Cloning Expression And Functional Study Of AXUD1 Protein Up-regulated By AXIN1 In Wnt Signal Transduction Passway

Axudl is a novel human gene induced by AXIN1 in Wnt signal transduction passway and identified in human colon-cancer cell. The axudl gene consists of five exons and encodes a nuclear protein with 63kD molecular weight. It was frequently down-regulated in lung, kidney, liver and colon cancers compared with their corresponding normal tissues. In addition, AXUD1 has a high homologue with transforming growth facotr beta(TGF-β) induced apoptosis protein 2 and 12 using BLAST exploration at GeneBank database. These suggest that axudl may have a tumor-suppressor function in these organs and may play an important role in TGF-β -induced apoptosis.In order to further investigate the role of axudl in human tumor carcinogenesis and the potential association between the axudl gene expression status and the stimulation of transforming growth factor beta in human cancers , the present study was performed in three aspects as follows: (1) cloning full length enconding region cDNA of axudl and construction of eukaryotic vector that expression the fusion protein of AXUD1 and influenza virus hemagglutin HA epitope tag;(2)exploring the time and dose effects of TGF-β 1 on the expression-of axudl gene in HepG2 hepatoma cells and SPC-A1 lung carcinomas cells , and studying theeffects of overexpression of AXUD1 on the expression of cell cycle and apoptosis related protein in HepG2 hepatoma cells;(3)construction and expression of human axudl in E. Coli M15.The following main results and conclusions can be obtained from the present study:1.The full length ecnoding region of human axudl cDNA from human peripheral blood lymphocytes was successfully cloned using one step RT-PCR method , and constructed into a eukaryotic expression vector which can be expressed a HA-AXUD1 fusion protein with AXUD1 and influenza virus hemagglutin HA epitope tag .The recombinant plasmid was identified by polymerase chain reaction, restriction endonuclease maping and sequencing, This expression vector might be instrumental to further study the function of AXUD1 protein in tumor cells.2. We showed here that axudl mRNA expression was elevated by TGF-β 1 treatment in a time and dose-dependent manner in HepG2 hepatoma cells and SPC-A1 lung carcinomas cells , and also indicated that de novo protein synthesis was required for this response using the protein synthesis inhibitor cycloheximide, suggesting that axudl is a primary target of TGF-β signalling.3. Overexpression of wide-type SmadS resulted in up-regulation of axudl mRNA expression in HepG2 cells, whereas axudl mRNA expression was compromised by dominant-negative Smad3(D407E) . On the other hand, axudl mRNA expression induced by TGF-β 1 was not inhibited by inhibitor of MEK1/2 or p38 MAPK. These results suggested that TGF-β 1 stimulated axudl expression required SmadS and was not dependent on activity of MAPK signal passway.4. In the absence of TGF-β 1, overexpression of the HA-AXUD1 fusion protein decreased the expression of Bax and resulted in a marked down-regulation of p53 protein expression. By contrast, it increased the expression of C-Myc. Overexpression of the HA-AXUD1fusion protein, however, did not affect the expression level of Bel-XL and Caspase-3. On the other hand, in the presence of TGF-β 1, the expression level of Bel-XL and C-Myc were decreased and the expression of Bax was increased in HepG2 cells transfected with pcDNAS. 1/ha-axudl plasmid, whereas no change of caspase-3 and p53 occurred. However, only C-Myc protein was slightly increased in cells transfected with vector alone and other proteins expression level were the same as conditions in the absence of TGF-β 1. In addition, HepG2 cells transfected with pcDNAS. 1/ha-axudl plasmid were arrested in Gl phase compared with the cells transfected with vector alone. These results suggested that AXUD1 protein functioned in a different way in the expression of cell cycle and apoptosis related proteins induced by TGF-β1 in tumor cells.5. Axudl cDNA fragment was cloned into procaryotic expression v...