敲低EN2诱导肝癌细胞凋亡并提高PTEN蛋白的表达

目的探讨EN2对肝癌细胞凋亡及细胞中PTEN蛋白表达的影响。方法用EN2 siRNA慢病毒感染肝癌细胞HuH-7,qRT-PCR和Western blot方法检测干扰效果。MTT方法测定细胞活力变化,PI单染法测定细胞周期分布,Annexin V-FITC/PI双染法测定细胞凋亡变化,Western blot测定细胞中激活型Caspase-3(Cleaved Caspase-3)、激活型Caspase-9(Cleaved Caspase-9)、第10号染色体同源缺失性磷酸酶-张力蛋白(PTEN)及细胞周期素B 1(Cyclin B 1)蛋白水平,JC-1法测定线粒体膜电位变化,Western blot测定胞浆中细胞色素C(Cytochrome C)蛋白水平。结果 EN2 siRNA慢病毒感染可明显下调肝癌细胞中EN2的表达。沉默EN2表达后的肝癌细胞活力降低,细胞凋亡率升高,细胞G2/M期比例升高,细胞中Cleaved Caspase-3、Cleaved Caspase-9、PTEN蛋白水平明显升高,Cyclin B1蛋白水平明显降低,线粒体膜电位下降,胞质中Cytochrome C蛋白水平升高。结论敲低EN2可以阻滞肝癌细胞周期,诱导肝癌细胞凋亡,其作用机制可能与PTEN、线粒体凋亡途径有关。

Knockdown of EN2 Induces Apoptosis and Enhances PTEN Protein Expression in Hepatocellular Carcinoma Cells

Objective To investigate the effect of EN2 on the apoptosis and PTEN protein expression in hepatocellular carcinoma cells. Methods Hepatoma cells HuH-7 was infected with EN2 siRNA lentivirus. qRT-PCR and Western blot methods were used to detect the interference effect. The changes of cell vitality was measured by MTT method, PI single staining method was used to determine cell cycle distribution, the apoptosis was measured by Annexin V-FITC/PI double staining, Western blot was used to measure the levels of Cleaved Caspase-3, Cleaved Caspase-9, PTEN and Cyclin B1 protein, the changes of mitochondrial membrane potential was measured by JC-1 method, and Western blot was used to measure the level of Cytochrome C protein in the cytoplasm. Results EN2 siRNA lentivirus infection could significantly reduce the expression of EN2 in hepatocellular carcinoma cells. After EN silence, the activity of hepatoma cells was decreased, the rate of apoptosis was increased, the proportion of G2/M phase in cells was increased, the levels of Cleaved Caspase-3, Cleaved Caspase-9 and PTEN protein were increased significantly, the level of Cyclin B1 protein was decreased significantly, the mitochondrial membrane potential was decreased, and the level of Cytochrome C protein in the cytoplasm was increased. Conclusion Knockdown of EN2 could block the cell cycle of liver cancer and induce the apoptosis of hepatoma cells, and the mechanism may be related to PTEN and mitochondrial apoptotic pathway.