Downregulation of BZW2 inhibits osteosarcoma cell growth by inactivating the Akt/mTOR signaling pathway

Abstract

Osteosarcoma is the most common malignant bone tumor in adolescents. The function of basic leucine zipper and W2 domains 2 (BZW2) in tumor progression has been reported. However, the role and mechanisms of BZW2 in osteosarcoma remain to be determined. The aim of the present study was to reveal the expression and biological functions of BZW2 in osteosarcoma and to elucidate the proximal mechanisms underlying these functions. The expression of BZW2 in osteosarcoma tissues and cell lines was assessed by qRT-PCR, western blotting and immunohistochemistry. BZW2 overexpression was detected in osteosarcoma cell lines. Clinically, BZW2 expression was higher in osteosarcoma tissues than in corresponding non-tumor tissues and was associated with advanced Enneking stage and tumor recurrence. The knockdown of BZW2 using siRNA inhibited osteosarcoma cell proliferation, colony-forming ability, and the cell cycle at the G2/M phase in vitro. Host signaling pathways affected by BZW2 were detected using a PathScan Intracellular Signaling Antibody Array kit. These data demonstrated that the knockdown of BZW2 suppresses protein phosphorylation in the Akt/mTOR signaling pathway. These observations suggest that BZW2 is upregulated and has a pro-tumor effect in osteosarcoma via activation of the Akt/mTOR signaling pathway and thus is a potential target for gene therapy.

Figure 1.

BZW2 was upregulated in osteosarcoma tissues and cell lines. (A and B) The mRNA and protein expression levels of BZW2 were assessed by qRT-PCR and western blotting in osteosarcoma cell lines (MNNG/HOS and U2OS) and a human normal osteoblast cell line (hFOB 1.19). (C) The grey intensity was calculated in the osteosarcoma cell lines and the osteoblast cell line. β-actin was used as an internal control. (D and E) The expression of BZW2 was detected in 20 pairs of osteosarcoma tissues and their adjacent non-tumor tissues. The expression of BZW2 was overexpressed in osteosarcoma tissues compared with non-tumor tissues.

Figure 2.

Knockdown of BZW2 inhibits osteosarcoma cell growth in vitro. (A-D) Levels of mRNA and protein expression were validated after si-BZW2 transfection in MNNG/HOS and U2OS cells by qRT-PCR and western blotting, respectively. (E and F) Cell Counting Kit-8 (CCK-8) assays were performed after siRNA transfection. (G-J) Colony formation assays were performed using BZW2-silenced osteosarcoma cells and control cells. Data are representative of results from three independent experiments’ *P<0.05.

Figure 3.

Knockdown of BZW2 inhibits G2/M cell cycle transition in osteosarcoma cells. Representative images of the cell cycle assay results for MNNG/HOS cells (A and B), and U2OS cells (D and E) after transfection with si-NC or si-BZW2. (C and F) Diagrams showing the results of the cell cycle assays for MNNG/HOS and U2OS cells; *P<0.05.

Figure 4.

Knockdown of BZW2 inhibits the Akt/mTOR signaling pathway in osteosarcoma cells. (A and D) MNNG/HOS and U2OS cell extracts were prepared and analyzed using the PathScan Intracellular Signaling Antibody Array kit (#7744). Images were captured using the Odyssey^® Infrared Imaging System (LI-COR). (B and E) Diagrams show the quantitative results for MNNG/HOS and U2OS cells. (C and F) Western blot analysis of the Akt/mTOR signaling pathway, including mTOR and Akt, in MNNG/HOS and U2OS cells transfected with si-NC or si-BZW2.

Figure 5.

Clinical significance of BZW2 in patients with osteosarcoma. (A) Representative IHC image of BZW2 expression in non-tumor adjacent tissues. (B) Representative IHC image of BZW2 expression in osteosarcoma tissues. (C) Representative IHC image of p-Akt expression in osteosarcoma tissues. Original magnification, ×50 and ×200.