TPA Response Elements Present in -416～ + 84 Position of NGAL Gene 5′Flanking Region in the Esophageal Cancer Cells EC109

Objective 　The TPA response elements in NGAL promoter region and the sequences surrounding its promoter region will be determined. Methods 　The fragment -416～ + 84 of 5′flanking region of NGAL gene was amplified from esophageal cancer cells by PCR and then cloned into pGL3-Basic (p GLB) and p GL3-Enhancer vector (p GL E), respectively. Then the constructed recombinant expression plasmid pGLB-416 or pGLE-416 was adopted to cotransfect with pRL-TKinto EC109 esophageal cancer cells stimulated with TPA (5ng/ ml) or not . Relative luciferase activity of whole cell extracts from cells was measured with Dual Luciferase Report Gene Assay System (DLR) to verify whether there are TPA response elements in the region and the potential positions were analyzed by bioinformatics. Results 　Relative luciferase activity of either pGLB-416 or pGLE-416 cotransfected with pRL-TKinto EC109 cells following stimulation with TPA increased sharply ( t test, P < 0. 01) by an average of 46. 82 or 46. 41-fold, compared with controls (empty vector pGLB or pGL E ), and by an average of 5. 17 or 7. 37-fold than that of unstimulated control. Analysis by bioinformatics showed that there are at least 4 putative TPA responsive elements in - 416～ + 84 position of 5′flanking region of NGAL gene. Conclusion 　The TPA response elements of NGAL gene located on -416～ + 84 position of its 5′flanking region in the esophageal cancer cells.