Study Of Expressional Profile And Regulational Mechanism Of NGAL Gene In Gastric Cancer Cells

Recently, many researches suggest that neutrophil neutrophil gelatinase-associated lipocalin (NGAL) was an important gene related with tumor, which was up-expressed in colon cancer, breast cancer, pancreatic cancer, lung cancer, skin cancer and esophageal cancer. However, the expression level of NGAL in gastric cancer remains unclear. In this study, we have detected the NGAL expression in gastric cancer tissues and cells, and investigated the regulational mechanism of NGAL gene expression in gastric cancer cells. This research will provide more clues for fully cognizing the oncobiology functions of NGAL.Materials and methods:1. Expression of NGAL protein was analyzed by immunohistochemistry in 55 paraffin sections of gastric cancer tissue and the corresponding sections of normal gastric glandular tissue.2. Expression of NGAL in BGC-823 and SGC-7901 cell lines was analyzed by western blotting and RT-PCR.3. The luciferase repoter gene expression vectors containing NGAL gene 5'flanking regulation region were constructed by subcloning the different length DNA fragments of NGAL gene 5'flanking regulation region into pGL3-Basic vector.4. In gastric cancer cell lines BGC-823 and SGC-7901, the key region of NGAL promoter was identified by Dual-Luciferase Reporter Assay System.5. By the use of mutation and deletion techniques, the TPA responsiveness of NGAL promoter was analyzed in gastric cell lines.6. By using western blotting and bioinformatics, we identified the transcriptional activator and approached the signal transduction pathways that mediate NGAL expression response to TPA in gastric cancer.Results:1. In immunohistochemical study, only weak positive or negative signal was presented in normal gastric gland epithelium cells. While in gastric cancer cells, strong positive staining could be observed, a whole-cytoplasm expression of NGAL was presented in the cancer cells.2. There was visible distinction of NGAL expression level in two kinds of gastric cancer cell lines and the expression level in BGC-823 cells was obviously stronger than that in SGC-7901 cells.3. By employing the Dual-Luciferase Reporter Assay System, we have found that the key element of NGAL promoter was located on the -110 to -79 segments from transcriptional start site in BGC-823 cells, but in SGC-7901 cells it was located on the -152 to -141.4. It was found that NGAL promoter has TPA responsiveness both in BGC-823 and SGC-7901 cells.5. In BGC-823 and SGC-7901 cells, the TPA response element of NGAL promoter remains in the region from -85 to -79.6. Results of bioinformational prediction and western blotting suggested that the phosphorylation of ERK might be involved in the TPA signal transduction and AP1 was the transcriptional activator mediating the TPA responsiveness of NGAL promoter in BGC-823 cells.Conclusions:1. NGAL gene was up-expressed in gastric cancer tissues and cells.2. There was significant difference of NGAL expression level between BGC-823 and SGC-7901 cells.3. In gastric cancer cells BGC-823 and SGC-7901, the core element of NGAL promoter was different.4. In gastric cancer cells, it was found that NGAL promoter has TPA responsiveness and the TPA response element located on the region from -85 to -79.5. AP1 might be the transcriptional activator mediating TPA responsiveness of NGAL promoter in gastric cancer cells.6. The phosphorylation of ERK might be involved in the signal pathway that mediates NGAL expression response to TPA.