RNA干扰介导的核转录因子-κB抑制蛋白激酶α和γ基因沉默对核转录因子-κB信号通路调节作用的影响

Influence of inhibitory κB protein kinase α and γ genes silencing induced by RNA interference on nuclear factor-κB signal pathway

目的观察RNA干扰介导的核转录因子-κB抑制蛋白（IκB）激酶（IKK）α和γ基因沉默对核转录因子-κB（NF—κB）信号通路的调节作用，进一步阐明其基因调控机制。方法设计IKKα及IKKγ靶向的小分子干扰RNA（siRNA），合成互补的寡核苷酸链，转染到RAW264．7小鼠巨噬细胞。观察经脂多糖（LPS）刺激后NF-κB的活化、IKKα及IKKγ基因表达的变化、NF—κB p65及p50核移位的变化以及前体蛋白NF—κB p105的表达情况。结果IKKα及IKKγ基因沉默后，可导致IKKα及IKKγ基因表达出现明显下调，同时NF-κBp65及p50的核移位受到抑制，胞质、胞核中NF-κB p65、p50及p105的表达显著下调，而且能抑制NF—κBp65、p50及p105蛋白的核移位。结论IKKα及IKKγ两种蛋白激酶参与NF—κB信号通路的调节，不仅能分别在抑制蛋白的泛肽化、组蛋白磷酸化等环节发挥作用，而且还能够通过相互之间的协同作用，弥补其中某种蛋白受到过度抑制时所引起的炎症信号通路的功能障碍。

Objective To investigate the role of inhibitory κB （κB） protein kinase （IKK） α and γ genes silencing induced by RNA interference （RNAi） in modulation of nuclear factor-κB （NF-κB） signal pathway, to elucidate further the regulatory mechanism of NF-κB gene. Methods IKKα and IKKγ argeting small interference RNA （siRNA） were designed to synthesize complementary oligonucleotide chains, then it was transfected into mouse macrophage cell line RAW264.7. The transfected cells were treated with lipopolysaceharide （LPS, 10 μg/ml）, then the expression of IKKα and IKKγ genes, the nuclear translocation changes in NF-κB p65 and p50 proteins, and the expression of precursor protein NF-κB p105 were detected at selected time points. Results It was shown that RNAi targeting IKKα and IKKγ could down-regulate the expression of IKKα and IKKγ genes, and at the same time, down-regulate the expression of NF-κB p65, p50 and p105 proteins both in cytoplasm and nucleus, and inhibit the nuclear translocation of NF-κB p65, p50 and p105 proteins. Conclusion These findings provide evidence for the involvement of the two protein kinases IKKα and IKKγ in the modulation of NF-κB signal pathway, not only they can inhibit the ubiquitin of protein and histone phosphorylation respectively, but also can redeem the functional impairment of the inflammation signal pathway induced by over inhibition of some proteins, through synergistic action of the two kinases.