Cloning And Initial Identification Of The Promoter Region Of NGAL

NGAL(neutrophil gelatinase associated lipocalin) is a new member of lipcalin family and was found for the first time in human neutrophils in 1993. The homology between NGAL and rat oncogene product 24p3 is very high, in addition, its expression is found remarkably enhanced in the colon and breast cancer tissues, and SHEEC and ovary cancer cell lines, suggesting NGAL is a new oncogene. However, until now, the over-expression mechanism of NGAL in tumor tissues is still unknown. For this, PCR technology, DNA recombination, nested deletion and dual-luciferase reporter gene assay are combined to identify and position the promoter region of NGAL, and at the same time Bioinformatics methods are used to predict the promoter trans-acting factors in the promoter region of NGAL, which will infer some essential datum for the full elucidation of the expression regulation mechanism of NGAL.Contents and methods1. Total DNA, isolated from SHEEC as template, and six pairs of primers were used for PCR reaction to attain a series of the different length sequences in the 5' flanking region of NGAL, that is, -1431-+84, -1137-+84, -945~+84, -657~+84, -416-+84 and -152-+84. These sequences were cloned into pGEM-T easy and subcloned into pGL3-Basic for the construction of dual-luciferase reporter gene assay plasmids pB1431,pB1137, pB945, pB657, pB416 and pB152, which may identify the distribution of the promoter of NGAL.2. Nested deletion and culture and dual-luciferase reporter assay were combined tofurther seek the relatively accurate position of the promoter region of NGAL. 3. Functional elements of the promoter region NGAL and trans-acting factors were predicted by Bioinformatics methods. Meanwhile, expression regulation mechanism of NGAL was discussed.Results1. Compared with pGL3-Basic, the relative luciferase activities of pB1431, pB1137, pB945, pB657, pB416 and pB 152 are enhanced (p<0.01 or p<0.05), suggesting the promoter region of NGAL is in the region of-152~+84.2. Eight deletion recombinants, pB 140 > pB78> pB5SK pB50> pB4K pB37> pB29 and pBlO, are attained from nested deletion on pB152. Compared with pGL3-Basic, the relative luciferase activities of pB152 and pB140 are remarkably enhanced (p<0.01 gHp<0.05), but the relative luciferase activities of pB78,pB59, pB50,pB41,pB37,pB29 and pBlO are not found obvious difference (p>0.05), which shows there is some functional element in the region of -140~-78, essential to the expression regulation of NGAL.3. Bioinformatics analysis shows there are probable eight sites in the region of -140~-78 of 5' flanking region of NGAL, which can bind trans-acting factor , including Ftz^ C/EBP a , NF-1, TTF-1, AP-1, MRF4JSCBP V, CRE-B, C/EBP y.Conclusion1. The promoter region of NGAL is in the 5' flanking region of-152~+84.2. Some transcription element probably exists in the 5' flanking region of 140~-78 of NGAL, essential to expression level of NGAL.3. Several trans-acting factors probably bind the sites in the 5' flanking region of 140—78 of NGAL. However, it is under study which factor on earth exerts influence on the expression of NGAL.