The establishment of supramolecular immunobead real-time PCR and the identification of Cox-2 as a metastasis-related marker in colorectal carcinoma

Abstract

Abstract. Cyclooxygenase-2 (Cox-2) is an inducible enzyme that converts arachidonic acid to prostaglandins, and it is hypothesized to induce carcinogenesis and metastasis in colorectal cancer. Our previous data also indicated that a higher expression level of Cox-2 was correlated with colorectal cancer metastasis. The Cox-2 protein was detected in the glandular cavity of colorectal cancer and the surrounding interstitial tissues. The usefulness of the Cox-2 gene as a gene therapy target and diagnostic marker remains unknown. In this study, a method using immuno-PCR and real-time PCR followed by supramolecular immunobead real-time PCR was established and used to detect the expression of Cox-2 in serum samples of nude mice with colorectal carcinoma. In addition, we established a Cox-2 gene stable knockdown colorectal cell line (SW480-EGFP-Cox-2 shRNA) using lentiviral vector-mediated RNA interference (RNAi) technology and established an imageable colorectal cancer metastasis mouse model. We found that the proliferation, invasion and tumorigenesis of SW480-EGFP-Cox-2 shRNA cells were attenuated compared with SW480 cells. In vivo experiments also demonstrated that angiogenesis in the Cox-2 knockdown colorectal cancer cells was decreased. The whole body optical imaging revealed that the SW480-EGFP-Cox-2 shRNA cells had an abrogated ability to develop metastases in the lymph nodes, lungs or liver in vivo. The improved immunobead PCR assay detected significantly lower Cox-2 protein levels in the serum samples of the SW480-EGFP-Cox-2 shRNA group compared with those of the SW480-EGFP-Cox-2-Ctrl shRNA group. In conclusion, our results indicated that the knockdown of Cox-2 expression suppressed the proliferation and invasion of colorectal cancer cells both in vitro and in vivo. This study also demonstrated that silencing Cox-2 in vivo reduced the metastastic potential of colorectal cancer. Thus, Cox-2 is a promising marker for the diagnosis of colorectal metastasis and a potential therapeutic target for colorectal cancer.