Livin基因反义寡核苷酸诱导肺腺癌A549细胞的凋亡

Antisense oligodeoxynucleotides of Livin mRNA induced the apoptosis in human lung adenocarcinoma A549 cells

目的:探讨Livin基因反义寡核苷酸(antisense oligodeoxynucleotides,ASODN)对肺腺癌细胞A549增殖和凋亡的影响。方法:用阳离子脂质体介导LivinASODN转染A549细胞后,MTT法检测对A549细胞增殖抑制率的影响;RT-PCR法和细胞免疫荧光化学检测LivinASODN转染前后,A549细胞中Livin mRNA和蛋白表达的变化;吖啶橙/溴乙锭(acridine orange/ethidium bromide,AO/EB)复合染色检测A549细胞的凋亡率。结果:在A549细胞中同时有Livinα和Livinβ的表达,Livin蛋白在细胞质和细胞核中均有分布。Livin基因ASODN可显著抑制A549细胞的增殖(P<0.01),且作用具有剂量依赖性。终浓度为400nmol/L的Lip-ASODN转染后,A549细胞Livin mRNA和Livin蛋白的表达均被明显抑制,细胞凋亡率达(31.25±5.75)%,与对照组(3.23±1.98)%相比差异有统计学意义(P<0.01)。结论:LivinASODN可下调Livin基因表达,有效抑制A549细胞的增殖,并促进A549细胞凋亡。因此,Livin基因有望成为肺癌基因治疗的新靶点。

Objective： To investigate the effect of antisense oligodeoxynucleotide （ASODN） of Livin mRNA on the proliferation and apoptosis of human lung adenocarcinoma A549 cells. Methods： Livin ASODN was transfected into A549 cells mediated by cationic liposome. The proliferation rates of A549 cells were assessed by MTT method. The transcription of Livin mRNA was detected by RT- PCR. The Livin protein expression was determined by immunohistochemistry and confocal laser scanning microscopy before and after transfection. The apoptotic ratios of A549 cells were examined by acridine orange/ethidium bromide （ AO/EB ） fluorescent staining. Results： It was observed that A549 cells expressed both Livin α and Livin β simultaneously, and the distribution of Livin protein could be observed in both cytoplasm and nuclei. The proliferation of A549 cells was inhibited significantly by Livin ASODN in a dose-dependent manner （P 〈 0.01 ）. After transfection of Lip-ASODN at a final concentration of 400 nmol/L, the expressions of Livin were inhibited at mRNA and protein levels and the apoptotic ratio reached （ 31.25 ± 5.75 ） %. The difference was significant compared with control group [ （3.23% ± 1.98 ）% , P 〈0.01 ]. Conclusion： Livin ASODN significantly down-regulates the expression of Livin mRNA and effectively inhibites the proliferation and induces the apoptosis of A549 cells. Therefore Livin gene may become a new target for gene therapy for lung cancer.