Zhang Ling, He Tao, Cui Hong, Wang Yunjian, Huang Changshan, Han Feng
Department of Hepatopancreatobiliary Surgery, Henan Tumor Hospital, Zhengzhou, Henan 450008, China.
Discov Med. 2012 Aug;14(75):115-24.
Alpha fetoprotein (AFP) is an oncoembryonal protein that is highly expressed in the majority of hepatocellular carcinomas. Previous studies have shown that AFP may be involved in multiple cell growth regulating, differentiating, and immunosuppressive activities. We investigated the effects of AFP gene silencing by siRNA on apoptosis and proliferation of hepatocellular carcinoma cell line EGHC-9901, which highly expresses AFP and may serve as an ideal model for investigation of AFP functions. siRNA expressing plasmid targeting the AFP gene was first established and subsequently transfected into hepatocellular carcinoma cell line EGHC-9901; cells were then divided into three groups: siRNA-afp, transfected with AFP-siRNA; siRNA-beta-actin, transfected with siRNA-beta-actin as the positive group; and vector control, transfected with empty vector as the blank control group. After G418 positive clone selection for a couple of weeks, Western blot and RT (reverse transcription)-PCR assay demonstrated that AFP expression was almost completely inhibited by siRNA-afp, which indicates that siRNA expressing plasmid targeting the AFP gene has been successfully established. Furthermore, MTT (methyl thiazolyl tetrazelium) assay showed that cells transfected with siRNA-afp proliferated at a significantly lower speed than the other two groups and flat plate clone formation assay also witnessed less clones with diameters of more than 75 μm in siRNA-afp immunofluorescence indicating that the apoptosis rate of cells transfected with siRNA-afp was significantly higher than the other two groups. Furthermore, flow cytometry manifested approximately 20% more cells of siRNA-afp within G1 phase than those of the negative group, indicating that inhibition of AFP expression may cause G1 phase arrest. Finally, Western blot and RT-PCR assay demonstrated that siRNA-afp induced a higher expression of caspase-3 than the other two groups whereas there was no difference in expression of caspase-8, caspase-9, and Bcl-2 between the three groups.
甲胎蛋白（AFP）是一种癌胚蛋白，在大多数肝细胞癌中高度表达。先前的研究表明，AFP可能参与多种细胞生长调节、分化和免疫抑制活动。我们研究了小干扰RNA（siRNA）沉默AFP基因对高表达AFP的肝癌细胞系EGHC-9901凋亡和增殖的影响，该细胞系可作为研究AFP功能的理想模型。首先构建了靶向AFP基因的siRNA表达质粒，随后将其转染至肝癌细胞系EGHC-9901；细胞随后分为三组：siRNA-afp组，转染AFP-siRNA；siRNA-β-肌动蛋白组，转染siRNA-β-肌动蛋白作为阳性对照组；载体对照组，转染空载体作为空白对照组。经过几周的G418阳性克隆筛选，蛋白质免疫印迹法（Western blot）和逆转录聚合酶链反应（RT-PCR）检测表明，siRNA-afp几乎完全抑制了AFP的表达，这表明靶向AFP基因的siRNA表达质粒已成功构建。此外，噻唑蓝（MTT）检测显示，转染siRNA-afp的细胞增殖速度明显低于其他两组，平板克隆形成实验也发现，siRNA-afp免疫荧光中直径大于75μm的克隆较少，表明转染siRNA-afp的细胞凋亡率明显高于其他两组。此外，流式细胞术显示，siRNA-afp组处于G1期的细胞比阴性组多约20%，表明抑制AFP表达可能导致G1期阻滞。最后，蛋白质免疫印迹法和RT-PCR检测表明，siRNA-afp诱导的半胱天冬酶-3（caspase-3）表达高于其他两组，而三组之间半胱天冬酶-8、半胱天冬酶-9和B细胞淋巴瘤-2（Bcl-2）的表达没有差异。
