Study On GK-Nur77Interaction And Its Regulation Of Lipid Metabolism In Liver

Interaction between GK and Nur77had been found in our previous research.Recent studies show that GK and Nur77play important role in liver lipid metabolism,so GK may regulate lipid metabolism through protein-protein interaction with Nur77.In this study, we research protein-protein interaction between GK and Nur77, revealthe function in liver lipid metabolism, provide ideas for pathogenesis and therapy ofdiseases about lipid metabolism. And to establish a basis for animal experiments, weconstruct a lentiviral vector for GK expression and another for GK RNA interference,and gain the lentiviral particles which can stably up/down-regulate the expression ofGK.Firstly we use co-IP to confirm the interaction between GK and Nur77, andinteraction region of GK. And then we use the luciferase reporter assay to examine theinfluence of Nur77transcriptional activity by GK. And then we choose genes whichcould be regulate by Nur77in lipid metabolism, co-transfect GK and Nur77expression vevtor in human liver cell line L02and mice, real-time PCR examinemRNA expression level of these genes in liver. In addition, GK CDS sequence hasbeen built to pCDH lentiviral vector and GK siRNA sequence has been built topSicoR lentiviral vector,293T cells package lentivuris, and lentivuris is tittered byFACS.The results of co-IP confirmed the interaction of GK and Nur77, and theN-terminal and C-terminal region of GK are interacting domain. GK could reduceNur77transcriptional activity, multiples of activation could be three times reduced to0.5times, and the inhibition does not depend on the activity of GK. Results ofreal-time PCR show Nur77inhibits expression of Srebp-1c、Fas、Gpam and Acaca,and GK could regain the expression from20%to90%. GK expression/interferencelentiviral particles pCDH-GK/pSicoR-GK were successfully packaged, titer of thelentivirus is7.8×10~6pfu/ml/3×10~7pfu/mL.These studies reveal the interaction of GK and Nur77, and the impact on theregulation functions of liver lipid metabolism, constructed lentivirus of GK toestablish a basis for following experiments.