Objective: To investigate the role of inhibitory KappaB (IKappaB) protein kinase (IKK) alpha and gamma genes silencing induced by RNA interference (RNAi) in modulation of nuclear factor-KappaB (NF-KappaB) signal pathway, to elucidate further the regulatory mechanism of NF-KappaB gene.
Methods: IKKalpha and IKKgamma 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 microg/ml), then the expression of IKKalpha and IKKgamma genes, the nuclear translocation changes in NF-KappaB p65 and p50 proteins, and the expression of precursor protein NF-KappaB p105 were detected at selected time points.
Results: It was shown that RNAi targeting IKKalpha and IKKgamma could down-regulate the expression of IKKalpha and IKKgamma genes, and at the same time, down-regulate the expression of NF-KappaB p65, p50 and p105 proteins both in cytoplasm and nucleus, and inhibit the nuclear translocation of NF-KappaB p65, p50 and p105 proteins.
Conclusion: These findings provide evidence for the involvement of the two protein kinases IKKalpha and IKKgamma in the modulation of NF-KappaB 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.