There are TPA Response Elements in -152～-60 Position of NGAL Gene 5′Flanking Region in The Esophageal Cancer Cells EC109

Overexpression of the neutrophil gelatinase-associated lipocalin (NGAL) gene is related to malignanttransformation of human immortalized esophageal epithelial cell induced by 12-O-tetradecanoyl phorbol13-acetate (TPA). However, the mechanisms of the transcriptional activation of this gene remain unclear. Therecent study indicated that there might be TPA response elements in the 5′flanking promoter region and thesequences surrounding it. In order to locate the position of TPA response elements, the NGAL fragments for- 152～+84, - 140～+84, - 78～+84, - 59～+84, - 50～+84, - 41～+84, - 37～+84, - 29～+84 and - 10～+84 wereobtained from esophageal cancer cells by PCR and nested deletion and cloned into pGL3-Basic (pGLB),pGL3-Enhancer (pGLE) and pGL3-promoter (pGLP) vector which are luciferase reportors, respectively. Theconstructed recombinant expression plasmids pGLB-152, pGLP-152, pGLE-152, pGLB-140, pGLB-78, pGLB-59,pGLB-50, pGLB-41, pGLB-37, pGLB-29 and pGLB-10 were adopted to cotransfect with pRL-TK into EC109esophageal cancer cells stimulated with TPA (5 μg/L) respectively or not. Relative luciferase activity of whole cellextracts from cells was measured with Dual Luciferase Report Gene Assay System (DLR) to verify the position ofTPA response elements. Results showed that the TPA response elements of NGAL are located on - 152～- 60region and have a strong response to TPA stimulation in the esophageal cancer cells. The analysis bybioinformatics showed that the potential TPA responsive element in - 152 ～- 60 position of NGAL is a newsequence. This suggested that there is the structural character responding to TPA induction in the gene. Theseresults will help to clarify the molecular mechanisms of NGAL overexpression in malignant transformation ofhuman immortalized esophageal epithelial cell induced by TPA.