siRNA沉默AFP基因对肝癌细胞系EGHC-9901凋亡的影响

Effects of AFP gene silenced by siRNA on apoptosis of hepatocellular carcinoma cell line EGHC-9901

目的建立稳定表达针对AFP基因siRNA质粒的肝癌细胞模型并探讨对其凋亡的影响。方法构建针对AFP基因的siRNAs表达质粒,脂质体法将该质粒转染肝癌细胞系EGHC-9901,G418筛选4~5周后Western blot及RT-PCR检测靶基因抑制效果,细胞分组：实验组（siRNA-afp）,转染AFP-siRNA质粒组;载体对照组,转染空载体组;空白对照组,未做任何处理组。免疫荧光检测细胞在无血清状态下凋亡情况,Western blot及RT-PCR检测Caspase-3、Caspase-8等凋亡相关蛋白表达。结果在体外成功构建针对AFP的siRNA表达质粒并在体外建立稳定表达该质粒肝癌细胞系EGHC-9901,转染后细胞表达AFP近乎完全抑制;免疫荧光表明促进细胞在无血清状态下的实验组凋亡率为32%±4%,对照组凋亡率为17%±3%,差异有统计学意义（P〈0.05）;RT-PCR及Western blot检测凋亡相关蛋白表明实验组Caspase-3表达较对照组高,差异有统计学意义（P〈0.05）,而Caspase-8、Caspase-9、bcl-2表达无显著差异（P〉0.05）。结论在体外成功建立稳定表达针对AFP基因的siRNA肝癌细胞系,抑制AFP表达可能通过上调Caspase-3表达促进其凋亡。

Objective To observe effects of AFP gene silenced by siRNA on apoptosis of hepatocellular carcinoma cell line EGHC-9901. Methods siRNA expressing plasmid aimed at AFP gene was firstly established and subsequently transfected into hepatocellular carcinoma cell line EGHC-9901 which had the high expression of AFP;after G418 positive clone selection for 4～5 weeks,expression of AFP mRNA and protein were respectively detected by semi-quantitative RT-PCR and Western blot.Then cells were divided into three groups： experimental group,AFP-siRNA transfected;positive control group,empty vector transfected;blank control group,untreated.Immunofluorescence was used to detect cell apoptosis in serum-free culture medium;RT-PCR and Western blot were applied again to detect expression of apoptosis-related proteins such as Caspase-3,Caspase-8. Results siRNA expressing plasmid aimed at AFP gene was successfully established and transfected into hepatocellular carcinoma cell line EGHC-9901;expression of AFP was almost completely inhibited.Immunofluorescence assay indicated that apoptosis rate of the experimental group（32%±4%） was significantly higher than that of positive control group（17%±3%）（P0.05）;Western blot and RT-PCR demonstrated that the expression of Caspase-3 was higher in experimental group than that in the control group （P0.05）,whereas there was no obvious difference in the expression of Caspase-8, Caspase-9,bcl-2 among the three groups（P0.05）. Conclusion siRNA expressing plasmid aimed at AFP gene was successfully established and stably transfected into hepatocellular carcinoma cell line EGHC-9901;AFP gene silenced by siRNA induces apoptosis,which may attribute to up-regulation of Caspase-3.