The Regulation Of Neutrophil Gelatinase-Associated Lipocalin Basal Expression In Lung Carcinoma Cells

NGAL (Neutrophil gelatinase-associated lipocalin), a member of the lipocalin family, wasoriginally found as a protein stored in specific granules of the human neutrophil. It has beenreported that NGAL showed a diverse functional pattern in various physiological andpathological conditions and in various tissues and cells. Previous researchers also demonstratedNGAL was overexpressed in human cancers and our previous work have identified that NGALmight be involved in the differentiation pathway and invasive progression of ESCC. However,the regulatory mechanisms of NGAL gene basal expression are still unknown. The aim of thisstudy is to find the transcriptional regulation factors of NGAL promoter in lung carcinoma cells.The study would help to further explore the roles of NGAL in the pathogenesy of lung cancersand provide significant clues to study the regulation mechanism of NGAL in other cancer cells.Materials and methods:1. Expression and distribution of NGAL were detected by immunohistochemistry staining in 23paraffin sections, including lung squamous carcinoma, adenocarcinomas, adenosquamouscarcinoma and bronchial alveolar cell carcinoma.2. mRNA and protein expression level of NGAL gene were detected by RT-PCR andimmunofluorescent staining in 95D and A549 cells.3. Several PCR primers and oligonucleotide of NGAL gene were synthesized. Then constructedmutational plasmids either by direct PCR amplification with mutated primers or by use of theGeneEditor^TM in vitro Site-Directed Mutagenesis System. The products were inserted intopGL3-Basic vector. Then cotransfected the resulting constructs with pRL-TK vector as aninternal control into 95D and A549 cells and examined relative luciferase activity of reportergene by using the Dual-Luciferase Reporter Assay System (DLR).4. The potential transcription factors located at the promoter region of NGAL gene werepredicted by bioinformatics methods.5. The expression of potential transcription factors were detected with Western blotting in 95Dand A549 cells.6. Oligonucleotides spanning from -152 to -114 and -112 to -74 of the NGAL promoter weresynthesized and Dig-ddUTP labeled as probes, respectivly. Then the binding reaction ofprobes and nuclear extracts from 95D and A549 cells examined by electrophoretic mobilityshift assay (EMSA). Supershift analysis was accomplished to find the essential transcriptionfactors binding with the regulatory element ofNGAL gene promoter.Results:1. In immunohistochemical study, results showed that 82.61％(19/23) of the cases were positive for NGAL staining. The lung adenocarcinomas and squamous carcinoma casesrevealed a diffuse cytoplasmic distribution of NGAL in the carcinoma cells. Thisdemonstrated that expression of NGAL in lung carcinomas.2. Results of RT-PCR and immunofluorescent staining both showed that NGAL was expressedin 95D and A549 cells.3. With the Dual-Luciferase Reporter Assay, we found that when truncated from -152 to -140,the promoter activity of NGAL was reduced about 70％, when the region between -106 and-79 was also deleted, then the remaining 30％activity was also lost, indicating that the-152～-141 and -106～-79 regions were essential for NGAL basal promoter activity, therefore it wasidentified as the core promoter and minimal promoter of NGAL.4. The results of bioinformatics prediction showed that the member of C/EBPs(CCAAT/enhancer binding proteins) and MRFs (Myogenic Regulatory Factors) may bind tothe region -152～-141 and -106～-79 of NGAL gene, respectively.5. The protein level of C/EBPs and MRFs was demonstrated by Western blotting. There wereonly C/EBPβand MyoD, Myf5 expression in 95D and A549 cells.6. In EMSA assay, Similar DNA-protein complexes were formed in two kinds of cells. Thenext supershift analysis, anti-C/EBPβand anti-MyoD antidodies made the DNA-proteincomplex disappear and later migrating bands appeared. These results suggested that C/EBPβand MyoD could bind NGAL core promoter in lung carcinoma cells.Conclusions: In lung carcinoma cells, the regions from - 152 to - 141 and - 106 to -79 of NGALpromoter were the core promoter and minimal promoter of NGAL gene. The transcriptionalfactors C/EBPβand MyoD could regulate NGAL basal promoter activity. The characteristics ofNGAL gene expression regulation in lung carcinoma cells were different from other tumour cells,indicated that the regulation of NGAL gene existed cell specificity.