RNAi-mediated knockdown of cyclooxygenase2 inhibits the growth, invasion and migration of SaOS2 human osteosarcoma cells: a case control study

Abstract

Background: Cyclooxygenase2 (COX-2), one isoform of cyclooxygenase proinflammatory enzymes, is responsible for tumor development, invasion and metastasis. Due to its role and frequent overexpression in a variety of human malignancies, including osteosarcoma, COX-2 has received considerable attention. However, the function of COX-2 in the pathogenesis of cancer is not well understood. We examined the role of COX-2 in osteosarcoma.

Methods: We employed lentivirus mediated-RNA interference technology to knockdown endogenous gene COX-2 expression in human osteosarcoma cells (SaOS2) and analyzed the phenotypical changes. The effect of COX-2 treatment on the proliferation, cell cycle, invasion and migration of the SaOS2 cells were assessed using the MTT, flow cytometry, invasion and migration assays, respectively. COX-2, vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) mRNA and protein expression were detected by RT-PCR and western blotting.

Results: Our results indicate that a decrease of COX-2 expression in human osteosarcoma cells significantly inhibited the growth, decreased the invasion and migration ability of SaOS2 cells. In addition, it also reduced VEGF, EGF and bFGF mRNA and protein expression.

Conclusions: The COX-2 signaling pathway may provide a novel therapeutic target for the treatment of human osteosarcoma.

Figure 1

Downregulation of COX-2 expression in 293T cells by shRNA transfection. (A) GFP expressed 48 h after the transfection of the control, shRNA1, shRNA2 and shRNA3 plasmid in 293T cells, under a fluorescent microscope, respectively. (magnification 200 ×). (B) COX-2 mRNA levels were detected by RT-PCR. (C) COX-2 protein levels were detected by western blotting. Data are presented as mean ± s.e.m. * P < 0.01, # P < 0.001, compared with untransfected 293T cells group or control plasmid transfected cells group.

Figure 2

COX-2 expression was inhibited by LV-COX-2siRNAi-1 in SaOS2 cells. (A) SaOS2 cells infected with LV-Control and LV-COX-2siRNAi-1. GFP expressed 48 h after the infection (magnification 40 ×). COX-2 (B), but not COX-1 (C) mRNA level was significantly inhibited by LV-COX-2siRNAi-1. Data are presented as mean ± s.e.m. # P < 0.001, compared with LV-Control and parental SaOS2 cell group.

Figure 3

Osteosarcoma cells proliferation were assessed by MTT assays. The growth of SaOS2 cells in 96-well plates applied to absorbance at 490 nm were detected on day 1, 2, 3, 4 and 5, respectively. Data are presented as mean ± s.e.m. # P < 0.001, compared with LV-Control and parental SaOS2 cell group.

Figure 4

Measurement of invasion and migration of SaOS2 cells. (A) Invading and migrating cells were stained with 0.2% crystal violet and visualized by microscopy. (magnification 100 ×). (B) Invasion and migration assay indicated LV-COX-2siRNA-1 significantly decreased the invasion or migration ability of the SaOS2 cells. Data are presented as mean ± s.e.m. # P < 0.001, compared with LV-Control and parental SaOS2 cell group.

Figure 5

Genes and proteins associated with angiogenesis were supressed by COX-2 gene knockdown. LV-COX-2siRNA-1 significantly inhibited the mRNA (A) and protein (C) expression of VEGFA, EGF, bFGF in SaOS2 cells. (B) VEGFA, VEGFB, VEGFC, EGF, bFGF protein expression in each group. Data are presented as mean ± s.e.m. * P < 0.01, # P < 0.001, compared with LV-Control and parental SaOS2 cell group.