Studies On The Role Of KCTD9and SHB In The Transduction Pathway Of NK Cell Activation

【Background and objective】Although multiple factors have been implicated in liver damage, it is generallyaceepted that immune-mediated machanisms play a critical role in HBV pathogenesis.Our previous studies have demonstrated that the activation of hepatic NK cellssignificantly contributed to virus-induced hepatocyte injury. by microarray analysis,potassium channel tetramerisation domain containing9(KCTD9) was found to be oneof the greatest upregulated proteins in PBMCs from patients with severe viralhepatitis B compared with patients withmild or moderate hepatitis. Then we foundthat KCTD9was highly expressed both in peripheral and hepatic NK cells inHBV-ACLF patients and positively correlated with the severity of liver injury. In vitro,NK92cells over-expressing KCTD9exhibited a marked increase in activation andcytotoxicity. By bioinformatics analysis, we found that there was a great positivefunctional correlation between KCTD9and adapter protein SHB which was known to be associated with IL-2receptor and PI3K pathway. According to these studies, weproposed that thers is a direct interaction between KCTD9and adapter protein SHB inNK cell activation. In this study, we constructed human KCTD9gene and SHB geneeukaryotic expression tagged vector. Then we validated whether there is an interactionbetween KCTD9and SHB in vitro【Methods】We constructed expression plasmid pHIS-hKCTD9and the pFLAG-hSHB byPCR (Polymerase Chain Reaction). The sequence of human KCTD9and SHB wereconfirmed by further sequencing. The pHIS-hKCTD9and the pFLAG-hSHBexpression plasmids were co-transfected into HEK293T cells, jurkat cells and NK92cells. And then we used co-immunoprecipitation to validate the interaction betweenKCTD9and SHB.【Results】The pHIS-hKCTD9and the pFLAG-hSHB expression plasmids weresuccessfully constructed and co-transfected into HEK293T cells, jurkat cells andNK92cells. Then we found that there was no interaction between KCTD9and SHBby using co-immunoprecipitation.【Conclusion】The preliminary data showed that there was no interaction between KCTD9andSHB. We have to screen another protein which can interact with KCTD9directly andenventually leading to the activation of NK cells.