Dissertation
Dissertation > Medicine, health > Basic Medical > Pathology > Pathophysiology

Nuclear Dot Protein NDP52 Bind to Tumor Necrosis Factor Receptor Associated Factor 6 and Their Clinical Significance of Research

Author SunLi
Tutor WangSiYing;YangXiaoMing
School Anhui Medical University,
Course Pathology and Pathophysiology
Keywords protein-protein interaction NDP52 TRAF6 NF-κB Hepatopoietin
CLC R363
Type Master's thesis
Year 2011
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Nuclear factor kappa B (nuclear factor kappa B, NF-KB) is a popular concern in recent years, a group of important factors, as can be induced and the ubiquitous transcription factor, a number of genes play a central role in transcriptional regulation, resulting in immunity, inflammation, cell survival, proliferation, differentiation and apoptosis, tumor formation has played a broad and important role. Currently, on the NF-KB signaling pathway in the development of hepatocellular carcinoma in the role of a research focus. NDP52 was ND10 composed of a member of the protein in the highest expression in liver tissue, can interact with other molecules eventually lead to cell life effects, and viral infections, inflammation of the formation of tumor development have a clear correlation. Study found that the protein and tumor necrosis factor receptor associated factor 6 (TRAF6) to participate in NF-KB signaling pathway in regulation, but regulatory mechanisms are not yet clear. TRAF6 NF-KB signaling pathway is an important positive regulatory factors to regulate downstream target gene expression. To further illustrate NDP52 and TRAF6 in NF-KB signaling pathway in the regulation mechanism, and its relationship with the development of liver cancer, we screened yeast two-hybrid cDNA library of adult liver are unknown interaction of NDP52 and TRAF6 as the research object, through a series of experiments show the interaction between the two, not to clarify the NF-κB signaling pathway in the mechanism of liver cancer molecular mechanisms and provide new clues.To take NDP52 and TRAF6 as the main object of study and explore the interactions between them to get the clinical functional significance.To get the unknown interaction of NDP52 and TRAF6 by screening adult liver cDNA library from yeast two-hybrid. To confirm the existence of physiological interactions between NDP52 and TRAF6 by exogenous co-immunoprecipitation, GST-Pull down experiments and fluorescence co-localization experiments.Transcription activity experiment shows that, NF-κB signaling plays a negative regulation role in TRAF6-mediated when NDP52 is over-expression, and this negative regulation gradually rises with the increasing in the amount NDP52.Except for TRAF6, other members TRAF1, TRAF2, TRAF3, and TRAF5 from the family NDP52 and TRAF6 also interact, and their interactions can affect the transcriptional activity of NF-κB. The experiments at different NDP52 hepatoma cells and other cancer cells were detected by Western blot. Immunofluorescence experiments confirmed that a NDP52 protein expressed in malignant melanoma (A375) and hepatoma cells (HCC cell) (7721) , and Distributed in the cytoplasm and nucleus, most of them are in the nucleus. Hepatocellular carcinoma immunohistochemistry results showed that the expression volume of NDP52 protein in HCC cell nucleus has a rising trend, at the same time , the expression is small in normal liver tissue. We used ELISA to detect the mount of NDP52 protein in hepatocellular carcinoma patient serum. ELISA results showed that the mount of NDP52 in patients with hepatocellular carcinoma in serum is higher than normal human serum.NDP52 and TRAF6 had physiological interactions. We found that NDP52 over expressed played a negative regulation role in NF-κB signaling TRAF6-mediated, and at the same time, this negative regulation gradually increased with the increasing amount of NDP52. Except for the TRAF6, other members TRAF1, TRAF2, TRAF3, and TRAF5 also interact, and their interactions can affect the transcriptional activity of NF-κB. We speculate that there are the same subcellular localization of in NDP52 with TRAF6. Detected by Western blot experiments at different NDP52 hepatoma cells and other cancer cells, we can see that NDP52 expression was existing in several hepatoma cells and several other non-liver cancer cells, and this showed that NDP52 is not the liver cell-specific expression protein.Hepatocellular carcinoma immunohistochemistry results showed that NDP52 protein expression are on the rise in the I, II and III-class of liver tissue nuclear, and the expression seems to be associated with the degree of tumor differentiation, and has less expression in the normal liver tissue. We detected 20 cases of hepatocellular carcinoma patients with serum NDP52 protein by ELISA, and the results showed that the NDP52 mount in patients with hepatocellular carcinoma in serum was higher than in normal human .The study showed that NF-κB signaling pathway was inhibited by NDP52, and this negative regulation is likely to be achieved at the level of TRAF6, which provide new clues to clarify the molecular mechanisms of NF-κB signaling pathway. Otherwise, this research clears subcellular localization in NDP52 and TRAF6 interaction, and we also found NDP52 related to liver cancer, which fill this gap of NDP52 with some diseases. At the same time, we rethink the role of the new features of the NDP52 form the study, and this laid a solid foundation for further NDP52 functional research.

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