摘要
目的:利用RNA干扰(RNAi)抑制宫颈癌Hela细胞环氧合酶-2(cyclooxygen-ase-2,COX-2)表达研究其对细胞周期的影响。方法:构建特异性COX-2小干扰RNA(siRNA)真核表达载体质粒Pgenesil-COX-2,以脂质体法将其转染至人宫颈癌Hela细胞。转染48h分别用RT-PCR和Western blot方法评价Pgenesil-COX-2对COX-2表达的抑制效应,48和96h时用流式细胞技术检测Hela细胞细胞周期,用RT-PCR方法检测CyclinE的表达变化。结果:转染48h后,Pgenesil-COX-2特异性抑制了Hela细胞COX-2的表达,与对照组比较差异有统计学意义,t=4.496,P=0.002。48及96h流式细胞仪分析显示,转染Pgenesil-COX-2后Hela细胞增殖活性降低,与对照组比较G0/G1期细胞增多,t值分别为3.217和2.495,P值分别为0.011和0.043;S期细胞减少,t=5.978和6.137,P值均为0.000。同时,CyclinE表达降低,t值分别为2.879和8.612,P值分别为0.020和0.000。结论:siRNA真核表达载体可特异性抑制Hela细胞COX-2基因的表达,抑制COX-2表达可能通过下调Cy-clinE的表达使细胞周期阻滞于G0/G1期。
OBJECTIVE：To investigate the influence of the expression of cyclooxygenase-2 （COX-2） down-regulated by RNA interference on the cell cycle of cervical cancer Hela cells. METHODS： An eukaryotic expression plasmid of siRNA targeting on COX-2, Pgenesil-COX-2 was constructed. Then Pgenesil-COX-2 was transfected into Hela cells with Lipofectamine TM2000 and 48 h after the transfection, reverse transcriptase-polymerase chain reaction （RT-PCR） and Western blot were used to assay the inhibition effect of Pgenesil-COX-2 on the expression of COX-2. The cell cycle of Hela cells was determined by flow cytometry 48 and 96 hours after the transfection. The change of the expression of CyclinE was detected by RT-PCR. RESULTS： After transfected with Pgenesil-COX-2 for 48 hours, the expression of COX-2 mRNA and protein in Hela cells was inhibited significantly （t= 4. 496, P= 0. 002）. After transfeeted for 48 and 96 hours, the proliferation of Hela cells was suppressed significantly. Compared with the control group, the percentage of Hela cells in G0/G1 phase was increased （t=3.217, P 0.011; t=2.495, P 0.043）, and in S phase decreased （t=5.978, P 0.000; t=6.137, P 0.000）. The expression of Cyclin E was decreased significantly （t=2. 879, P=0. 020; t =8. 612, P =0. 000）. CONCLUSIONS： By RNA interference through an eukaryotic expression plasmid, Pgenesil COX-2 can ef fectively inhibit the expression of target gene. Silencing COX-2 gene can arrest the cell cycle at G0/G1 phase by down-regulation of the expression of CyclinE gene.
出处
《中华肿瘤防治杂志》
CAS
2007年第20期1535-1538,共4页
Chinese Journal of Cancer Prevention and Treatment
