论文部分内容阅读
Objective:We explored the interfering effect of COX-2 gene expression and the influence on the malignant proliferation of A2 cells by RNAi after quenching COX-2 in vitro. Methods:COX-2 was selected as the subject. Three COX-2 siRNA expression vectors with human U6 promoter were constructed and three vectors and the vacant vector (pEGFP) were transfected into A2 cells with lipofectamine respectively. The cell strains transfected were constructed. 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 studied by cell growth curve and clonogenic assay in vitro. Results: 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 named 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, 48 and 72 h after transfected. The results of RT-PCR and Western blot showed the three siRNA expression vectors produced effects and the expression of COX-2 was inhibited in different extent. In contrast to A2 cells, the levels of COX-2 mRNA of A2-3, A2-7 and A2-10 cells reduced 15.6%, 20.4% and 64.2% respectively; the levels of COX-2 protein of A2-3, A2-7 and A2-10 cells 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 colonial formation rate reduced but the growth of A2-3 and A2-7 cells had not obvious changes in contrast to the controls (A2 and A2-p). Conclusion: Silencing the COX-2 gene in vitro by RNAi technique can significantly inhibit the malignant proliferation of A2 cells.
Objective: We explored the interfering effect of COX-2 gene expression and the influence on the malignant proliferation of A2 cells by RNAi after quenching COX-2 in vitro. Methods: COX-2 was selected as the subject. Three COX-2 siRNA expression Vectors with human U6 promoter were constructed and three vectors and the vacant vector (pEGFP) were transfected into A2 cells with lipofectamine respectively. 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 studied by cell growth curve and clonogenic assay in vitro. Results: The three siRNA and U6 promoter cloned into pEGFP plasmid were validated by PCR, restriction endonucleases identification, DNA sequencing and BLAST alignment. The cell stain transfected were named 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, 48 and 72 h after transfected. The results of RT-PCR and Western blot showed the three siRNA expression vectors produced effects and the expression of COX-2 was inhibited in different extent. In contrast to A2 cells, the levels of COX-2 mRNA of A2-3, A2-7 and A2-10 cells reduced by 15.6%, 20.4% and 64.2% respectively; the levels of COX-2 protein of A2-3, A2-7 and A2-10 cells reduced by 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 colonial formation rate reduced but the growth of A2-3 and A2-7 cells had not obvious changes in contrast to the controls (A2 and A2-p). Conclusion: Silencing the COX-2 gene in vitro by RNAi technique can significantly inhibit the malignant proliferation of A2 cells.