Identification of an inhibitory cis-element in the promoter region of human homeobox gene NKX3.1

BACKGROUND. NKX3.1 is a prostate-specific homeobox gene related strongly to prostate development and prostate cancer. To study its transcriptional regulation, a 1,040 bp promoter of human NKX3.1 gene was cloned into the upstream of the luciferase reporter gene in pGL3-basic plasmid and a 180 bp region extending from -391 to -212 in the upstream of NKX3.1 gene was identified presenting an inhibitory regulation for NKX3.1 expression in our previous experiments. In this work, it is aimed to identify precisely the functional cis-element within the 180 bp region involved in the inhibitory regulation of the NKX3.1 promoter and to identify its specific binding protein. METHODS. 5'-deletion mutation, substitution mutation, and electrophoresis mobility shift assay (EMSA) were carried out to identify precisely the functional cis-element contributing to the inhibitory regulation of NKX3.1 and its binding ability to nuclear extracts from prostate cancer cell line LNCaP. RESULTS. A 20-bp inhibitory element located between -362 and -343 in the upstream of NKX3.1 gene was identified by deletion and substitution mutation analysis and proven to be a functional inhibitory cis-element by EMSA. CONCLUSIONS. We have identified a functional inhibitory cis-element between -362 and -343 in the upstream of NKX3.1 gene; it is likely to play an important role in downregulating NKX3.1 gene transcription.