| BRD7 is a novel gene cloned in nasopharyngeal carcinoma (NPC) by cDNA representative differential analysis (cDNA RDA) (GenBank accession number: AF 152604), which encodes a bromodomain protein. Molecular mechanism of the bromodomain proteins involved in the tumorigenesis is still unknown. To elucidate the role of those proteins in carcinogenesis will offer a new strategy for the prevention and therapy of human tumor .In previous studies, we found that BRD7 was characteristic of suppressing the malignant phenotype of nasopharyngeal carcinoma cell line HNE1, and preventing the progression of cell cycle from GO/G1 stage to S stage when transfected in HNE1 cell. These results suggested that BRD7 could reverse the malignant phenotype of NPC cells by affecting the cell cycle. We also identified six putative proteins interacted with BRD7 by yeast two hybrid system screening of a human fetal brain cDNA library, which were BRD2, BRD3, p-IKB kinase, KIAA1375, IL-7 and 8, - A P3 . According to the length of the fragments inserted to pACT2 vector, the number of positive clones and the intensity of interaction, we inferred that BRD2 and BRD3 werethe most likely proteins interacted with BRD7. Besides, both BRD2 and BRD3 have two bromodomains and belong to bromodomain containing protein family, BET family. BRD2, known as Ring 3, is a cell cycle dependent transcription mediator, which shows distinct protein kinase activation. BRD3 is highly homology to BRD2. It has been reported that those proteins which interacted with each other could be functionally highly related and involved in the same signal transduction pathway and process of tumorigenesis. We inferred that BRD2 and BRD3 could play important roles in the nuclear translocation and activation of BRD7 protein.In this study, the subcellar location of BRD7 protein in HNE1 and Hela cell lines was studied by immunofluorescent assay. The interaction of BRD7 and BRD2, BRD3 was testified by coimmunoprecipitation. The preliminary functional study of BRD2 and BRD3 was performed.The main results and findings are as follows:1. Expression and location pattern of BRD7 protein in different cell lines studied by immunofluorescenceWe constructed the recombinant of pCMV-Myc-BRD7, then transfected respectively into Hela and HNE1 cells mediated by lipofectin. The anti-Myc antibody was used to detect the Myc tag of BRD7 fusion protein. The fluorescnce microscope was used to observe the expression and subcellar location of BRD7 protein. We found that only in the cytoplasm had green fluorescence observed in almost all Hela cells and approximate 50 percent10HNE1 cells. But BRD7 is a nucleus protein expressed both in nuclear and cytoplasm in primary cultures of normal nasopharyngeal epithelial (PNNE) in previous experiments and bioinformatical analysis. So we could draw a conclusion that there may be a translocation obstacle from the cytoplasm to the nucleus in HNE1 and Hela cells.2. The tissue distribution of BRD7 in the biopsies of NPC and normal Nasopharyngeal epithelialNPC is derived from the naspharyngeal mucosa epithelial, and the low differential spuama carcinoma is most commonly seen. In order to ascertain the tissue distributions of BRD7 gene in NPC and normal naspharyngeal epithelial, in-situ hybridation was performed. It was shown that both in NPC and normal nasopharyngeal epithelial, BRD7 was expressed only in the naspharyngeal mucosa epithelia and tumor tussues, but not in lymphocyte and interstitial cell, which supported that BRD7 could play an important role in the tumorigenesis of nasopharyngeal epithelial.3. Further identification of the protein-protein interaction between BRD7 and BRD2, BRD3In order to elimilate the false positive results, yeast two-hybrid system was used to specially confirm the protein-protein interactions in yeast.There are still few false positive interacting proteins existed in the results of two-hybrid system for its own limitation, and the protein-protein interactions in yeast may not really occur...
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