Abstract
COX-2 was highly expressed in many tumor tissues and was involved in the initiation and development of tumors. The RNAi technique is a method to inhibit gene expression economically, quickly, efficiently and specifically. This study used RNAi technique to explore the interfering effect of COX-2 gene expression and the influence on the malignant proliferation of A2 cells after quenching COX-2 in vitro . Three COX-2 siRNA expression vectors with human U6 promoter were constructed. The COX-2 siRNA vectors and the vacant vector (pEGFP) were transfected into A2 cells with lipofectamine respectively. The cell strains transfected were selected. The change of COX-2 expression levels was examined by Western blot and RT-PCR. The effects on the proliferation of A2 cells after silencing COX-2 were detected by cell growth curve and clonogenic assay in vitro . The three siRNA and U6 promoter cloned into pEGFP plasmid were validated by PCR, restriction endonucleases identification, DNA sequencing and BLAST alignment. The cell strains transfected were coded as A2-3, A2-7, A2-10 and A2-p respectively. Green fluorescence was seen in A2-p cells and not in A2-3, A2-7 and A2-10 cells in 24 h, 48 h and 72 h after transfected. The results of RT-PCR and Western blot showed the three siRNA expression vectors acted effectively and the expression of COX-2 was inhibited in different extent. In contrast to A2 cells, COX-2 mRNA levels of A2-3, A2-7 and A2-10 cells were reduced 15.6%, 20.4% and 64.2% respectively, and COX-2 protein expressions of them were reduced 23.7%, 36.7% and 60.2% respectively. The results of cell growth curve and clonogenic assay showed the growth of A2-10 cell slowed and the clonal formation rate was reduced. However the growth of A2-3 and A2-7 cells had no obvious changes vs controls (A2 and A2-p). Silencing COX-2 gene in vitro by RNAi technique can significantly inhibit the malignant proliferation of A2 cells.
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