Investigation On The Molecular Mechanism Of Bmi-1 Promoted Migration And Invasion

Background:Breast cancer is a highly invasive and metastatic disease with a poor prognosis if diagnosed at an advanced stage, which is often the case. However, the mechanisms by which tumor cells become metastatic remain poorly understood. To provide insight that will enable the development of new therapeutic strategies, it is crucial to elucidate the molecular mechanisms that promote the invasive and metastatic properties of breast cancer cells.Bmi-1 (B-cell-specific moloney murine leukemia virus insertion site 1), a member of the polycomb group transcription repressors, is known to play an important role in carcinogenesis as it was originally identified as an oncogenic partner of c-Myc in murine lymphomagenesis (1-5). Previous studies demonstrated that Bmi-1 regulates the differentiation andclonogenic self-renewal of I-type neuroblastoma cells (6). Other studies also have revealed that Bmi-1 is involved in the regulation of stem-cell-associated genes to control cell self-renewing and differentiation.Objective:To investigate the molecular mechanism of Bmi-1 promoted migration and invasion.Methods:Chemically synthesized siRNA targeting the Bmi-1 gene was transfected into MDA-MB-231 cells, which have high invasive and metastatic potential, using Lipofectamine RNAiMAX Reagent. The expression of Bmi-1 mRNA and protein was detected by quantative Real-time PCR and Western Blot, respectively.The effect of Bmi-1 Knockdown on MDA-MB-231 cells migration and invasion was analysied by Transwell migration assay and Matrigel invasion assay. A pair of primers of Bmi-1 was designed according to the mRNA sequence in GenBank, and then a 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 selecte 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.Results:Transfected with Bmi-1 siRNA-269 significantly down regulated the expression of Bmi-1 mRNA and protein as compared with the blank group, negative control group. MDA-MB-231 cells transfected with Bmi-1 siRNA-269 had lower levels of invasion and migration capacity than cells in the blank group, negative control group. 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 (P<0.05). 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.Conclusions:SiRNA-mediated silencing of the Bmi-1 gene could significantly inhibit cell migration and invasion in human breast cancer cell line MDA-MB-231. 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 migration in human breast cancer BT474 cells.