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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 investigate the effect of siRNA targeting survivin on the chemosensitivity of hepatocellular carcinoma cells. Methods siRNA eukaryotic expression vector was constructed and stably transfected into HepG2 hepatocellular carcinoma cells. The effect of mitomycin C (MMC) on the tumor cells after transfection was observed. Results sequencing confirmed that siRNA eukaryotic expression vector was constructed successfully. The results of RT-PCR showed that siRNA inhibited survivin gene expression at mRNA level by 73%. The survival rate of HepG2 and HepG2 / Silence (-) cells treated with siRNA was measured by MTT assay. The survival rate of MMC group was significantly lower than that of control group (0.824 ± 0.322) (P <0.05). When survivin expression was effectively inhibited, the survival rate of hepatocellular carcinoma cells (0.520 ± 0.017) at 12 h was significantly lower than that of the control group (0.741 ± 0.005), and peaked at 48 h (P <0.05). Western blotting showed that the OD value of survivin protein expression without MMC was 34 273 ± 323, and the OD value of survivin protein expression decreased significantly after adding MMC for 24, 48 and 48 h (21 415 ± 142, 16 771 ± 122 and 13 672 ± 133), all with statistical difference (all P <0.05). Flow cytometry showed that the apoptotic rates of HepG2 / Silence (+) plus MMC group at 12, 24 and 48 h were significantly different from that of the control group. Caspase-3 activity assay showed that Caspase-3 activity of HepG2 / Silence (+) cells were 0.19 ± 0.05, 0.33 ± 0.12, 3.79 ± 0.27 and 9.34 ± 0.86 at 0, 12, 24, The difference was significant (all P <0.05). Conclusion siRNA targeting survivin not only effectively inhibits the expression of survivin in hepatocellular carcinoma cells, but also enhances the sensitivity of cells to MMC. Its mechanism is through increasing the activity of caspase-3 and increasing the apoptosis of hepatoma cells.