High-mobility group box 2 is associated with prognosis of glioblastoma by promoting cell viability, invasion, and chemotherapeutic resistance

Abstract

Background: The expression profile of high-mobility group box 2 (HMGB2) in patients with glioblastoma multiforme (GBM) and its clinical signature with underlying mechanisms were not fully explored.

Methods: HMGB2 protein levels were measured in 51 GBM patients by immunohistochemical studies. To clarify the precise role of HMGB2 on cell invasion and viability of 3 GBM cell lines, we did in vitro and in vivo analyses with lentivirus vectors and small interfering RNA. Transwell invasion assays and wound-healing assays were used to analyze the invasion of GBM cells. Expression of p53 and matrix metalloproteinase 2/tissue inhibitors of metalloproteinase 2 (MMP2/TIMP2) protein was analyzed by Western blot.

Results: HMGB2 protein expression was significantly higher in GBM than in controlled brain tissues (P < .0001). HMGB2 overexpression was significantly correlated with shorter overall survival time, which was the only independent prognostic factor for overall survival in a multivariate analysis (P = .017). HMGB2 knockdown by small interfering RNA decreased cell viability and invasion in vitro and significantly decreased tumor volume in vivo, which might be involved in the change of p53 expression and the balance of MMP2/TIMP2. Moreover, silencing of HMGB2 could significantly increase the sensitivity of GBM cells to temozolomide chemotherapy.

Conclusions: Our present data suggest that HMGB2 expression is a significant prognostic factor and might play an important role in cell invasion and temozolomide-induced chemotherapeutic sensitivity of GBM. This study highlights the importance of HMGB2 as a novel prognostic marker and an attractive therapeutic target of GBM.

Keywords: glioblastoma; high-mobility group box 2; invasion; prognosis; temozolomide.

Fig. 1.

The growth-suppressive effect of HMGB2 silencing on GBM cells in vitro and in vivo. (A and B) The effect of HMGB2 knockdown via siRNA silencing. The cells were transfected with siControl or siHMGB2 for 72 h and subjected to immunoblot analysis and quantitative PCR of HMGB2 expression. (C) HMGB2 knockdown via siRNA silencing inhibited the proliferation of SHG66 (left), U87 (middle), and U251 (right) cells. The cells were transfected with siControl or siHMGB2 for 24–96 h and subjected to cell proliferation assay by CCK-8. (D and E) HMGB2 knockdown inhibited the growth of U87 cells in vivo. The representative images for xenograft tumor on the nude mouse are shown in Fig. 1E (top panel), and the tumor growth curve or tumor weight is shown in Fig. 1D and E (n = 4). *P < .05, **P < .01, ***P < .001.

Fig. 2.

HMGB2 silencing suppressed migration and invasion in GBM cells. (A) HMGB2 knockdown suppressed the invasive abilities of GBM cells. The cells were transfected with siControl or siHMGB2 for 72 h and subjected to cell invasion assay by transwell assay. The representative image for SHG66 cells is shown in Fig. 2A (left), and data from 3 independent experiments are expressed as the mean ± SE (right). **P < .01, significantly different from the siControl group. (B and C) HMGB2 knockdown suppressed the migration abilities of GBM cells. As described, the cells were subjected to cell migration assay by wound-healing assay. The representative images for GBM cells are shown in Fig. 2B, and data from 3 independent experiments are expressed as the mean ± SE (Fig. 2C). *P < .05, **P < .01, significantly different from the siControl group.

Fig. 3.

The effect of HMGB2 on invasion-related biomarkers. (A) Cells were transfected with siControl or siHMGB2 for 72 h and subjected to immunoblot analysis for expression of indicated proteins. (B) Cells were infected with pCDH-vector (control) or pCDH-HMGB2 (HMGB2) for 72 h and then subjected to immunoblot analysis for expression of indicated proteins.

Fig. 4.

HMGB2 silencing sensitized GBM cells to TMZ chemotherapy. (A) The transfection efficiency of pCDH-HMGB2. The cells were infected with pCDH-vector (control) or pCDH-HMGB2 (HMGB2) for 72 h and subjected to immunoblot analysis. (B and C) The cells were transfected with siHMGB2 (B) or infected with pCDH-HMGB2 (C) for 24 h, treated with TMZ (12.8 μM) for 48–96 h, and then subjected to cell proliferation assay by CCK-8. *P < .05, **P < .01, ***P < .001.

Fig. 5.

HMGB2 was overexpressed in GBM and negatively correlated with patient survival. (A and B) IHC staining of human GBM tissues using HMGB2-specific antibody, as described in Material and Methods. HMGB2 protein is expressed in nucleus (A, right panel). Classification of samples according to the extent of staining of HMGB2 expression (n = 51). (C) Immunoblot analysis to determine expression of HMGB2 in GBM and controlled brain tissues. T: GBM tumor tissues (n = 10); C: controlled brain tissues (n = 8). (D) Quantification of HMGB2 expression in GBM and controlled brain tissues. (E) The extent of staining in the low- and high-score group of HMGB2. Low: low-score group of HMGB2 with staining extent < 50%; High: high-score group of HMGB2 with staining extent ≥50%. (F and G) Correlation analysis of HMGB2 protein expression (F, n = 51) and mRNA levels (G, n = 180) and patient OS. **P < .01.