Study Of Transcriptional Regulation Of NGAL Gene In Esophageal Carcinoma Cells

Esophageal squamous cell carcinoma (ESCC) is a familiar malignant cancer in China, but the pathogenesis of ESCC is unclear so far. In 2000 our work have identified that neutrophil gelatinase-associated lipocalin (NGAL), also known as lipocalin 2, was overexpressed in the progression of malignant transformation from human immortalized esophageal epithelial cell by HPV E6E7 gene and TPA induction, but the function of NGAL in ESCC was unknown at that time. Recently, it is reported that protein product of NGAL as a member of the lipocalin family is involved in diverse cellular processes, including transport of small hydrophobic molecules, protection matrix-metalloproteinase-9 (MMP-9) activity from degradation and regulation of immune response, in which NGAL is the carrier of informational molecules. Furthermore, it is reported that NGAL tightly binds bacterial catecholate-type ferric siderophores and acts as a potent bacteriostatic agent by sequestering iron. And NGAL protein played an important role in tumor invasion and the dysregulated-differentiation property of cancer cells. These results indicated that NGAL showed a diverse functional pattern in various pathological and physiological conditions or in the various tissues and cells. However, there is little information regarding the mechanisms of transcriptional regulation of NGAL. This is the key problem for restricting to farther study NGAL function. Therefore, considered the expression pattern of NGAL in ESCC, in this study, we examined the molecular mechanisms of the full transcriptional regulation repertoire of NGAL using human EC109 esophageal carcinoma cells as a model.First, the DNA fragments from -1431～+84 to -152～+84 of 5'flanking of NGAL gene were amplified from the genomic DNAs of esophageal cancer cell line SHEEC by using polymerase chain reaction (PCR). The PCR products were subcloned into pGL3-Basic vector and pGL3-Promoter vector. A serious of firefly luciferase reporter gene vectors pGLB-1431～152 and pGLP-1431～152 were constructed. And then the deletions for -152～+84 and the mutations for -140～+84 of 5'flanking region of NGAL gene were identified by nested deletion and directed site mutation, respectively, according the analysis of bioinformatics.