Cloning and Characterization of the NPCEDRG Gene Promoter

NPCEDRG is a novel tumor suppressive gene that localizes to 3p21.3, a chromosomal region frequently associated with loss of heterozygosity (LOH) in a number of malignancies including nasopharyngeal carcinoma (NPC). Its transcriptional down-expression has been shown in the cell lines and primary tumor tissues of NPC. Reintroduction of NPCEDRG into CNE2, a cell line derived from NPC, was effective to induce cell differentiation, control cell growth, and regulate the cell cycle. Little is known about the transcriptional mechanisms controlling NPCEDRG gene expression. In this article, we describe the NPCEDRG gene structure and the transcriptional expression of NPCEDRG; we found that NPCEDRG was expressed weakly in most of NPC cell lines. Using 5' rapid amplification of complementary DNA ends (5'-RACEs), we found that the NPCEDRG gene has several transcription start sites (TSSs) due to the existence of alternatively spliced variants, and the specific TSS of NPCEDRG was located -25 nucleotides upstream of the translation start site. We amend that Human NPCEDRG CDS containing 516 bp but not the 510 bp reported previously. To characterize the NPCEDRG promoter, transient luciferase and/or EGFP reporter assay were carried out with the constructs including various lengths of the 5' flanking region of the NPCEDRG gene. The results demonstrated that the basal promoter is located at the region from -215 to -8 nucleotides, and the optimal promoter is located at the region from -625 to -8 nucleotides upstream of the translation start site. In silico analysis suggested that the promoter region contained potential binding sites for SP1, c-Myb, AREB6, Nkx2-5, and so on. These results provide important clues to elucidate the regulation of NPCEDRG gene expression and function. Further studies are apparently required for the identification of the transcription factors, essential for NPCEDRG expression, which would lead to better understanding of the molecular mechanism of NPCEDRG expression in nasopharyngeal epithelial cells.