Analysis Of Regulational Mechanism And Signal Transduction Of NGAL Gene In Gastric Cancer Cells

Neutrophil gelatinase-associated lipocalin, which was originally found as a protein stored in specific granules of the human neutrophi, has been reported to play an important role in various cell functions such as inflammation, immune reaction, cell differentiation, cell apoptosis, cell reconstruction, and even the happening and development of various tumors.Our previous results have shown that NGAL was over-expressed in the gastric cancer. Then, by employing the Dual-Luciferase Reporter Assay System, we found that the expression of NGAL can be induced by TPA and the TPA-induced activity of the report gene was increased by 3-9 times than the base vector, which implied that TPA response element (TRE) exists in the NGAL promoter. These findings suggested that the expression of NGAL in gastric carcinoma cells might be specifically up-regulated by TPA which is coincident with our data in the research of esophageal cancer. Moreover, we found that the core promoter region of NGAL gene was located in the segments from–110 to–79 by using mutation technique. Recently, we have also purified and identified the factors respond to TPA induction and bind to–107 to–82 region of NGAL promoter from esophageal carcinoma cells by using methods including DNA affinity chromatography, MOLDI-TOF-MS and bioinformatics. These candidate new TPA responsive factors include C19, KLF10, KLF15, RXRβand so on.In current study, to further identify the transcript factors which bind to the TRE of the NGAL promoter, we performed various technologies, such as molecular cloning, dual luciferase report gene array, semi-quantitative RT-PCR, and western blotting. Results showed that the promoter activities of NGAL were increased in gastric carcinoma cells which were transfected with the expression plasmid of C/EBPβ, AP-1, C19, KLF10, KLF15, KIAA1949 or RXRβ, respectively. Among these factors, the induction activity of C/EBPβwas the highest, showing 8.76 times higher than the control. We proposed that these factors might be the nucleus proteins respond to the TPA induction of NGAL gene. Finally, the pathways analysis by using pathway inhibitors showed that MEKâ†'ERK1/2 pathway plays an important role in the TPA induction of NGAL expression.