Effect of cycloxygenase-2 silencing on the malignant biological behavior of MCF-7 breast cancer cells

Abstract

The aim of the present study was to investigate the effect of cyclooxygenase-2 (COX-2) silencing on the malignant biological behavior of MCF-7 breast cancer cells. COX-2 short hairpin RNA (shRNA) and unassociated sequences were synthesized and a shRNA lentiviral vector was constructed. The vector was transfected into MCF-7 breast cancer cells, in which clones with stable expression were screened out. The expression of COX-2 mRNA and protein was silenced using RNA interference (RNAi). Quantitative polymerase chain reaction, western blotting, a mononuclear cell direct cytotoxicity assay (MTT assay), a cell invasion assay and scratch tests were performed to investigate the downregulation of COX-2 mRNA and protein expression, the proliferative activity and growth rate of MCF-7 breast cancer cells, the glioblastoma multiforme (GBM) penetrating capacity, the cell movement and migratory capacity, and vascular endothelial growth factor (VEGF)-A and VEGF-C protein expression. The results revealed that the sequence-specific shRNA significantly downregulated the expression of COX-2 at the mRNA and protein levels. Furthermore, the downregulation of COX-2 expression markedly decreased the invasive and metastatic capacities of the cells, suppressed the proliferation, decreased the rate of growth, decreased the capacity of GBM penetration and migration, and decreased the protein expression of VEGF-A and VEGF-C, the two key factors that regulate tumor angiogenesis and lymphangiogenesis. In conclusion, the RNAi technique effectively silenced COX-2 gene expression and inhibited MCF-7 breast cancer cell proliferation, invasion and metastasis by decreasing VEGF-A and VEGF-C expression, which regulates tumor angiogenesis and lymphangiogenesis. Therefore, an RNAi technique that targets COX-2 presents a promising prospect for breast cancer gene therapy.

Keywords: RNA interference; breast cancer; cyclooxygenase-2; invasion; proliferation.

Figure 1

(A) Vector used was a no load virus. COX-2-shRNA-1, 2 and 3 represent three pairs of COX-2 shRNA, whose plasmids were effectively transfected into MCF-7 breast cancer cells (magnification, ×100). (B) Detection of COX-2 interference in each group by quantitative polymerase chain reaction. (C) Detection of COX-2 interference in each group by western blotting. COX-2, cyclooxygenase-2; shRNA, short hairpin RNA.

Figure 2

Expression of COX-2 (A) mRNA and (B) protein in MCF-7 breast cancer cells in the COX-2-shRNA group was significantly lower than that of the blank and mock groups. COX-2, cyclooxygenase-2; shRNA, short hairpin RNA.

Figure 3

MTT assay revealed that the cell proliferation rates in the COX-2-shRNA group were significantly lower than that of the blank and mock groups. COX-2, cyclooxygenase-2; shRNA, short hairpin RNA.

Figure 4

(A) Number of MCF-7 breast cancer cells penetrating the plasma membrane in the COX-2-shRNA group was significantly lower than that of the mock and blank groups 24 h after transfection (magnification, ×100). (B) The invasive capacity of the MCF-7 breast cancer cells was significantly decreased following transfection in the COX-2-shRNA group when compared with that of the blank and mock groups. COX-2, cyclooxygenase-2; shRNA, short hairpin RNA.

Figure 5

(A) Cell migration of MCF-7 breast cancer cells in the COX-2-shRNA group was significantly decreased (magnification, ×100). (B) Cell migration of MCF-7 breast cancer cells 24 h after transfection. COX-2, cyclooxygenase-2; shRNA, short hairpin RNA.

Figure 6

VEGF-A and -C protein expression of breast cancer MCF-7 cells in the COX-2-shRNA group was significantly lower than that of the blank and mock groups. COX-2, cyclooxygenase-2; VEGF, vascular endothelial growth factor; shRNA, short hairpin RNA.