小干扰RNA抑制核因子-κB活化对HepG2细胞凋亡的影响

Effect of siRN A-mediated inhibition of nuclear transcription factor-kappa B on apoptosis of hepatocarcinoma cells

目的 探讨小干扰RNA（SiRNA）抑制核转录因子（NF）-κB活化对肝癌细胞凋亡的影响.方法 化学合成NF-κB siRNA和阴性对照siRNA,脂质体法转染HepG2细胞,用巢式RT-PCR和荧光定量PCR检测NF-κB mRNA表达情况;免疫组织化学法、酶联免疫吸附法、Western b10t检测NF-κB蛋白表达情况;用磷脂结合蛋白V-异硫氰酸荧光素法检测细胞凋亡,分析NF-κB表达抑制和细胞凋亡间关系.多个样本均数间的比较先行方差齐性检验,方差齐时行单因素方差分析;计数资料比较采用确切概率法分析.结果 NF-κBp65 mRNA在HepG2细胞相对表达量为1.13±0.03,在正常肝细胞L02为0.29±0.07,两者比较,t=27.02,P〈0.05,差异有统计学意义.利用NF-κB siRNA干扰可下调NF-κB表达,且呈剂量、时间依赖;NF-κB siRNA转染HepG2细胞72h后,NF-κBmRNA和蛋白表达分别下降了93%和62%,抑制NF-κB表达使HepG2细胞凋亡增加85%. 结论 NF-κB在肝癌细胞中高表达,NF-κB SiRNA能特异性抑制其在肝癌细胞中活化并促进癌细胞凋亡发生.

Objective To investigate the effect of siRNA-mediated inhibition ofNF-κB on apoptosis of hepatocarcinoma cells. Methods Specific small interfering RNA Targeting NF-κB gene was synthesized and transfected into HepG2 cells by liposomes. Nested RT-PCR and quantitative RT-PCR were used to dectect the mRNA expression of NF-κB. Immunohistochemistry, enzyme-linked immunosorbent assay and Western blot were performed to examine the protein expression of NF-κB.Annexin V-FITC was used to test cell apoptosis. Results The expressin of NF-κB in HepG2 cells （1.13±0.03）was significantly higher（t=27.02, P〈0.05） than that in normal hepatocytes （0.29±0.07）.The down-regulation of NF-κB expression was depended on the dosage of siRNA and the time after transfection. 72 h after siRNA transfection,NF-κB expression reduced by 93% and 62% at the mRNA and protein levels, respectively. The apoptosis of HepG2 cells increased by 85% with NF-κB inhibition. Conclusions NF-κB is abnomally active in HepG2 cells and NF-κB inhibition mediated by siRNA promotes HepG2 cells apoptosis. It suggested that NF-κB could be a potential target for HCC prevention gene therapy.