BRMS1 suppresses glioma progression by regulating invasion, migration and adhesion of glioma cells

Abstract

Breast cancer metastasis suppressor 1 (BRMS1) is a metastasis suppressor gene in several solid tumors. However, the expression and function of BRMS1 in glioma have not been reported. In this study, we investigated whether BRMS1 play a role in glioma pathogenesis. Using the tissue microarray technology, we found that BRMS1 expression is significantly decreased in glioma compared with tumor adjacent normal brain tissue (P<0.01, χ(2) test) and reduced BRMS1 staining is associated with WHO stages (P<0.05, χ(2) test). We also found that BRMS1 was significantly downregulated in glioma cell lines compared to normal human astrocytes (P<0.01, χ(2) test). Furthermore, we demonstrated that BRMS1 overexpression inhibited glioma cell invasion by suppressing uPA, NF-κB, MMP-2 expression and MMP-2 enzyme activity. Moreover, our data showed that overexpression of BRMS1 inhibited glioma cell migration and adhesion capacity compared with the control group through the Src-FAK pathway. Taken together, this study suggested that BRMS1 has a role in glioma development and progression by regulating invasion, migration and adhesion activities of cancer cells.

Figure 1. Representative images show BRMS1 immunohistochemical staining.

A Positive BRMS1 staining in normal brain tissue (NB); B Positive BRMS1 staining in adjacent normal brain tissue (AB); C Negative BRMS1 staining in benign tumor (BT); D Negative BRMS1 staining in malignant tumor (MT); E A significant difference in BRMS1 staining was observed between normal brain tissue and glioma tissue (GT) (P<0.01, χ² test) and between tumor adjacent normal brain tissue and glioma tumor (P<0.01, χ² test). F Whole-cell protein extracts were further prepared from four paired tumor adjacent normal glioma tissues and glioma tissues. The BRMS1 protein level was determined by Western blot analysis. G Western blot analysis of BRMS1 expression in normal human astrocytes NHA and glioma cell lines, including SHG44, C6, U251, T98G, U87. H BRMS1 staining was dramatically decreased in malignant tumor compared with benign tumor (P<0.05). All experiments were carried out in triplicate. Data are shown as mean ± SD. *P<0.05, **P<0.01, ***P<0.001. Original magnification (A–D) ×40.

Figure 2. Overexpression of BRMS1 suppresses cell invasion but not cell proliferation in glioma cells.

A Twenty-four hours after transfection, the expression of BRMS1 in U251 and U87 glioma cells was evaluated by western blot. B, C CCK-8 cell proliferation assay was performed after BRMS1 overexpression in U251 and U87cells. D, E Matrigel cell invasion assay was performed after the overexpression of BRMS1 in U251 and U87 cells. The graph shows the percentage of cells invaded in 5 fields of view compared with the control group. F BRMS1 inhibits MMP-2 activity in U251 and U87 cells by zymography. G Western blot analysis of the relative protein levels of MMP-2, uPA and NF-κB in BRMS1 overexpression and control group of U251 and U87 cells. H Quantitative analysis of relative protein level of MMP-2, uPA, p27 and NF-κB in glioma U251 and U87 cells. All experiments were carried out in triplicate. Data are shown as mean ± SD. *P<0.05, **P<0.01.

Figure 3. Overexpression of BRMS1 inhibits glioma cell migration.

A, B Wound-healing assay was done on monolayers of U251and U87 glioma cells after 24 hour of transfection. The photographs were taken at 0 and 24 hours after wounds were made. C, D Cell migration assay was performed after overexpression of BRMS1 in glioma U251 and U87 cells. The graph shows the percentage of cells migrated in 5 fields of view compared with the control group. All experiments were carried out in triplicate. Data are shown as mean ± SD. **P<0.01; ***P<0.001.

Figure 4. Overexpression of BRMS1 inhibits glioma cells adhesion ability.

A, B Cell attachment assay after BRMS1 overexpression in U251 and U87 cells. The graph shows the percentage of attached cells compared with the control group. C Western blot analysis of the relative protein levels of Src and FAK in BRMS1 overexpression and control group for both U251 and U87 cell lines. D Quantitative analysis of relative protein level of Src and FAK in glioma U251 and U87 cells. All experiments were carried out in triplicate. Data are shown as mean ± SD. *P<0.05, **P<0.01.