Bmi-1 promotes the aggressiveness of glioma via activating the NF-kappaB/MMP-9 signaling pathway

Abstract

Background: The prognosis of human glioma is poor, and the highly invasive nature of the disease represents a major impediment to current therapeutic modalities. The oncoprotein B-cell-specific Moloney murine leukemia virus integration site 1 protein (Bmi-1) has been linked to the development and progression of glioma; however, the biological role of Bmi-1 in the invasion of glioma remains unclear.

Methods: A172 and LN229 glioma cells were engineered to overexpress Bmi-1 via stable transfection or to be silenced for Bmi-1 expression using RNA interfering method. Migration and invasiveness of the engineered cells were assessed using wound healing assay, Transwell migration assay, Transwell matrix penetration assay and 3-D spheroid invasion assay. MMP-9 expression and activity were measured using real-time PCR, ELISA and the gelatin zymography methods. Expression of NF-kappaB target genes was quantified using real-time PCR. NF-kappaB transcriptional activity was assessed using an NF-kappaB luciferase reporter system. Expression of Bmi-1 and MMP-9 in clinical specimens was analyzed using immunohistochemical assay.

Results: Ectopic overexpression of Bmi-1 dramatically increased, whereas knockdown of endogenous Bmi-1 reduced, the invasiveness and migration of glioma cells. NF-kappaB transcriptional activity and MMP-9 expression and activity were significantly increased in Bmi-1-overexpressing but reduced in Bmi-1-silenced cells. The reporter luciferase activity driven by MMP-9 promoter in Bmi-1-overexpressing cells was dependent on the presence of a functional NF-kappaB binding site, and blockade of NF-kappaB signaling inhibited the upregulation of MMP-9 in Bmi-1 overexpressing cells. Furthermore, expression of Bmi-1 correlated with NF-kappaB nuclear translocation as well as MMP-9 expression in clinical glioma samples.

Conclusions: Bmi-1 may play an important role in the development of aggressive phenotype of glioma via activating the NF-kappaB/MMP-9 pathway and therefore might represent a novel therapeutic target for glioma.

Figure 1

Ectopic expression of Bmi-1 enhances the migration and invasion of glioma cells. A, Western blot of Bmi-1 protein expression in A172-vector, A172-Bmi-1, LN229-vector and LN229-Bmi-1 cells; β-actin was used as a loading control (upper). The fold changes of Bmi-1 expression were analyzed by densitometry quantification (lower). B, Wound healing assay of A172-vector, A172-Bmi-1, LN229-vector and LN229-Bmi-1 cells. C, Representative micrographs (left) and quantification (right) of cell migration in the Transwell migration assay (without matrigel). D, Representative micrographs (left) and quantification (right) of cell invasion in the Transwell matrix penetration assay (with matrigel). E, Representative micrographs from the three-dimensional spheroid invasion assay on the 4^th day after cells were planted; these experiments were repeated at least three times with similar results. Vector: pMSCV-vector. Error bars represent the mean ± SD of three independent experiments; ** P < 0.01.

Figure 2

Bmi-1 activates MMP-9 in glioma cells. A, Real-time PCR quantification of MMP-9 mRNA expression levels in vector-control cells and Bmi-1-overexpressing cells (Bmi-1). MMP-9 expression levels are presented as the fold changes relative to that in vector-control cells and normalized to GAPDH. B, ELISA for secreted MMP-9 protein in cell supernatants. C, Gelatin zymography assay of MMP-9 gelatinase activity in cell supernatants. Vector: pMSCV-vector. Error bars represent the mean ± SD of three independent experiments; ** P < 0.01.

Figure 3

Knockdown of Bmi-1 reduces the migration and invasion of glioma cells. A, Western blot analysis of Bmi-1 protein expression in vector-control cells and Bmi-1-shRNA-transduced glioma cell lines (Bmi-1-RNAi); β-actin was used as a loading control (upper). The fold changes of Bmi-1 expression were analyzed by densitometry quantification (lower). B, Wound healing assay of vector-control cells and Bmi-1-shRNA-transduced glioma cell lines. C, Representative micrographs and quantification of cell migration in the Transwell migration assay (without matrigel). D, Representative micrographs and quantification of cell invasion in the Transwell matrix penetration assay (with matrigel). E, Representative micrographs from the three-dimensional spheroid invasion assay on the 4^th day after cells were planted. The experiments were repeated for at least three times with similar results. RNAi-Vector: pSuper-retro-puro-vector. Error bars represent the mean ± SD of three independent experiments; ** P < 0.01.

Figure 4

Knockdown of Bmi-1 transcriptionally downregulates MMP-9 expression and activity. A, Quantification MMP-9 mRNA expression levels in control cells and Bmi-1 RNAi-transfected cells; normalized to β-actin. B, ELISA quantification of MMP-9 protein in cell supernatants. C, Gelatin zymography assay of MMP-9 gelatinase activity in cell supernatants. RNAi-Vector: pSuper-retro-puro-vector. Error bars represent the mean ± SD of three independent experiments; ** P < 0.01

Figure 5

Bmi-1 induces NF-kappaB transcriptional activity. A, Luciferase reporter assay of NF-κB transcriptional activity in vector-control, Bmi-1 overexpressing (Bmi-1) and Bmi-1 silenced glioma cells (Bmi-1-RNAi). B, Real-time PCR analysis of NF-κB-regulated gene expression in vector-control, Bmi-1 overexpressing and Bmi-1 silenced glioma cells; GAPDH was used as the control gene. C, Left, schematic illustration of luciferase reporter gene construction using cloned fragments of the human MMP-9 promoter. Right, transactivation activity of luciferase reporter genes driven by MMP-9 promoter fragments (as indicated on the left) in vector-control, Bmi-1 overexpressing and Bmi-1 silenced glioma cells. Luciferase activity was normalized to Renilla luciferase activity. Vector: pMSCV-vector, RNAi-Vector: pSuper-retro-puro-vector. Error bars represent the mean ± SD of three independent experiments; * P < 0.05, ** P < 0.01.

Figure 6

Bmi-1 promotes an aggressive phenotype in glioma cells via activation of the NF-kappaB-MMP-9 pathway. Bmi-1-overexpressing cells were treated with a NF-kappaB inhibitor JSH-23 at 30μM or MMP-9 inhibitor at 50μM. A, Quantification of cell migration in the Transwell assay (without matrigel). B, Quantification of cell invasion in the Transwell matrix penetration assay (with matrigel). C, Representative micrographs of the 3-D spheroid invasion assay. The experiments were repeated for at least three times with similar results. D, ELISA analysis of MMP-9 secretion. Error bars represent SD of three independent experiments; ** P < 0.01.

Figure 7

Clinical relevance of Bmi-1 expression in human gliomas. A, Bmi-1 levels in association with MMP-9 and NF-kappaB expression in 127 primary human glioma specimens. Two representative cases, diagnosed as WHO grade I (upper row) and grade III (lower row), are shown. The insets are enlarged images derived from the original pictures. B, Percentages of all specimens (upper) and specimens in grade IV (lower) showing low- or high-Bmi-1 expression relative to the levels of MMP-9 and nuclear or cytoplasmic NF-kappaB p65 localization, analyzed by IHC staining.