泛素特异性肽酶22对肝癌细胞凋亡的影响及机制研究

Effect of USP22 on apoptosis of hepatocellular carcinoma cells and its mechanism

目的探讨泛素特异性肽酶22(USP22)对肝癌细胞凋亡的影响及其可能的分子机制。方法采用RT-PCR和Western blotting分别检测人正常肝Chang liver细胞和人肝癌HepG2细胞中USP22 mRNA及蛋白表达。利用脂质体转染USP22-siRNA靶向沉默USP22基因,并采用RT-PCR和Western blotting检测其沉默效果;流式细胞仪检测USP22基因沉默对HepG2细胞周期和细胞凋亡的影响,Western blotting检测USP22基因沉默对HepG2细胞中活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved caspase-3)、活化的含半胱氨酸的天冬氨酸蛋白水解酶9(Cleaved caspase-9)、细胞周期素D1(Cyclin D1)和细胞周期蛋白依赖性激酶2(CDK2)表达的影响。结果 USP22基因在人肝癌HepG2细胞中表达明显高于人正常肝Chang liver细胞(P<0.05);siRNA干扰HepG2细胞后,与对照组相比,干预组中USP22 mRNA和蛋白的表达均显著降低(P<0.05),而阴性组差异无统计学意义(P>0.05)。靶向沉默USP22基因后,与对照组相比,干扰组中G_0/G_1期细胞所占比例显著升高,S期和G_2/M期细胞所占比例显著下降,细胞凋亡率明显升高,差异均有统计学意义(P<0.05);阴性组与对照组间G_0/G_1期、S期、G_2/M期细胞所占比例和细胞凋亡率相比,差异无统计学意义(P>0.05)。与对照组相比,干预组中Cleaved caspase-3、Cleaved caspase-9、Cyclin D1和CDK2蛋白的相对表达量均显著降低(P<0.05),阴性组与对照组间差异无统计学意义(P>0.05)。结论 USP22基因沉默可诱导肝癌HepG2细胞凋亡,其作用机制可能与周期蛋白Cyclin D1、CDK2及凋亡蛋白Cleaved caspase-3、Cleaved caspase-9的表达下降有关。

Objective To investigate the effect of ubiquitin specific peptidase 22（ USP22） on apoptosis of hepatocellular carcinoma cells and its possible molecular mechanism. Methods RT-PCR and Western blotting were used to detect the expression of USP22 mRNA and protein in human normal Chang liver cells and human hepatoma HepG2 cells. The USP22 gene was silenced by transfection of USP22-siRNA with liposome,and the silencing effect was detected by RT-PCR and Western blotting. The effects of USP22 gene silencing on HepG2 cell cycle and apoptosis were investigated by flow cytometry,and Western blotting was used to detect the effect of USP22 gene silencing on the expressions of Cleaved caspase-3,Cleaved caspase-9,Cyclin D1 and CDK2 proteins. Results The expression of USP22 gene in human hepatocellular carcinoma HepG2 cells was significantly higher than that in human normal Chang liver cells（ P 0. 05）. After siRNA interference with HepG2 cells,the expressions of USP22 mRNA and protein in the intervention group were significantly lower than those in the control group（ P 0. 05）,but there was no significant difference in the negative group（ P 0. 05）. After the siRNA interfered with HepG2 cells,compared with the control group,the expressions of USP22 mRNA and protein in the intervention group were significantly decreased（ P 0. 05）,but the difference in the negative group was not significant（ P 0. 05）. After silence of the USP22 gene,compared with the control group,the proportion of G_0/G_1 phase cells was significantly increased,the proportions of S phase and G_2/M phase cells were decreased significantly,and the rate of apoptosis was increased significantly,whose differences were statistically significant（ P 0. 05）; but there was no significant difference in the percentage of G_0/G_1 phase,phase S and phase G_2/M cells and apoptosis rate between the negative group and the control group（ P 0. 05）. Compared with the control group,the relative expressions of Cleaved caspase-3,Cleaved caspase-9,Cyclin D1 and CDK2 proteins in the intervention group were significantly reduced（ P 0. 05）,and the difference between the negative group and the control group was not significant（ P 0. 05）. Conclusion The gene silencing of USP22 can induce apoptosis of HepG2 cells in hepatocellular carcinoma,and its mechanism may be related to the decline in expression of cell cycle correlated proteins Cyclin D1,CDK2,and apoptotic proteins Cleaved caspase-3 and Cleaved caspase-9.