Abstract
BackgroundThe 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.MethodsA172 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.ResultsEctopic 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.ConclusionsBmi-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.
Highlights
The prognosis of human glioma is poor, and the highly invasive nature of the disease represents a major impediment to current therapeutic modalities
We demonstrated that the ability of Bmi-1 to stimulate the invasive phenotype in glioma cells was mechanistically associated with activation of NF-kappaB and subsequent upregulation and activation of Matrix metalloproteinase-9 (MMP-9)
The 3-D spheroid invasion assay demonstrated that Bmi-1-overexpressing glioma cells displayed cellular morphologies typical of a highly invasive phenotype, as the cells presented increased numbers of outward projections compared to the control cells (Figure 1E)
Summary
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; the biological role of Bmi-1 in the invasion of glioma remains unclear. B-cell-specific Moloney murine leukemia virus integration site 1 protein (Bmi-1) acts as a repressor of the expression of certain genes by forming complexes with multiple other Polycomb group (PcG) family members, such as RING1, HPC2 and Mph2[12]. Numerous experimental studies have indicated that Bmi-1 plays an important role in the development and progression of cancer and essentially functions as an oncogene [15,16]. Various studies have revealed that Bmi-1 is required for the maintenance of the self-renewing proliferation of several normal and cancer stem cells, including neural crest stem cells and mammary stem cells [19,20]
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