Molecular Cloning and Characterization of Human Homeobox Gene Nkx3.1 Promoter

Nkx3.1 is a prostate-specific homeobox gene related strongly to prostate development and prostate cancer. To study its regulation of transcription, 1.06 kb 5' flanking region of Nkx3.1 gene and its 5' deletion mutants (861, 617, 417 and 238 bp) were obtained by PCR and cloned into pGL(3)-basic, a promoter-less luciferase reporter vector, to examine their promoter activities driving the reporter gene transcription. pRL-TK, a Renilla luciferase reporter vector was used as internal control, and pGL(3)-control and pGL(3)-basic were used as positive and negative control respectively. The promoter activities were determined by dualluciferase reporter assay 48 h after pGL(3) constructs were cotransfected with pRL-TK into prostate cancer cell LNCaP. The results showed that dual-luciferase reporter assay (M(1)/M(2)) of pGL(3)-1.06 kb cotransfection with pRL-TK was 2.7, which was about 1.5-fold higher than that of pGL(3)-control cotransfection with pRL-TK and 50-fold higher than that of pGL(3)-basic cotransfection with pRL-TK. The results also showed that the relative activities (M(1)/M(2)) were 0.71, 0.84, 0.44 and 2.07 respectively for pGL(3)-861 bp, pGL(3)-617 bp, pGL(3)-417 bp, pGL(3)-238 bp, the last one still had 80% promoter activity compared with pGL(3)-1.06 kb, which showed that deletion from 1.06 kb to 238 bp had small effects on promoter activity. The conclusion was that the 238 bp fragment containing a TATA box and two CAAT boxes had strong promoter activity. However, the deletion from 1.06 kb to 861 bp reduced activity 3.8-fold while the deletion from 417 bp to 238 bp enhanced activity 4.7-fold, which indicated that these deleted sequences might contain some important positive or negative regulatory elements. It will be important to identify the elements within the Nkx3.1 promoter that contribute to regulation of the gene transcription in the future studies.