RNA干扰Livin联合顺铂对食管癌细胞Eca-109增殖和凋亡的影响

摘要

Objective To explore the influence of RNA interference(RNAi) targeting Livin combined with cisplatin treatment on proliferation and apoptosis of esophageal cancer Eca-109 cells and its related mechanism. Methods shRNA-Livin system was constructed using lentiviral vector, which was subsequently transfected into Eca-109 cells. Eca-109 cells were divided into 5 groups:normal control group, negative control group, RNAi group, cisplatin group and combination group (RNAi combined with cisplatin treatment). Spectrophotometry, 2 , 3-bis (2-methoxy-4-nitro-5-sulfophenyl) 5 [(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) colormetric assay, flow cytometry (FCM), terminal deoxynucleotidyl transferase-mediated Dutp nick end labeling (TUNED and transmission electron microscopy (TEM) were employed to detect the expression of cysteinyl aspartate specific pro-tease(Caspase)-3 and Caspase-9 ,cellular proliferation,cell cycle,apoptosis and morphological changes in Eca-109 cells,respectively. Results After transfection,Caspase-3 and Caspase-9 expression in Eca-9 cells were obviously increased (P<0. 01), while cellular survival rate was markedly decreased (P<0. 01). Eca-109 cells showed a main S phase arrest and significantly increased apoptotic rate after RNAi. Typical apoptotic cells were observed by TEM. Compared to cells in cisplatin group,cellular features above in combination group were particularly obvious. Conclusion RNAi targeting Livin combined with cisplatin treatment can inhibit proliferation and induce apoptosis of Eca-109 cells via up-regulating Caspase-3 and Caspase-9 expression.%目的 探讨RNA干扰Livin联合顺铂对食管癌细胞Eca-109增殖和凋亡的影响及相关作用机制.方法 利用慢病毒载体构建shRNA-Livin系统并转染至食管癌细胞Eca-109,将Eca-109细胞分为5组:正常对照组、阴性对照组、干扰组、顺铂组、联合组(干扰+顺铂).采用分光光度法、二甲氧唑黄比色法(XTT)、流式细胞术(FCM)、原位末端转移酶标记技术(TUNEL)、透射电镜(TEM) 分别检测食管癌细胞的半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)、Caspase-9表达,细胞增殖、细胞周期、凋亡及形态学改变情况.结果 转染后,Eca-109 细胞Caspase-3、Caspase-9表达明显增强(P<0.01),细胞存活率明显降低(P<0.01);经RNA干扰后,细胞周期主要被阻滞于S期,细胞凋亡率明显增加;TEM下可见典型的凋亡细胞.与顺铂组比较,联合组细胞的上述表现尤为明显.结论 Livin靶向RNA干扰联合顺铂通过上调Caspase-3、Caspase-9的表达从而抑制 Eca-109 细胞的增殖和促进其凋亡.