siRNA降低COX-2基因表达对肝癌细胞系HepG2增殖的影响

siRNA-mediated downregulation of COX-2 gene expression alters the proliferation of hepatocellular carcinoma HepG2 cells

目的:探讨小干扰RNA降低环氧化酶-2(cyclooxygenase-2,COX-2)基因对肝癌细胞HepG2增殖与细胞外信号调节蛋白激酶(ERK)表达的影响.方法:将人肝癌细胞株HepG2细胞分为4组:COX-2 siRNA干预组、阴性对照siRNA组、空脂质体组、空白对照组.脂质体转染法将COX-2 siRNA转染入HepG2细胞;MTT法测定转染后24,48,72h的增殖抑制率;流式细胞仪测定转染后24h的细胞周期分布;半定量RT-PCR与Western blot检测转染后24h肝癌细胞COX-2和ERK1/2的表达.结果:COX-2 siRNA干预组的增殖抑制率明显高于阴性对照组和空脂质体转染组(67.08%vs2.45%,1.56%,均P<0.01);COX-2 siRNA干预组G1期细胞明显增多,与空白组、空脂质体组及阴性对照组相比,均具有显著差异(72.80%vs50.27%,50.97%,53.13%,均P<0.05);RT-PCR显示:COX-2siRNA干预组COX-2,ERK1,ERK2 mRNA表达(0.58,0.32,0.48)明显降低,与空白组(0.93,0.76,0.72)、空脂质体组(0.89,0.64,0.67)及阴性对照组(0.83,0.71,0.65)比较,差异具有统计学意义(均P<0.05),Western blot显示以上各项指标表达趋势与RT-PCR相同.结论:小干扰RNA可降低肝癌细胞HepG2的COX-2基因表达,抑制肝癌细胞的生长.COX-2促进肝癌生长可能与ERK1/2通路调控有关.

AIM： To explore the effects of siRNA-mediated downregulation of COX-2 gene expression on the proliferation of hepatocellular carcinoma HepG2 cells.METHODS： HepG2 cells were divided into four groups： COX-2 siRNA intervention group, control siRNA intervention group, empty liposome group and blank control group. COX-2 siRNA was transfected into HepG2 cells by lipofection. Cell proliferation inhibition was measured at 24, 48 and 72 h after transfection by MTT （thiazolyl blue） assay. The changes in cell cycle distribution were detected at 24 h after transfection by flow cytometry. The expression levels of COX-2 and ERK1/2 mRNAs and proteins were detected at 24 h after transfection by serniquantitative RT-PCR and Western blot, respectively.RESULTS： The proliferation inhibition rate achieved in the COX-2 siRNA intervention group was higher than those in the control siRNA intervention group and empty liposome group （67.08% vs 2.45% and 1.56%, both P 〈 0.01）. The percentage of HepG2 cells arrested at G1 phase （DNA presynthetic phase） was increased in the COX-2 siRNA intervention group as compared to the control siRNA intervention group, empty liposome group and blank control group （72.80% vs 50.27%, 50.97% and 53.13%, all P 〈 0.05）. Semiquantitative RT-PCR analysis showed that the expression levels of COX-2, ERK1 and ERK2 mRNAs in the COX-2 siRNA intervention group were significantly decreased as compared to the control siRNA intervention group, empty liposome group and blank control group （0.58 vs 0.83, 0.89 and 0.93; 0.32 vs 0.71, 0.64 and 0.76; 0.48 vs 0.65, 0.67 and 0.72; all P 〈 0.05）. Similar results were obtained for protein expression as revealed by Western blot analysis. CONCLUSION： siRNA can downregulate the expression of COX-2 gene in hepatocellular carcinoma HepG2 cells and inhibit their proliferation. COX-2 promotes the development of primary hepatic carcinoma perhaps through regulation of ERK1/2 pathway.