Li Wei-Ying, Wang Hui, Lai Bai-Tang, Yang Xue-Hui, Zhang Chun-Yan
Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing 101149, China.
Zhonghua Zhong Liu Za Zhi. 2007 Dec;29(12):904-8.
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.
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.
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.
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.
体外研究在不同靶点干扰后COX-2基因表达的抑制及其对人肺腺癌A549细胞恶性增殖的影响。
选择COX-2的第3、7和10外显子作为靶点，构建3种含人U6启动子的COX-2 siRNA表达载体。分别用脂质体将3种siRNA表达载体和2种空载体转染入表达COX-2的A549细胞。构建转染后的细胞株，采用蛋白质免疫印迹法（Western blot）和逆转录-聚合酶链反应（RT-PCR）检测COX-2表达水平的变化。通过体外细胞生长曲线和集落形成实验研究不同靶点干扰后对A549细胞增殖的影响。
通过聚合酶链反应（PCR）、限制性内切酶酶切、DNA测序和BLAST比对验证了3种小干扰RNA（siRNAs）和U6启动子，并克隆到pEGFP载体中。转染后的细胞株分别命名为A549-3、A549-7、A549-10、A549-p和A549-pU6。A549-p细胞在转染后24、48和72小时显示绿色荧光蛋白（GFP）表达，而A549-3、A549-7、A549-10、A549-p和A549-pU6细胞未显示。RT-PCR和Western blot结果显示，在3个靶点（第3、7和10外显子）干扰后COX-2表达受到抑制。与A549细胞相比，A549-3、A549-7和A549-10细胞中COX-2 mRNA水平分别降低了10.6%、33.4%和61.2%。A549-3、A549-7和A549-10细胞中COX-2蛋白水平分别降低了26.7%、44.7%和56.2%。细胞生长曲线和集落形成实验结果显示A549-10细胞生长减慢，集落形成率降低。另外两个靶点对A549细胞生长无明显影响。
RNA干扰对体外A549细胞的恶性增殖有显著抑制作用，以COX-2第10外显子为干扰靶点时效果最为显著。
