphere-formation assays used to detect the effect of BAF170 knockdown on breast cancer stem cell phenotype.12.RNA-seq combined with bioinformatics analysis was used to detect the signaling pathways involved in the regulation of BAF170 in breast cancer cells.13.Cell function experiments after PI3 K inhibitor treatment to explore BAF170 regulates breast cancer cell proliferation through PI3 K.Results: 1.BAF170 is abnormally expressed in breast cancer cell lines.Western Blot showed that BAF170 was highly expressed in the eight breast cancer cell lines compared to normal breast cell MCF-10.Therefore,the abnormal expression of BAF170 in breast cancer cells has research significance.2.Knockdown of BAF170 inhibits cell proliferation,cell cycle and inducesapoptosis in breast cancer cell lines.MTT results: Compared with the sh Ctrl group,the proliferation of breast cancer cell lines MCF-7 and T-47 D decreased after BAF170 knockdown,and the difference was more significant with the increase of culture time.The results of colony formation experiments showed that the number of colonies forming in BAF170 knockdown group was significantly reduced by long-term culture of breast cancer cells at a lower cell density.The results of Ed U embedding experiments showed that after BAF170 knockdown,the proportion of S phase cells in breast cancer cells was significantly reduced.Flow cytometry showed that after BAF170 knockdown,the ratio of G0/G1 in MCF-7 and T-47 D cells increased and the proportion of cells in S phase decreased(consistent with the results of Ed U insertion experiment).Apoptosis was measured by double staining: the apoptosis rate of breast cancer cell lines after BAF170 knockdown was significantly higher than that of sh Ctrl group.3.Knockdown of BAF170 inhibits the proliferation of breast cancer cells in vivo.Immunodeficient mice were randomly divided into two groups.The experimental model was established by subcutaneous injection of breast cancer cells in mice.The results of xenograft assay showed that the weight and volume of BAF170 knockdown tumors were significantly lower than the control group.BAF170 promotes breast cancer cells proliferation in vivo.4.BAF170 can regulate the cancer stem cell phenotype in breast cancer cells.The results of Mammosphere-formation assays showed that in the breast cancer cell line,the number of cell microspheres formed by the BAF170 knockdown group was significantly less than that of the control group,and the volume of single microspheres was also smaller than that of the control group.The results of flow cytometry to detect cell surface antibodies showed that the proportion of cell subsets of stem cell phenotype CD44+/CD24-was significantly reduced after BAF170 knockdown.5.BAF170 regulates the proliferation of breast cancer cells by PI3 K signaling pathway.Firstly,RNA-seq combined with bioinformatics analysis revealed that BAF170 is involved in and regulates gene expression in the PI3 K signaling pathway in breast cancer cells.Secondly,the results of the bioinformatics analysis were verified by QPCR.Finally,the PI3 K inhibitor LY294002 was used to block the expression of PI3 K in wild-type breast cancer cells,and the effect of PI3 K inhibitor on the proliferation of breast cancer cells was confirmed by cell function experiments.The results showed that after using PI3 K inhibitor,the proliferation of breast cancer cells decreased,the proportion of S phase in cells decreased,and the apoptosis rate increased.Conclusion: The expression of the SWI/SNF complex subunit BAF170 was higher in breast cancer cell lines than in normal breast cells.Knockdown of BAF170 inhibits cell proliferation,cell cycle and induces apoptosis in breast cancer cells.In breast cancer cell lines,BAF170 has a regulatory effect on cancer stem cells.BAF170 regulated the proliferation of breast cancer cells through PI3 K signaling pathway.