The Regulatory Mechanisms Of PCAN1 Gene Expression By NKX3.1 In Prostate Cancer LNCaP Cells

NKX3.1 is an androgen regulated prostate-specific homeobox gene that is thought to play important roles in normal prostate development and cancerogenesis.Human NKX3.1 gene maps to chromosome 8p21,the full length ofNKX3.1 gene is about 4.2 kb,encodes a predicted protein of 234 amino acids with homology to the Drosophila NK family of homebox gene.NKX3.1 is a novel homeoprotein and function as nucleus transcription factor.Studies show that human chromosome 8p21 is a region with high loss of heterozygosity(LOH)in about 80%human prostate cancer.The NKX3.1 gene is proposed to have tumor suppressor function and loss of NKX3.1 protein expression is closely related with the initiation of prostate carcinogenesis and with prostate tumor progression.PCAN1(prostate cancer gene 1,also known as GDEP)is highly expressed in prostate epithelial tissue and frequently mutated in prostate tumors.The expression of PCAN1 is undetectable in highly undifferentiated DU145 and PC-3 prostate cancer cell lines and weakly detected in more differentiated LNCaP cell line.This gene is localized to chromosome 4q21,a region of the genome that experiences frequent loss of heterozygosity in prostate cancer.It is mutated in 35%of the tumor samples. Therefore,PCAN1 has been proposed to have tumor suppressing function in prostate cancer.NKX3.1 and PCAN1 are both prostate-specific genes strongly related to prostate development and prostate cancer.So far,little is known about the regulatory mechanisms of their expression.In the present study,we found that NKX3.1 upregulated PCAN1 gene transcription in LNCaP prostate cancer cells.To understand the regulatory mechanisms,the eukaryotic expression plasmid of NKX3.1 cDNA (pcDNA3.1-NKX3.1)was constructed and the expression of NKX3.1 mRNA and protein were detected by RT-PCR and Western blotting respectively after the plasmid was transfected into prostate cancer LNCaP and PC-3 cells.The eukaryotic expression plasmid of siRNA targeting NKX3.1(pRNAT-NKX3.1 RNAil)was constructed and stably transfected into LNCaP cells.The silence of NKX3.1 was detected by RT-PCR and Western blotting.A 2.6 kb fragment upstream of the PCAN1 gene was obtained by PCR to form the PCAN1 promoter-luciferase report plasmid (pGL_3-pPCAN1)and the promoter activity was examined by dual luciferase reporter assays.After cotransfection with pGL_3-pPCAN1 and pcDNA3.1-NKX3.1 into prostate cancer LNCaP and PC-3 cells,the effects of NKX3.1 on PCAN1 promoter activity and PCAN1 mRNA expression were detected by dual luciferase reporter assays and RT-PCR respectively.RT-PCR was used to detect the effects of NKX3.1 knock-down by siRNA on PCAN1 mRNA expression in LNCaP cells.Analysis of the 2.6 kb sequence with Matlnspector 2.2 revealed potential binding sites of some important transcription factors,including five potential NKX3.1 transcription factor binding sites(NBSs).To investigate the binding activities of these five NBSs with NKX3.1 transcription factor, we carried out CHIP,dual-luciferase reporter assays and EMSA.After confirming that NBS1 and NBS3 in the PCAN1 promoter were functional NKX3.1 binding sites,the NBS1 or both NBS1,3 deletion plasmids of pGL_3-pPCAN1 were then constructed and the positive regulation of NKX3.1 on PCAN1 promoter activity was analyzed by dual luciferase reporter assays.The results showed that the eukaryotic expression plasmid of NKX3.1 cDNA (pcDNA3.1-NKX3.1)was successfully constructed and could be effectively expressed in PC-3 and LNCaP cells.The expression of NKX3.1 could be knocked down by the eukaryotic expression plasmid of siRNA targeting NKX3.1(pRNAT-NKX3.1 RNAil) in stably transfected LNCaP cell strain.A 2.6 kb fragment of 5'flanking region of PCAN1 gene was cloned and the PCAN1 promoter-luciferase reporter plasmid (pGL_3-pPCAN1)was constructed.Analysis of pGL_3-pPCAN1 presented a promoter activity in prostate cancer LNCaP cells.The PCAN1 promoter activity and mRNA expression in LNCaP cells were enhanced by pcDNA3.1-NKX3.1 cotransfection while the mRNA expression of PCAN1 was decreased after the expression of endogenous NKX3.1 in LNCaP cells was knocked down by the siRNA.Two functional NKX3.1 binding sites upstream of the PCAN1 gene(NBSI:-1848bp～-1836bp,NBS3:-803bp～-791bp)were identified,which were involved in the positive regulation by NKX3.1 of PCAN1 gene expression.In a word,our research suggested that two functional NKX3.1 binding sites located at-1848bp to-1836bp and -803bp to -791bp upstream of the PCAN1 gene were involved in the positive regulation of PCAN1 gene expression by NKX3.1.Both NKX3.1 and PCAN1 are related to prostate development and prostate cancer.Our findings will contribute to the understanding of molecular regulatory mechanisms of PCAN1 gene expression in prostate development and cancer.