靶向livin的小分子干扰RNA联合表阿霉素对乳腺癌细胞ZR-75-30凋亡的影响

RNAi-mediated gene silencing of livin synergistic with epirubicin enhance apoptosis of human breast cancer cells

目的利用RNA干扰技术阻断livin基因的表达，观察其联合表阿霉素对乳腺癌细胞ZR-75-30凋亡的作用。方法体外化学合成靶向livin的小分子干扰RNA（siRNA），在脂质体（1ipofectamine^TM2000）的介导下转染人乳腺癌细胞ZR-75-30。荧光共聚焦显微镜下观察转染效率，半定量RT-PCR和蛋白质印迹法检测转染前后ZR-75-30细胞livin基因表达的变化，流式细胞术检测转染靶向livin的iRNA联合表阿霉素作用的ZR-75-30细胞的凋亡率。结果靶向livin的siRNA可以有效特异地抑制ZR-75-30细胞livin基因的表达，在mRNA水平上其表达抑制率约为53．66％；在蛋白质水平其表达抑制率约为58．32％，转染靶向livin的siRNA36h后，靶向livin的siRNA联合表阿霉素可以诱导（15．18±0．05）％的细胞凋亡。结论靶向livin的siRNA能有效、特异地阻断livin基因的表达，阻断livin基因表达联合表阿霉素可促进ZR-75-30细胞的凋亡。

Objective To observe the effects of silencing of livin gene expression combined with anthracycline chemotherapy on the apoptosis of human breast cancer cells. Methods Double stranded RNA （dsRNA） targeting the livin gene was chemically synthesized in vitro and transfected into human breast cancer cells of the line ZR-75-30 mediated by lipofectamne^TM2000. The transfection efficiency was observed by fluorescence confocal microscopy. Reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting were used to detect the expression of livin at mRNA and protein levels. ZR-75-30 cells transfected with dsRNA targeting livin for 24 h were treated with 50 μg/ml epirubicin for 12 h, flow cytometry was used to detect the apoptosis rate of the cells. Results The livin mRNA expression rates of the livin-siRNA transfected group was （29.68±2.7 ） %, with an inhibitory rate of 53.66%, significantly lower than those of the negative control and blank control groups [（52.01±2. 9 ）% and （51.95± 3.1）% respectively, both P 〈 0.01 ]. The livin protein expression rate of the livin-siRNA group was （27.80±2.1） %, significantly lower than those of the negative control siRNA group and blank control group [（53.80±3.0） % and （55.12±2.8 ） % respectively, both P 〈 0.01 ]. 36 h after the treatment of siRNA against livin combined with epirubicin the apoptosis rate was （ 15.18± 0.05 ） %, significantly higher than those of the negative control group and blank control group [ （2.78± 0.08）% and （2.65± 0. 12）% respectively, both P 〈 0. 01 ]. Conclusions Sequence specific siRNA targeting livin synergistic with epirubicin is capable of enhancing the apoptosis rate of human breast cancer cells. Silencing of livin gene expression with siRNA combined with anthracycline chemotherapy may hold great promise as a novel therapy for livin expressing breast cancer.