RNA干扰环氧化酶-2基因表达对A549细胞体外恶性增殖的影响

Effects of RNA Interference in COX-2 Gene Expression on Malignant Proliferation of A549 Cells in vitro

目的研究不同靶点对环氧化酶-2（COX-2）基因表达的抑制及其对A549细胞体外恶性增殖的影响。方法以COX-2第3、7和10外显子为靶点，构建3个带有人U6启动子的COX-2小干扰RNA（siRNA）表达载体。用脂质体lipofectamine介导，分别将3个siRNA表达载体及2个空载体转染到cox-2表达阳性的A549细胞，建立转染细胞株。采用逆转录聚合酶链反应（RT—PCR）和Western blot方法检测COX-2表达水平的变化。通过细胞生长曲线、集落形成试验研究COX-2不同干扰靶点对A549细胞体外增殖的影响。结果3个siRNA和u6启动子序列正确并准确克隆到pEGFP载体。转染细胞株分别命名为A549—3，A549—7、A549—10、A549-p和A549-pU6。转染后24、48、72h，A549-p细胞均有绿色荧光表达，而A549-pU6、A549-3、A549—7和A549—10细胞均未观察到绿色荧光。RT—PCR和Western blot结果显示，以COX-2第3、7和10外显子作为靶点进行干扰，COX-2表达均受到抑制。与A549细胞比较，A549—3、A549-7、A549-10细胞的COX-2 mRNA表达量分别降低10．6％、33．4％和61．2％，COX-2蛋白表达量分别降低26．7％、44．7％和56．2％。细胞生长曲线、集落形成试验的结果显示，A549—10细胞生长减慢，集落形成率减少，而3外显子和7外显子两个干扰靶点对A549细胞生长没有明显的影响。结论以COX-2第10外显子为靶点干扰效果最好，对A549细胞的体外恶性增殖有明显的影响。

Objective To investigate the inhibition of COX-2 gene expression and its effects on malignant proliferation of human lung adenocarcinoma A549 cells after interfering at different target sites in vitro. Methods The 3rd, 7th and 10th exon of COX-2 were selected as the targets and three COX-2 siRNA expression vectors with human U6 promoter were constructed. Three siRNA expression vectors and two vacant vectors were transfected into A549 cells expressing COX-2 with lipofectamine, respectively. The transfected cell strains were constructed and the change of COX-2 expression levels was examined by Western blot and RT-PCR. The effects on the proliferation of A549 cells after interfering at different target sites were studied by cell growth curve and colony formation assay in vitro. Results The three siRNAs and U6 promoter were validated by PCR. restriction endonuclease digestion, DNA sequencing and BLAST alignment, and cloned into the pEGFP vector. The cell strains transfected were named as A549-3, A549-7, A549-10, A549-p and A549-pU6 respectively. A549-p cells showed expression of GFP and A549-3, A549-7, A549-10, A549-p and A549-pU6 cells did not show at 24, 48 and 72 hours after transfection. The results of RT-PCR and Western blot showed an inhibition of COX-2 expression after interfering at three target sites（3rd, 7th and 10th exons）. In contrast to A549 cells, the levels of COX-2 mRNA of A549-3, A549-7 and A549-10 cells were reduced by 10.6%, 33.4% and 61.2%, respectively. The levels of COX-2 protein of A549-3, A549-7 and A549-10 cells were reduced by 26.7%, 44.7% and 56.2%, respectively. The results of cell growth curve and colony formation assay showed a slowing down of the growth of A549-10 cells and reduction of their colony formation rate. The other two targets had no apparent effect on the growth of A549 cells. Conclusion There is a significant inhibiting effect of RNA interference on the malignant proliferation of A549 cells in vitro, and the most striking effect can be seen when the 10th exon of COX-2 is taken as the interference target.