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.
Highlights
Previous studies have shown that cyclooxygenase‐2 (COX‐2) expression is enhanced in various solid tumors, including breast, lung and colorectal cancer, and is closely associated with tumor proliferation, invasion and metastasis, and COX‐2 is considered a promising target in antitumor gene therapy [1,2]
The COX‐2‐short hairpin RNA (shRNA) plasmid was transfected into the MCF‐7 breast cancer cells, screened and amplified via G418 for further study
Western blotting indicated that the expression of the COX‐2 protein in the MCF‐7 breast cancer cells of the COX‐2‐shRNA group was significantly lower than that of the blank and mock groups (P
Summary
Previous studies have shown that cyclooxygenase‐2 (COX‐2) expression is enhanced in various solid tumors, including breast, lung and colorectal cancer, and is closely associated with tumor proliferation, invasion and metastasis, and COX‐2 is considered a promising target in antitumor gene therapy [1,2]. As an important method for investigating gene function, the RNA interference (RNAi) technique is a rapid, economical and highly efficient technique for silencing gene expression [3,4]. A short hairpin RNA (shRNA) lentiviral vector was constructed and its effect on malignant biological behaviors, including proliferation, invasion and metastasis, of breast cancer cells was investigated. The function of COX‐2 in the carcinogenesis and development of breast cancer was verified, which permitted further study regarding these mechanisms
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