Survivin-ASODN对肝癌SMMC-7721细胞凋亡的促进作用及其机制

Promotion effects of survivin-ASODN on apoptosis of SMMC-7221 cell and its mechanism

目的:应用反义寡核苷酸(ASODN)技术靶向抑制生存素(survivin)基因,研究其对肝癌SMMC-7721细胞凋亡的影响,阐明survivin反义寡核苷酸(survivin-ASODN)促进SMMC-7721细胞凋亡的作用机制。方法:设计合成survivin-ASODN序列(FAM荧光素标记)。利用脂质体介导法分别用浓度为100、200、300、400和600nmol·L-1 survivin-ASODN转染SMMC-7721细胞(ASODN转染组),并设空白对照组、空脂质体对照组和正义寡核苷酸(SODN)对照组。应用流式细胞术(FCM)检测不同浓度survivin-ASODN体外转染SMMC-7721细胞24、48和72h后SMMC-7721细胞凋亡率及细胞周期;Western blotting法检测survivin蛋白表达水平。结果:与各对照组比较,荧光素标记的ASODN转染SMMC-7721细胞24h后,细胞生长开始受到抑制,细胞凋亡率增加,呈现剂量-时间依赖性(P<0.05);作用48h后,ASODN转染组细胞在G1期前出现明显的亚二倍体凋亡峰,G0/G1期细胞明显减少(P<0.05),G2/M期细胞显著增加(P<0.05),细胞被阻滞于G2/M期。与各对照组比较,不同浓度ASODN转染组SMMC-7721细胞survivin蛋白表达水平下降(P<0.05),且随作用时间的延长而降低(P<0.05)。结论:survivin-ASODN通过抑制survivin蛋白表达、改变细胞周期进程等机制促进SMMC-7721细胞凋亡,并且呈现时间-剂量依赖性。

Objective To study the influence of survivin targetedly inhibited with antisense oligonucleotide（ASODN）technique on the apoptosis of hepatoma carcinoma cells SMMC-7221,and to clarify the mechanism of promotion effect of survivin-ASODN on the apoptosis of SMMC-7721 cells.Methods The ASODN sequence of survivin marked by FAM fluorescein was designed and synthized.The SMMC-7721 cells were transfected by different concentrations（100,200,300,400,and 600nmol·L-1）of survivin-ASODN（ASODN transfection groups）,at the same time blank control group and blank liposome control group and sense oligonucleotide（SODN）control group were set up.The apoptotic rates and the changes of cell cycle of the SMMC-7721cells 24,48,and 72h after transfected with different concentrations of survivin-ASODN were detected by FCM.The expression levels of survivin were measured by Western blotting method.Results Compared with each control group,24h after transfection,the apoptotic rates of survivin-ASODN transfected SMMC-7221 cells were increased,the growth of cells was inhibited（P〈0.05）,and the effects had time-dose dependent tendency.48h after transfection,the hypodiploid apoptotic peak appeared in ASODN transfection groups before G1 phase,the number of the cells at G0/G1 phase was decreased（P〈0.05）and the number of the cells at G2/M phase wsa increased（P〈0.05）.Compared with each control group,the survivin expression levels in the SMMC-7721cells in ASODN transfection groups were decreased（P〈0.05）,and the effects of survivin-ASODN was time-dose dependent（P〈0.05）.Conclusion Survivin-ASODN can block the expression of survivin in SMMC-7721 cells and inhibit the proliferation of SMMC-7721cells by changing the cell cycle and increasing apoptosis in a time-dose dependent manner.