靶向survivin的siRNA诱导肝癌细胞化疗敏感性的研究

Study of siRNA targeting survivin on inducing sensitization of heaptocarcinoma cells to chemotherapy

目的探讨靶向survivin的siRNA对肝癌细胞化疗敏感性的影响。方法构建siRNA真核表达载体,稳定转染肝癌细胞HepG2后观察其对丝裂霉素(MMC)作用的转基因后肿瘤细胞的效应。结果测序证实siRNA真核表达载体构建成功。RT-PCR检测到siRNA在mRNA水平抑制survivin基因表达率达73%。MTT法观测siRNA作用下MMC对HepG2,HepG2/Silence(-)细胞的存活率,结果显示,仅在48 h时,加MMC组细胞的存活率(0.505±0.015)显著低于对照组(0.824±0.322)(P<0.05)。当survivin的表达被有效抑制时,肝癌细胞存活率(0.520±0.017)在12 h点显著低于对照组(0.741±0.005),且48 h点达到高峰(P<0.05)。免疫印迹法显示,未加MMC前survivin蛋白表达的OD值为34 273±323,加入MMC12,24,48 h survivin蛋白表达的OD值均显著降低(分别为21 415±142,16 771±122和13 672±133),均有统计学差异(均P<0.05)。流式技术检测HepG2/Silence(+)加MMC组12,24,48 h细胞凋亡率与对照组比较,差异有统计学意义。Caspase-3活性检测显示HepG2/Silence(+)细胞在MMC作用下0,12,24,48 h Caspase-3活性分别为0.19±0.05,0.33±0.12,3.79±0.27和9.34±0.86;各时点间差异具显著性(均P<0.05)。结论靶向survivin的siRNA不仅有效抑制肝癌细胞中survivin的表达,而且增强了细胞对MMC的敏感性,其作用机制系通过增强细胞内caspase-3活性,增加肝癌细胞的凋亡而实现的。

Objective To study the influence of siRNA targeting survivin on chemotherapy sensitivity of HCC cells.Methods siRNA eukaryotic expression vector was generated.After the vector stablely transfected into HepG2 cells,the effects of chemotherapy drugs on HCC cells were observed.Results The recombinant plasmid Psilence（＋）-survivin was successfully constructed.Survivin mRNA expression inhibition ratio reached 73% detected by RT-PCR.MTT methods detected that siRNA treated HCC cells were affected by MCC,the survival rate of HepG2,HepG2/Silence（-） cells was inhibited only at 48 h（0.505±0.015） compared to control groups untreated with MCC（0.824±0.322）（P〈0.05）.When the survivin gene was inhibited,the survival rate of the HepG2/Silence（＋） cells（0.520±0.017）was inhibited at 12 h compared to control groups（0.741±0.005） and reached peak at 48 h（P〈0.05）.The Westen-blot detection indicated that OD value of survivin protein expression in untreated with MMC was 3 4273±323 and decreased to 2 1415±142,1 6771±122,1 3672±133 at 12 h,24 h and 48 h after treated with MMC,the difference was significant（P〈0.05）.The apoptosis ratio of HepG2/Silence（＋）＋MMC groups at 12 h,24 h and 48 h increased significantly compared to control groups.The caspase-3 activity detection indicated that the caspase-3 activity in HepG2/Silence（＋） cells treated with MMC at 12h,24h and 48 h was 0.19±0.05,0.33±0.12,3.79±0.27,9.34±0.86 respectively,and the difference between the all time points were significant（all P〈0.05）.Conclusions The siRNA targeting survivin can not only suppress the expression of survivin in HCC cells,but can also enhance the chemotherapy sensitivity.This effect is accomplished by enhancing the caspase-3 activity and increasing the apoptosis ratio in HCC cells.