Objective       To construct a stable breast cancer cell line with over-expression of human Bmi-1 gene and investigate the effects of Bmi-1 over-expression on the invasion and metastasis in human breast cancer BT474 cells.       Methods       A pair of primers of Bmi-1 was designed according to the mRNA sequence in GenBank

and then an eukaryotic expression vector GV144-Bmi-1 was constructed. Human breast cancer BT474 cells were transfected with GV144-Bmi-1 and then treated with G418 to screen the cells with stable expression of Bmi-1. The mRNA and protein expression of Bmi-1 was detected by QRT-PCR and Western blotting. The invasion and metastatic ability of the BT474 cells with or without Bmi-1 over-expression were determined by Matrigel invasion assay and Transwell migration assay. The expression of MMP-2 and -9 was also detected at the mRNA and protein levels.       Results       The eukaryotic expression vector GV144-Bmi-1 was successfully constructed. A cell line with stable over-expression of Bmi-1 was obtained in BT474 cells. qRT-PCR and Western blotting indicated that the expression of Bmi-1 was significantly higher in the GV144-Bmi-1 over-expression group than in the control group (P0.05). The cells with stable over-expression of Bmi-1 showed significantly enhanced migration ability with more cells moved from the upper chamber into the lower

and obviously increased expression of MMP-2 and -9 at the mRNA and protein levels (P0.05).       Conclusion       A stable cell line with a persistent over-expression of Bmi-1 gene is established. Bmi-1 over-expression increases the ability of invasion and metastasis in human breast cancer BT474 cells.