Livin反义寡核苷酸诱导人肾癌细胞凋亡及其机制的研究

Antisense oligonucleotide targeting livin induces apoptosis of human renal carcinoma cell 786-O in vitro and its mechanism

目的探讨Livin反义寡核苷酸(ASODN)对人肾癌细胞株786-O Livin mRNA及蛋白表达的抑制作用以及对细胞凋亡的影响。方法设计合成全硫代磷酸化修饰的Livin ASODN,脂质体辅助转染至786-O细胞内。采用RT-PCR检测转染后Livin mRNA的变化,细胞免疫化学、激光扫描共聚焦显微镜观察Livin蛋白在细胞内的分布、转染后表达的变化及细胞形态的变化,流式细胞仪检测细胞凋亡率的变化,激酶法测定Caspase3活性的变化。结果转染Livin ASODN后,RT-PCR显示786-O细胞表达Livin mRNA减少(P<0.01),激光扫描共聚焦显微镜下见细胞表达Livin蛋白减少、细胞形态发生变化,流式细胞仪检测细胞凋亡率显著增加(P<0.01),Caspase3活性明显增加(P<0.01)。结论Livin ASODN能明显下调786-O细胞Livin基因的表达,诱导细胞凋亡。抑制Caspase3活性是Livin抑制细胞凋亡的途径之一。

Objective To investigate the effect of antisense oligonucleotides （ASODN） targeting livin on the inhibition of livin mRNA and protein expression and the apoptosis of human renal carcinoma cell line 786-0 cells. Methods Specific phosphorothioate antisense oligodeoxynucleotides targeting livin were synthesized and then transfected into 786-0 cells. The expressions of livin mRNA were detected by RT-PCR. Expression and location of livin protein were observed by confocal laser scanning microscope （CLSM）. Apoptosis rate of 786-O cells was investigated by flow cytometer. The activity of Caspase-3 was detected by colorimetric assay. Results After the transfection of ASODN, the expression of livin mRNA was decreased （ P 〈 0.01 ）, the fluorescence signal of livin protein observed by CLSM was significantly decreased, the apoptosis rates detected by flow cytometry were significantly increased （P 〈 0.01 ）, and the caspase-3 activity was enhanced （P 〈 0.01 ）. Conclu- sion Livin ASODN could remarkably down-regulate the expression of livin gene in 786-O cells line and induce tumor cell apoptosis. These events are linked to the activation of caspase-3.