Experimental Research On Transcription Regulation Of Mouse Hepcidin Gene By NF-κ B

Hepcidin, the first liver-specific antimicrobial peptide of mammal, is cysteine-rich and has wide spectrum of antimicrobial activities. Search of the data bases found homologous liver cDNAs in species from fish to human. Moreover, Hepcidin is also a key component of iron homeostasis, implicated in iron metabolism disorders. Its double role heats up the relative research. Researchers hope to regulate Hepcidin expression precisely with the purpose of setting up Hepcidin-targeting therapy for iron metabolism disorders. As a result, the would-be regulating molecules of Hepcidin expression catch great attention. Investigation pointed out the presence of HNF3β, C/EBP, and NF-κB regulatory element binding sites located upstream of the first start codon. Although C/EBPαwas proved likely to be a regulator of Hepcidin gene transcription, further research is needed to explore the role of other transcription factors in regulating Hepcidin expression.A possible role of NF-κB in regulating Hepcidin expression in acute phase response is predicted according to the following findings: 1. Putative NF-κB binding sequences were found in promoter region of Hepcidin gene and preserved in many species.2. As a type II acute-phase protein, Hepcidin expression is obviously upregulated by LPS. 3. NF-κB is implicated in development of inflammation: NF-κB is activated by LPS, and consequently bind to specific DNA sequence to regulate gene transcription. 4. NF-κB activation has close relation with iron accumulation in liver. And iron overload in liver is the direct reason for upregulation of Hepcidin expression. Thus, NF-κB may function in signaling pathway involved in Hepcidin upregulation by iron overload in liver.5. It is known that NF-κB is involved in tumor genesis and development, and that Hepcidin is over expressed in liver adenocarcinoma. Can there be a link between NF-κB and Hepcidin expression? To answer this question, we designed and synthesized probes for NF-κB binding sequences in Hepcidin promoter to explore the possibility and molecular mechanism of Hepcidin transcription regulation by NF-κB.The commonly-used methods to research on NF-κB regulation on target gene are the following:(1) EMSA(Electrophoretic Mobility Shift Assay) to explore the possible DNA binding site of transfection factor.(2) South-western to detect molecular weight of transfection factor binded to probe.(3) Gel super-shift assay to find the effective subunit of the binding transfection factor.Though these methods are classical, mature and systemized, their disadvantages are to be overcome:(1)The exact transfection factor that regulates transfection of target gene can not be decided after the experiments.(2)In vitro cell experiments and in vivo experiments are needed to confirm the results.So, if we inhibit the expression of transfection factor, and then detect expression of target gene, we can therefore confirm the results of the previous experiments.RNAi (RNA interference) is one of the most powerful tools to research on gene function, which is highly stable, highly efficient and highly specific in inhibiting gene expression. Using RNAi to inhibit NF-κB expression specifically at cell level, our project aimed at exploring transfection regulation of Hepcidin by NF-κB. Obj.0ective1. To explore the possibility of NF-κB regulation on Hepcidin gene transcription in acute phase response.2. To investigate the molecular mechanism of NF-κB regulation on Hepcidin transcription.Materials and Methods1. Research on transcription regulation of Hepcidin using mouse model⑴ Building mouse model for acute phase response5-week-old mice were given LPS intraperitoneally to induce acute phase response. After they were sacrificed, the livers were used for total RNA and nuclear protein extraction. RT-PCR, Northern blot and dot blot were performed to detect the relationship between Hepcidin expression and time after/dose of LPS injection. The serum iron concentrations were detected at different LPS injecti...