Construction of luciferase reporter gene vector for human MUC5AC gene promoter and analysis of its transcriptional activity.

Abstract

To clone the human mucin (MUC) 5AC gene promoter and construct its luciferase reporter vector for human MUC5AC gene and analyze its transcriptional activity. The 1 348 bp DNA sequence at the human MUC5AC gene 5 end was analyzed by the Vector NTI software. After the target sequence from human A549 cells genomic DNA was amplified by PCR method, and the product of PCR was sequenced. By promoter deletion analysis, 3 promoter segments with different lengths were amplified by PCR, then the products were identified by DNA sequencing, and 4 promotor segments were inserted into pGL3- enhancer vectors. Site-specific mutagenesis technique was used to establish mutants of specificity protein (SP)-l and nuclear factor-kappa B (NF-kappaB) site in MUC5AC gene promoter. The relative luciferase activities were detected in the transfected A549 cells. Sequence analysis indicated that there were many cis-acting elements in the regions of 1 348 bp DNA sequence at the human MUC5AC gene 5 end. The 4 reporter gene vectors with promoter segments with different lengths were constructed successfully. Dual-luciferase assay revealed the 372 bp fragment including activity with the minimal fragment. Neutrophil elastase (NE) could increase the expression of luciferase reporter gene plasmid containing mutated NF-kappaB version (P<0.05 vs. control) of MUC5AC promoter in the transfected A549 cells. The induction by NE decreased markedly when the SP-l element in MUC5AC promoter were mutated. This research may provide an important basis for the further study of human MUC5AC gene promoter activity and regulation of gene expression. There is an up-regulative element of gene transcription in the region of -324 to -64 bp in MUC5AC gene upstream. SP-l site of the promotor mediates NE-induced MUC5AC expression in human A549 cells.