短发夹状RNA对人肝癌细胞环氧合酶-2的抑制作用

Silencing cyclooxygenase-2 gene by construction of eukaryotic expression vector expressing short hairpin RNA in human hepatocellular carcinoma cells

目的:构建针对人环氧合酶-2(COX-2)基因编码区的短发夹状RNA(shRNA)真核表达载体质粒,观察其在不同时间点对人不同肝癌细胞株COX-2表达的影响.方法:以人COX-2mRNA编码区作为RNA干扰靶点,构建shRNA真核表达载体质粒WBH1和WBH2,应用阳离子脂质体分别转染人肝癌细胞株HepG2和Bel7402,利用逆转录聚合酶链反应和Westernblot法分别观察两株细胞转染后24,48,72,和96hCOX-2mRNA和蛋白的表达变化,检测抑制效果.结果:质粒在HepG2细胞和Bel7402细胞中的转染率分别约为60%和54%.WBH1导入细胞24,48,72和96h后,逆转录聚合酶链反应检测COX-2mRNA表达抑制率,HepG2细胞分别为18.5%,88.6%,52.8%和42.4%(P<0.01).Bel7402细胞分别为9%,45.1%,70.1%和56.3%(P<0.01).Westernblot法检测蛋白表达抑制率,HepG2细胞分别为10.3%,80.5%,45.3%和39.0%(P<0.01);Bel7402细胞分别为8.3%,40.2%,66.4%和35.6%(P<0.01).质粒WBH2对COX-2的表达无影响(P>0.05).结论:针对人COX-2的shRNA能高效特异的抑制不同肝癌细胞株的COX-2表达.HepG2细胞和Bel7402细胞分别以48h和72h抑制效果最明显.56.3%(P<0.01).Westernblot法检测蛋白表达抑制率,HepG2细胞分别为10.3%,80.5%,45.3%和39.0%(P<0.01);Bel7402细胞分别为8.3%,40.2%,66.4%和35.6%(P<0.01).质粒WBH2对COX-2的表达无影响(P>0.05).结论:针对人COX-2的shRNA能高效特异的抑制不同肝癌细胞株的COX-2表达.HepG2细胞和Bel7402细胞分别以48h和72h抑制效果最明显.

AIM： To construct the eukaryotic expression plasmids of short hairpin RNA （shRNA） specific to human cyclooxygenase-2 （COX-2） gene and observe their inhibitory effects on COX-2 expression in human hepatocellular carcinoma cells. METHODS： Plasmids named WBH1 and WBH2, containing the different sequences of human COX-2 mRNA coding region, were constructed. The expression of COX-2 was assayed by reverse transcription-polymerase chain reaction （RT-PCR） and Western blot, respectively, 24, 48, 72 and 96 h after HepG2 and Bel7402 cells were transfected with liposomes. RESULTS： The transfection rates in HepG2 and Bel7402 cells were about 60% and 54% respectively. RT-PCR showed that the inhibition efficiencies of the plasmid WBH1 were 18.5%,88.6%, 52.8%, 42.4% in HepG2 cells and 9%, 45.1%, 70.1%, 56.3% in Bel7402 ceils, respectively, 24, 48, 72 and 96 h after transfection （P 〈 0.01）. Western blot demonstrated that the inhibition efficiencies of the plasmid WBH1 were 10.3%, 80.5%, 45.3%, 39.0% in HepG2 cells and 8.3%, 40.2%, 66.4%, 35.6% in Bel7402 cells, respectively （P 〈 0.01）. The plasmid WBH2 had no significant inhibitory effect on COX-2 expression （P 〉 0.05）. CONCLUSION： COX-2 expression in human hepatocellular carcinoma cells can be inhibited significantly by construction of eukaryotic expression vector expressing the shRNA. The maximal inhibition occurs at the 484 and 724 h after transfection, respectively.