短发夹RNA靶向抑制环氧化酶-2基因表达对肺癌细胞A_(549)增殖的影响  

Effects of COX-2 gene expression inhibited by shRNA on proliferation of pulmonary carcinoma A_(549)cells

作　　者：杨义明[1] 刘预[2] 涂植光[3] 甘晓协[2] 刘靳波[3] 陈亚芹[3] 

机构地区：[1]重庆医科大学检验系临床检验诊断学省部共建教育部重点实验室 重庆市重点实验室,400016 [2]重庆市肿瘤研究所临床检验中心 [3]重庆医科大学检验系 临床检验诊断学省部共建教育部重点实验室 重庆市重点实验室,400016

出　　处：《中华检验医学杂志》2007年第10期1172-1175,共4页Chinese Journal of Laboratory Medicine

基　　金：重庆市自然科学基金(2006BB5271)

摘　　要：目的评价短发夹 RNA(short hairpin RNA,shRNA)靶向肺癌细胞 A_(549)环氧化酶-2(COX-2)基因表达的抑制作用,并观察对肺癌细胞 A_(549)生长和细胞周期的影响。方法将抑制 COX-2基因的 shRNA DNA 模板插入到真核表达载体 pGenesil-1的 U_6启动子下游,构建靶向抑制 COX-2基因的重组表达载体 pshRNA-COX-2。将试验分为3组:未转染肺癌细胞 A_(549)组、阴性对照 HK 组和pshRNA-COX-2转染组。用脂质体 Lipofectamine^(TM)2000转染肺癌细胞 A_(549)9,逆转录(RT)-PCR 和免疫印迹法(Wb)分别检测 COX-2基因 mRNA 和蛋白表达情况,流式细胞术检测细胞周期,细胞计数检测细胞生长变化。结果与阴性对照组相比,pshRNA-COX-2重组表达载体抑制 COX-2 mRNA 及蛋白表达的抑制率分别为72.2%和48.6%;G_0-G_1,期细胞由63.7%上升为71.0%,S 期细胞由26.5%下降为20.0%;细胞生长明显减慢。结论 pshRNA-COX-2 重组表达载体能显著抑制肺癌细胞 COX-2表达,引起 G_0-G_1期细胞增多,S 期细胞减少,从而抑制肺癌细胞生长。Objective To investigate the inhibiting effects of shRNA （short hairpin RNA,shRNA） targeting COX-2 gene in pulmonary carcinoma A549 cells, and its effects on the cells cycle and growth of pulmonary A549 cells. Methods shRNA targeting COX-2 was inserted into the downstream of U6 promoter in eukaryotic expression vector pGenesil-1 to construct recombinant expression plasmid pshRNA-COX-2. The cultured cells were divided into three groups：untransfected A549 cells group, negative control group HK, and pshRNA-COX-2 group. Pulmonary carcinoma A549 cells were transfected with LipofectamineTM 2000. Expression of COX-2 mRNA and protein were detected with reverse transcriptional polymerase chain reaction （RT-PCR） and Western blot respectively. Cell cycle analysis and cell proliferation were detected with flow cytometry and cell count respectively. Results Compared with negative control group HK, recombinant expression vector pshRNA-COX-2 resulted in the reduction of COX-2 mRNA and protein by 72.2% and 48. 6% respectively; cells in G0-Gl phase increased from 63. 7% to 71.0%, cells in S phase decreased from 26. 5% to 20. 0%, and the growth of A549 cells was slow significantly. Conclusion Recombinant expression vector pshRNA-COX-2 can significantly inhibit the expression of COX-2 gene, and result in the increase of cells in G0-G1 phase and decrease of cells in S phase, and suppress proliferation of pulmonary carcinoma Am cells.

关 键 词：肺肿瘤 基因表达 短发夹RNA 

分 类 号：R734.2[医药卫生—肿瘤]