Molecular cloning and analysis of the human PCAN1 (GDEP) promoter

Abstract

AbstractHuman PCAN1 (prostate cancer gene 1) is a prostate-specific gene that is highly expressed in prostate epithelial tissue, and frequently mutated in prostate tumors. To better understand the regulation of the PCAN1 gene, a 2.6-kb fragment of its 5′ flanking region was obtained by PCR. Its promoter activity was examined via the dual-luciferase reporter assay after it had been cloned into a pGL3-basic vector generating pGL3-p2.6kb and transfected into LNCaP cells. pGL3-basic and pGL3-control were respectively used as the negative and positive controls. Sequence analysis with the MatInspector database showed that some possible binding sites for the transcriptional factors, NKX3.1, P53, SP1, cEBP and the PPAR/RXR heterodimers may locate on a 2.6-kb region upstream of the PCAN1 gene. To examine the relevant regulation of PCAN1, pGL3-p2.6kb was transfected into the prostate cancer cell line LNCaP, which was treated with R1881 (10−7∼10−9 mol/l), 17β-estradiol (17β-E2, 10−7∼10−9 mol/l), all-trans-retinoic acid (all-trans-RA, 10−5∼10−7 mol/l) or 9-cis-retinoic acid (9-cis-RA, 10−5∼10−7 mol/l), and eukaryotic expression plasmids of NKX3.1, p53, Sp1, Pten, PPARγ or cEBPα were cotransfected with pGL3-p2.6kb into LNCaP cells. pRL-TK, a Renilla luciferase reporter vector, was cotransfected into all the transfection lines as an internal control. The activities of pGL3-p2.6kb (PCAN1 promoter) were analyzed via the dual-luciferase reporter assay 48 h after transfection. The results showed that 9-cis-RA enhanced the PCAN1 promoter activity in a dose-dependent manner, while R1881, 17β-E2 and all-trans-RA had no significant effect on PCAN1 promoter activities. Cotransfection with pGL3-p2.6kb and the expression plasmids of NKX3.1, p53, Sp1 or Pten respectively resulted in 1.66-, 2.48-, 2.00-and 1.72-fold 2.6 kb PCAN1 promoter activity increases relative to the controls, which were cotransfected with pcDNA3.1(+), while cotransfection of PPARγ and cEBPα yielded no significant effect on PCAN1 promoter activities. These results could be applied for further study of the function and transcription regulation of the PCAN1 gene in prostate development and carcinogenesis.