环氧合酶2基因沉默诱导人肝癌细胞凋亡的作用观察

Cyclooxygenase-2 gene silence induces apoptosis of human hepatocellular carcinoma cells

目的观察作用于环氧合酶2(COX-2)mRNA的发卡状COX-2(COX-2 shRNA)真核表达质粒对人肝癌HepG2细胞增殖和凋亡的影响。方法设计靶向COX-2基因的RNA干扰序列,构建COX-2 shRNA表达载体,用阳离子脂质体Lipofectamine 2000转染体外培养的HepG2细胞。半定量RT-PCR检测COX-2 mRNA表达水平的变化,筛选有效抑制COX-2基因表达的序列。采用CCK-8细胞计数法检测COX-2 shRNA对细胞增殖的影响,流式细胞仪测定细胞凋亡率和细胞周期,Western blotting检测细胞内COX-2和Bcl-2蛋白表达水平。结果测序结果显示成功构建了2个表达COX-2 shRNA的质粒载体,转染HepG2细胞后COX-2 mRNA表达下降至阴性对照组的41.66%和77.79%。选择沉默效率较佳者转染细胞并经G418筛选,获得稳定表达COX-2 shRNA的细胞,胞内COX-2 mRNA表达水平下降了75.30%,细胞增殖活性下降了29.33%。在稳定表达COX-2 shRNA、错义序列以及未转染的细胞中,细胞凋亡率分别为17.83%、4.63%和4.47%,G0/G1期细胞比例分别为73.93%、61.2%和57.630%,而S期细胞比例分别为13.43%、26.03%和26.90%。Western blotting检测显示,表达COX-2 shRNA的HepG2细胞内COX-2蛋白水平下降至表达错义序列组的31.25%(P<0.01),Bcl-2蛋白水平下降至表达错义序列组的54.93%(P<0.01),而转染错义质粒组与未转染组之间比较差异无统计学意义。结论构建的真核表达载体pSilencer 2.1-U6-COX-2 shRNA能有效沉默人肝癌HepG2细胞内COX-2基因的表达,并具有抑制细胞增殖、促进细胞凋亡和细胞周期阻滞、降低胞内抗凋亡蛋白Bcl-2水平的作用。

Objective To specifically silence COX-2 gene expression in cultured human hepatocellular carcinoma cells HepG2 by construction and transfection of COX-2 hairpin RNA（shRNA） expression vectors,and investigate the effects of knocking off COX-2 on cell proliferation and apoptosis.Methods The inserted sequences coding shRNA homologous to COX-2 gene were designed and COX-2 shRNA expression vectors were constructed and transfected into HepG2 cells with Lipofectamine 2000.The inhibitory efficiency of COX-2 shRNA was determined by detection of COX-2 mRNA levels in transfected cells using semi-quantitative RT-PCR.Cell viability was analyzed by cell counting kit-8（CCK-8） assay.Indexes of apoptotic cells and cell cycle distribution were analyzed by flow cytometry.Western blotting was used to detect the changes in protein levels of COX-2 and Bcl-2 in HepG2 cells.Results Plasmid sequencing showed the successful construction of two shRNA expression vectors.The expressive levels of COX-2 mRNA in HepG2 cells transfected with two COX-2 shRNA expression vectors decreased to 41.66% and 77.79%,respectively,of that in cells transfected with control scrambled shRNA expression vector.The vector which efficiently suppressed the expression of COX-2 mRNA was transfected into HepG2 cells,and G418 was used to select a population of cells which may stably express the shRNA.The expression of COX-2 mRNA decreased by 75.30% and the cell viability decreased by 29.33% in cells which stably expressed COX-2 shRNA.Flow cytometry analysis showed a higher proportion of apoptotic cells in COX-2 shRNA group than in untransfected and scrambled shRNA groups（17.83% vs 4.47% and 4.63%）.The proportion of cells in G0/G1 phase was 73.93%,57.63% and 61.2%,while in S phase was 13.43%,26.90% and 26.03%,respectively,in COX-2 shRNA group,untransfected and scrambled shRNA groups.Western blotting revealed that the protein levels of COX-2 and Bcl-2 in HepG2 cells which expressed COX-2 shRNA reduced to 31.25%（P0.01） and 54.93%（P0.01） of scrambled shRNA expression vector respectively.Conclusion Recombinant expression vector pSilencer 2.1-U6-COX-2 shRNA may efficiently silence COX-2 gene expression,inhibit cell proliferation,promote apoptosis,arrest cell cycle and decrease the Bcl-2 protein level in human hepatocellular carcinoma cells HepG2.