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

Abstract

Objective To investigate the role of inhibitory κB (IκB) 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 targeting 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 lipopolysaccharide (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. Key words: gene silencing; RNA interference; nuclear factor-κB; inhibitory κB kinase; lipopolysaccharide; macrophage