Cloning Of Human Homeobox Gene NKX3.1 Promoter And Identification Of The Regulatory Region

NKX3.1 is an androgen regulated prostate-specific homeobox gene that is thought to play the important roles in normal prostate development and cancerogenesis. In mice NKX3.1 is exclusively expressed in prostate epithelium and its targeted disruption leads to aberrations in prostate ductal morphogenesis and secretary protein production, and epithelial hyperplasia and dysplasia. Notably NKX3.1 mutant mice display the pathologic changes of prostatic intraepithelial neoplasia (PIN) that is the presumed precursor to prostate cancer in human, which implies that loss of NKX3.1 expression correlates with the initiation of prostate carcinogenesis. In human NKX3.1 expression is generally restricted to the prostate and it is androgen-regulated. Since NKX3.1 gene maps to the chromosomal region 8p21, a region with high loss of heterozygosity in about 90% human prostate cancer, the gene has been proposed to have tumor suppressor function. Loss of NKX3.1 protein expression is closely related with the initiation of prostate carcinogenesis and with prostate tumor progression. But no mutations in the NKX3.1 gene have been found in prostate tumor specimens. Its second allele is inactivated by some mechanisms other than mutations in the coding region. The strong association of NKX3.1 with prostate development and prostate cancer makes this gene an attractive molecular target for further study. The NKX3.1homeobox gene provides an excellent model to explore the relationship between embryogenesis and oncogenesis. So far little is known about the regulatory mechanisms of NKX3.1 gene expression as well as relevant regulatory elements and factors. We have firstly cloned the 1040bp-5' flanking region of NKX3.1 gene to identify its regulatory regions and binding proteins. It will provide an insight into the regulatory mechanisms of NKX3.1 gene expression in further study.PART ONE Cloning and Identification of Human Homeobox Gene NKX3.1 PromoterObjective: The aim of this part is to clone 1040bp-promoter fragment upstream of NKX3.1 gene and to determine its promoter activity.Methods and results: According to the sequence of NKX3.1 gene in Genbank, a pair of primer was designed for PCR. 1040bp-promoter fragment upstream of NKX3.1 gene was amplified by PCR and inserted into pGL3-basic, a promoter-less luciferase reporter vector, to form 1040bp promoter-luciferase reporter plasmid (pGL3-1040) that proved to be right by the restriction enzyme digestion and DNA sequencing. Prostate cancer cell line LNCaP was cotransfected with pGL3-1040 and pRL-TK using lipofectaminTM 2000, and then the expression of luciferase reporter driven by 1040bp-promoter of NKX3.1 was tested by assay of luciferase activity. The results showed that the 1040bp-fragment presented a strong promoter activity that was 1.5-fold stronger than that of pGL3-promoter and 50 fold stronger than that of pGL3-basic. NKX3.1 is an androgen-regulated gene. To observe whether or not 1040bp-promoter is regulated by androgen, the transfected LNCaP and PC-3M were treated by 10-7 M concentration of R1881, and then checked for luciferase activity. The results showed that the activity of 1040bp-promoter was increased by 78% at 8 hours after LNCaP cells were transfacted with pGL3-1040bp promoter and treated with R1881 of 10-7 M. The activity of 1040bp promoter was increased by 31% at 8 hours after PC-3M cells were cotransfacted with pGL3-1040bp promoter and androgen receptor expressed plasmid, comparing with that PC-3M cells were transfected only with pGL3-1040bp promoter. The activity of 1040bp promoter wasincreased by 69% at 8 hours after PC-3M cells were cotransfacted with pGL3-1040bp promoter and AR, and treated with R1881 of 10-7 mol/L, comparing with that PC-3M cells were cotransfacted with pGL3-1040bp promoter and AR without treatment of R1881.The results indicated that 1040bp promoter was regulated by androgen in certain extent. To check the activity of 1040bp-promoter in various cells, 1040bp promoter-green fluorescence protein (GFP) reporter plasmid was constructed and then transfected in...