The impact of COX-2 on invasion of osteosarcoma cell and its mechanism of regulation

Abstract

Background: Recently, cyclooxygenase-2 (COX-2) has become an important new target in the field of tumor metastasis. However, the relationship between COX-2 gene expression and the behavior of osteosarcoma metastasis is largely unknown. The study is to investigate how antisense oligonucleotides (ODNs) of COX-2 inhibit the invasion of human osteosarcoma cell line OS-732 and their mechanism of regulation.

Methods: A COX-2 antisense oligonucleotide was designed, synthesized, and transfected into OS-732 human osteosarcoma cells. RT-PCR and western blotting were performed to determine the transfection efficiency. A modified Boyden-transwell assay was used to measure the inhibition rate of tumor cell invasion. In OS-732 cells transfected with COX-2 antisense ODNs, RT-PCR was used to examine the mRNA expression of urokinase-type plasminogen activator (uPA) and that of its receptor, uPAR.

Results: Both the mRNA and protein expression levels of COX-2 were significantly reduced when cells were transfected with COX-2 antisense ODNs, which significantly reduced the invasive ability of OS-732 cells in a dose-dependent manner. The expression levels of uPA and uPAR were also significantly reduced (p < 0.01).

Conclusion: COX-2 antisense ODNs significantly inhibited the invasion of OS-732 cells, primarily by decreasing the mRNA expression of uPA and uPAR.

Keywords: Antisense oligonucleotide; COX-2; Invasion; Osteosarcoma.

Figure 1

The expression of COX-2 mRNA (12 hours after transfection) a. COX-2 mRNA (488 bp) (1. The control group, 2. Lipofectin group, 3. C1 group: 100 nmol/L, 4. C2 group: 200 nmol/L, 5. C3 group: 400 nmol/L, 6. C4 group: 800 nmol/L); b. GAPDH as an internal control (330 bp).

Figure 2

The protein expression of COX-2 by western blotting (0.The control group, 1. Lipofectin group, 2. COX-2 AS-ODN: 100 nmol/L, 3. COX-2 AS-ODN: 400 nmol/L).

Figure 3

The influence of COX-2 antisense oligonucleotides on osteosarcoma cell invasion.

Figure 4

The number of trans-membrane cells (×200). a. The control group, b. The COX-2 AS-ODN transfection group (100 nmol/L), c. The COX-2 AS-ODN transfection group (800 nmol/L).

Figure 5

RT-PCR amplification: a. uPA, b. uPAR, group 0, 1, 2 and 3 (COX-2 AS-ODN at 0, 200, 400 or 800 nmol/L).