Localization Of Swine Protein Akirin2 And Its Impact On IL-1β And IL-6 MRNA Expression

Akirin gene got involved in innate immune response of mammalian and insect. In drosophila, expression of antimicrobial peptides (like Attacin, Cecropin and Diptericin) was decreased after RNA interference against Akirin, and RNAi-mediated knock-down of Akirin in whole flies led to enhanced sensitivity to Gram-negative bacterial infection. Two homologs of the Akirin gene were present in mouse: Akirin1 and Akirin2, and Akirin2 got involved in mouse innate immune system. In Akirin knock-down mouse embryonic fibroblasts(Akirin2-/-MEFs), Akirin2 participated in NF-κB pathway. When Akirin2-/- MEFs were stimulated with TLR ligands( lipopolysaccharide LPS), IL-1βand TNF, respectively, production of IL-6 was severely impaired compared to that of control group. In response to these stimuli, neither degradation of IκBa nor induction of NF-κB DNA was found. These data indicated that mouse Akirin2 acted together with or downstream of NF-κB, and located in nucleus. Subcellular localization showed that Akirin had a strict localization in nucleus. On this basis, in order to investigate whether porcine Akirin2 can affect the expression of IL-1βand IL-6, and the expression of NF-κB in NF-κB pathway, this research conducted following experiments, and the results are as follows:.1. pEGFP-Akirin2 vector was transfected into swine umbilicus veins endothelial cells (SUVECs), and positive clone was selected by G418. MTT results showed that over-expression of Akirin2 gene did not affect the growth of SUVECs .2. Subcellular localization of Akirin2 protein in SUVECs was analysed by using laser confocal microsopy, and the results showed that this protein was localized in the cell nucleus.3. LPS was used to stimulate SUVECs that transfected with pEGFP-Akirin2, and the mRNA expression of IL-1βand IL-6 was analyzed by using quantative real-time PCR post 12h and 24h. In response to LPS stimulation, the mRNA expression of IL-1βand IL-6 was increased dramatically. This indicated that Akirin2 may upregulate IL-1βand IL-6 mRNA expression. NF-κB mRNA expression was analyzed by using the same method, and the result showed that mRNA expression of NF-κB did not be affected. These data indicated that pocine Akirin2 acted together with or downstream of NF-κB.