Molecular cloning and analysis of the human PCAN1 (GDEP) promoter.
Human 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 pGL(3)-basic vector generating pGL(3)-p2.6 kb and transfected into LNCaP cells. pGL(3)-basic and pGL(3)-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, pGL(3)-p2.6 kb was transfected into the prostate cancer cell line LNCaP, which was treated with R1881 (10(-7) approximately 10(-9) mol/l), 17beta-estradiol (17beta-E(2), 10(-7) approximately 10(-9) mol/l), all-trans-retinoic acid (all-trans-RA, 10(-5) approximately 10(-7) mol/l) or 9-cis-retinoic acid (9-cis-RA, 10(-5) approximately 10(-7) mol/l), and eukaryotic expression plasmids of NKX3.1, p53, Sp1, Pten, PPARgamma or cEBPalpha were cotransfected with pGL(3)-p2.6 kb into LNCaP cells. pRL-TK, a Renilla luciferase reporter vector, was cotransfected into all the transfection lines as an internal control. The activities of pGL(3)-p2.6 kb (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, 17beta-E(2) and all-trans-RA had no significant effect on PCAN1 promoter activities. Cotransfection with pGL(3)-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 PPARgamma and cEBPalpha 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.
Liu
Wenwen
W
Institute of Biochemistry and Molecular Biology, School of Medicine, Shandong University, Jinan, 250012, China.
Chen
Weiwen
W
Zhang
Pengju
P
Yu
Chunxiao
C
Kong
Feng
F
Deng
Jingti
J
Zhang
Jianye
J
Jiang
Anli
A
D000077556:Alitretinoin; D001483:Base Sequence; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D005786:Gene Expression Regulation; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011467:Prostate; D011471:Prostatic Neoplasms / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection; D014212:Tretinoin / Q000378:metabolism
D009363:Neoplasm Proteins; C435944:PCAT4 protein, human; D014157:Transcription Factors; D000077556:Alitretinoin; D014212:Tretinoin; D008156:Luciferases