The Effects Of Silencing Bmi-1 Gene On The Proliferation, Metastasis And Invasion Of Lung Cancer Cell Line A549

Objective: Bmi-1 (B-cell-specific Moloney murine leukemia virus insertion site 1), identified as an oncogene, belongs to the polycomb group family and highly expressed in several malignant tumors, is required for the proliferation and self-renewal of normal and tumor stem cells. In lung adenocarcinoma cell line A549, it has been found Bmi-1 is over-expressed and the proposed Bmi-1 signaling pathway INK4a/ARf locus has been found deleted in this cell line. The aim of the study was to observe the effects of silencing Bmi-1 gene expression on the proliferation, invasion, and metastasis of A459 cells and tried to elucidate its possible alternative ink4a locus-independent pathway of Bmi-1-regulated proliferation, invasion and metastasis of this cell line.Methods: The most effective sequence from four Bmi-1 siRNA sequences designed by our lab was chosen as a target sequence and one random sequence chosen as a negative control, has been published previously. In short, the chemically synthesed siRNA or control sequences were connected to a retrovirus expressing vector, pSUPERretro-Neo plasmid, and was transfected into A549 cells. The stably transfected cells were cultured and passed. The level of Bmi-1 mRNA expression was analyzed with reverse-transcriptional polymerase chain reaction (RT-PCR), and its protein products were assessed with western blotting in A549 cells. The proliferation of A549 cells were checked with MTT and trypan blue exclusion methods. Flow cytometry was used to test cell cycle. Plate colony forming assay was used to analyze single-cell proliferative capability. Trans-well assay was used to examine cell migration. In vitro invasion assay was used to examine cell invasion capacity. The potency of tumorigenesis and metastasis of A549 cells was observed in nude mouse through hypodermic inoculation and direct tail vein injected of A549 cells respectively. The level of PTEN( Phosphatase and tensin homologue deleted on chromosome 10),total-AKT(ATP-dependent tyrosine kinase), pho-AKT and cyclin D1 protein was analyzed by Western blotting. The expression of MMP(matrix metalloproteinase) -2 and MMP-9 in A549 cells were analyzed by Gelatin zymography method. The expression of VEGF in A549 cells was determined by ELISA.Results: The endogenous expression of Bmi-1 gene both in mRNA and the protein levels were effectively silenced by retroviruses-mediated siRNA. Silencing Bmi-1 gene inhibited the proliferation of A549 cells without inducing cell death in vitro. G1 phase accumulation and decreased number of cells at the S phase was observed in Bmi-1 siRNA expressing A459 cells (A549-siRNA-Bmi-1) cells compared to wild type A459 cell (A549-wt) and negative control vector transfected A459 cells (A549-ctr) cells (P<0.05). Colony forming test demonstrated the single-cell proliferative capability of A549-siRNA-Bmi-1 cell was significantly inhibited. In vitro silencing Bmi-1 gene repressed migration and invasion potency of A549 cells. In vivo down regulation of Bmi-1 could weaken the ability of tumorigenesis and metastasis of A549 cells. Compared with A549-wt and A549-ctr cells the expression of cyclin D1 and pho-AKT in A549-siRNA-Bmi-1 cell was decreased, and silencing Bmi-1 gene expression could up-regulated PTEN expression, inhibited the levels of MMP-2, MMP-9 and VEGF,while the total-AKT was not affected.Conclusion: Silencing Bmi-1 gene could inhibit the proliferation and invasion of A549 in vitro,weaken the tumorigenesis and metastasis of A549 cells in vivo. In addition to the classical INK4a/ARf locus signaling pathway in regulating cell proliferation, Alternative pathway, such as AKT activation and cyclin D1 expression, pho-AKT activation may be related to PTEN up-regulation, Bmi-1modulate the invasion and metastasis might be through regulating MMPs and VEGF, but not EMT (epithelial-mesenchymal transition), this alternative pathway might exist for the signaling of Bmi-1 mediated effects on the invasion and metastasis of this lung cancer cell line.