【摘要】 目的检测人同源盒NKX3.1基因内含子及5’上游10 kb调控区对启动子活性的影响,分析其雄激素反应性和组织特异性,为进一步研究该基因表达调控机制奠定基础。方法利用基因重组技术分别构建5’上游10 kb调控区/内含子-NKX3.1基本启动子-荧光素酶报告基因质粒。转染雄激素依赖性前列腺癌细胞株LNCaP后,检测荧光素酶表达活性,观察内含子及5’上游调控区对NKX3.1启动子活性的影响;加入雄激素类似物R1881刺激,进一步检测其雄激素反应性;通过转染不同细胞分析其组织特异性。结果荧光素酶报告基因分析表明,NKX3.1基因5’上游-7 681～-6 483 bp可以增强其启动子活性2.6倍,但该增强作用并不具有组织特异性。内含子及5’上游10 kb其他区域对NKX3.1启动子活性未见明显的增强作用。R1881刺激并不能使内含子及5’上游10 kb调控区活性明显增强。结论 NKX3.1基因内含子及5’上游10 kb调控区不具备雄激素反应性,5’上游-7 681～-6 483 bp可以增强NKX3.1启动子活性,但其组织特异性增强元件并不存在于该片段。更多还原

【Abstract】 Objective To further illustrate regulation mechanisms on the expression of homeobox gene NKX3.1,the intron and the 10 kb upstream region of NKX3.1 gene were cloned and their effects on NKX3.1 promoter activity were determined to find the androgen response element and the tissue-specific enhancer.Methods According to the sequence of NKX3.1 gene in Genbank,a NKX3.1 promoter-luciferase reporter plasmid was constructed by PCR technology.Further,an intron-promoter-luciferase reporter plasmid and 10 fragments-promoter-luciferase reporter plasmids were constructed separately.After recombinated plasmids were transfected into prostate cancer cell line LNCaP,the effects of the intron or 10 fragments in the upstream of NKX3.1 gene on the promoter activity were tested by luciferase reporter assay.Androgen response region was analyzed by stimulating LNCaP cells with R1881.Prostate-specific enhancer was analyzed by the transfection of plasmids in different cells.Results The region between-7 681~-6 483 bp enhanced the activity of NKX3.1 promoter to 2.6 fold,but it was not tissue-specific.The intron and other 9 fragments could not increase the NKX3.1 promoter activity obviously.R1881 didn’t enhance the activities of intron and 10 kb upstream region of NKX3.1.Conclusion The intron and 10 fragments of NKX3.1 gene were not androgen-responsible.A functional positive regulatory element existed from-7 681~-6 483 bp,but it was not tissue-specific.更多还原