Effects Of Silencing FBI-1 Gene On Proliferation And Apoptosis Of Human Breast Cancer Cells And Its Mechanism

Objective:The purpose of this study is to investigate the expression of FBI-1 in breast cancer cells,and to study the effect of silencing FBI-1 gene expression on the proliferation,apoptosis of breast cancer cells and its possible mechanism.Methods:1.Quantitative real-time PCR(qRT-PCR)and Western blot analysis were applied to detect FBI-1 expression in human normal mammary epithelial cell line MCF-10 A,breast cancer cell MCF-7 and MDA-MB-231.2.The FBI-1 gene of human breast cancer cell lines MCF-7 and MDA-MB-231 were stably transfected with shRNA lentivirus interference technique and verified by qRT-PCR and Western blot.3.The effects of silencing FBI-1 on the proliferation of MCF-7 and MDA-MB-231 cells were detected by CCK-8 assay and plate colony forming assay.The effects of silencing FBI-1 on the cell cycle andapoptosis of MCF-7 and MDA-MB-231 cells were detected by flow cytometry.4.qRT-PCR and Western blot were used to detect the expression levels of survivin and NF-?Bp65,and to explore the mechanism of silencing FBI-1 on proliferation and apoptosis of breast cancer cells.Results:1.qRT-PCR and Western blot assay showed that the expression of FBI-1 was higher in breast cancer cells MCF-7 and MDA-MB-231 than what in human normal mammary epithelial cells MCF-10 A,and the difference was statistically significant(P<0.05).2.By using RNAi technology,we successfully constructed the lentiviral vector of FBI-1 and transfected into human breast cancer cell lines MCF-7 and MDA-MB-231.The silencing effect of shRNA was detected by qRT-PCR and Western bolt,respectively.The results showed that the Lv-shFBI-1-2 virus particles had the best silencing effect,and the difference was statistically significant(P<0.05).The Lv-shFBI-1-2lentivirus infected breast cancer cell lines MCF-7 and MDA-MB-231 were used for the following experiments.3.Silencing FBI-1 can inhibit the proliferation of breast cancer cells:CCK8 showed that the proliferation ability of transfection group MCF-7/shRNA-FBI-1 cells were significantly lower than that in empty plasmid control group MCF-7/shRNA-NC and empty control group MCF-7 cells,the difference was statistically significant(P<0.05).Plate clone formation assay showed that the cell clone formation of MCF-7/shRNA-FBI-1 was(37.000±4.885)%,which were lower than(59.918±3.214)% in MCF-7 and(54.753±4.057% in MCF-7/shRNA-NC,the difference were statistically significant(P<0.05).MDA-MB-231 cells come to the same conclusion.4.Silencing FBI-1 can block the cell cycle of breast cancer: flow cytometry results showed that cell percentage of G0/G1 stage in MCF-7and MCF-7/shRNA-NC cells were(59.257±2.222)% and(60.210±0.795)%,which were lower than(72.993±0.401)% in MCF-7/shRNA-FBI-1 cells,similarly,the proportion of G0/G1 phase cells in MDA-MB-231 cells and MDA-MB-231/shRNA-NC cells were lower than that of MDA-MB-231/shRNA-FBI-1 cells,the difference were statistically significant(P<0.05),visible FBI-1 gene silencing could arrest MCF-7 and MDA-MB-231 cells in G0/G1 phase.5.Silencing FBI-1 induces apoptosis of breast cancer cells: the results of flow cytometry showed that the apoptosis rate(5.140±0.478)%in MCF-7/shRNA-FBI-1 cells were higher than that(3.683±0.608)% in MCF-7 cells and(2.840±0.221)% in MCF-7/shRNA-NC(2.840±0.221)cells,similarly,the apoptosis rate of MDA-MB-231/shRNA-FBI-1 cells were higher than that in MDA-MB-231 cells and MDA-MB-231/shRNA-NC cells,the difference were statistically significant(P<0.05).6.Study on related mechanism: qRT-PCR results showed that the mRNA relative expression of survivin and NF-?Bp65 in MCF-7/shRNA-FBI-1were lower than that in MCF-7 and MCF-7/shRNA-NC,the difference were statistically significant(P<0.05).Western blot results showed that the protein relative expression of survivin and NF-?Bp65 in MCF-7/shRNA-FBI-1were lower than that in MCF-7 and MCF-7/shRNA-NC,the difference were statisticallysignificant(P<0.05).MDA-MB-231 cells reached the same conclusion.Conclusions:1.FBI-1 was highly expressed in human breast cancer cells.2.Silencing FBI-1 can inhibit the proliferation of human breast cancer cell line MCF-7 and MDA-MB-231,block cell cycle and induce cell apoptosis.3.The effect of silencing FBI-1 on proliferation and apoptosis of human breast cancer cells may be related to the inhibition of the expression of survivin and NF-?B pathway.