Role And Mechanism Of FBI-1 Protein In Breast Cancer Stem Cells And Chemotherapy Resistance

OBJECTIVE: FBI-1 is a necessary raw carcinoma gene in normal cellular evolution,and has a special role in tumor formation.Tumor stem cells are tumor formation and development and key factors in recurrence,which is closely related to chemotherapy resistance.Therefore,we identify whether FBI-1 can be used as a prognosis indicator of a chemotherapy prognosis by FBI-1 in breast cancer stem cells and chemotherapy resistance.METHODS:1.Mammosphere formation experiments were performed using MDA-MB-231 cells,and the expression of FBI-1 was highly increased in Mammosphere.Knock the FBI-1 and Mammosphere formation experiments with the control cells,and the detection of the decrease FBI-1 significantly reduced the size and quantity of Mammosphere.Then,the flow cytometry is used to detect the change in CD44 +/CD24-CD44+/CD24 in the parent cell.2.Use Trans Sew L to migrate and invasive experiments,and detect whether the FBI-1 is significantly reduced to reduce the migration and invasion of breast cancer cells.The tumor mouse model was established to detect the growth of tumors.After 8 weeks later,mice were sacrificed,collected tumors and lung tissues,and the number of pulmonary metastasis extracted under microscope was treated.3.Establish MCF-7 drug-resistant cell lines and then approved whethe r the cell line has obtained drug resistance to the cell line with the MTT method.Changes in FBI-1 protein expression in MCF-7/ADR cells were detected by Western Blot,and the FBI-1 was expressed in drug resistance cells.Then,FBI-1 in the drug-resistant cells MCF-7/ADR was knocked,and the changes in cells were detected by MTT experiments.4.Use Real-Time PCR and Western Blot to detect the change in PTEN m RNA and protein expression in the MCF-7 cells of FBI-7 cells.The cells were carried out by anti-FBI-1 antibodies for chromatin immunoassate(CHIP)experiments.The 8pairs of primers were designed according to the sequence of the PTEN promoter and the published studied,and the obtained immunoprecipitate was carried out using this 8 pair of primers,and the FBI-1 was analyzed in the PTEN promoter and its binding region.5.After tapping FBI-1 in MCF-7/ADR cells,the cells were subjected to second infections with PTENSHRNA and control lentiviral infections,and used Western Blot experiments to identify the kn ockout effect of two proteins.MTT experiments were performed to detect control cells,FBI-1 knocking,dual knockdown cells on the sensitivity of doxorubicin.Results: 1.Through Western analysis,it was found that the expression of FBI-1was raised in Mammosphere.The expression of silencing FBI-1 makes Mammosphere’s formation and the number of tumor stem cells significantly reduced.2.Cell migration and invasion experiments show that knocking FBI-1 can reduce migration and invasiveness of MCF-7 and MDA-MB-231 cells.3.Nude mice tumor experiments have shown that knocking FBI-1 can not only inhibit tumor growth,but also reduce the metastasis of tumor to lung cancer.4.The results of WEStern showed significant increase in FBI-1 in MCF-7 drug resistant cell line MCF-7/ADR.It was found that knocking FBI-1 was able to enhance the sensitivity of the drug-resistant cell line to dorthysin drugs.5.The results of Real-Time PCR and Western indicate that the Knocking FBI-1enhances the expression of PTEN at m RNA and protein levels.FBI-1 is likely to be in a region of the PTEN promoter +400 to 900 bp.6.MTT experiments show that the effect of FBI-1 on the sensitivity of the FBI-1can be suppressed after the PTEN.Conclusion: FBI-1 can affect the pulmonary metastasis of breast cancer stem cells and breast cancer using cytology and nude mice experiments;at the same time,it is found that the expression of FBI-1 in resistive cells increases,and the resistance can be enhanced after the FBI-1 Sensitivity of cells on doxorubicin.By studying We found that FBI-1 can directly bind to the promoter of the PTEN gene to inhibit the expression of the PTEN gene.These findings show that FBI-1 effects in tumor stem cells and chemotherapy resistance.This provides a clue for a further mechanism study.