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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。
Objective To detect the effect of NKX3.1 gene intron and the 10 kb regulatory region upstream of 5 'on promoter activity in human homeobox and to analyze the androgen responsiveness and tissue specificity of NKX3.1 gene in human homeobox and to lay a foundation for further study on the regulatory mechanism of this gene. Methods The 5 'upstream 10 kb regulatory region / intron - NKX3.1 basic promoter - luciferase reporter plasmid was constructed by gene recombination technique. After transfection with androgen-dependent prostate cancer cell line LNCaP, the activity of luciferase was detected, and the effect of intron and 5 'upstream regulatory region on the activity of NKX3.1 promoter was observed. Androgen R1881 was added for further testing It is androgen-responsive; its tissue specificity is analyzed by transfection of different cells. Results The luciferase reporter gene analysis showed that the promoter activity of NKX3.1 gene upstream of -7 681 ~ -6 483 bp increased 2.6-fold, but the enhancement was not tissue-specific. Introns and other 10 kb regions upstream of 5 'showed no obvious enhancement of NKX3.1 promoter activity. R1881 stimulation did not significantly increase the activity of introns and the 10 kb regulatory region upstream of 5 '. CONCLUSION: The intron of NKX3.1 gene and the 5 kb upstream regulatory region of 10 kb do not possess androgen responsiveness. The 5 'upstream -7 681 ~ -6 483 bp can enhance the activity of NKX3.1 promoter, but its tissue specific enhancer Does not exist in the fragment.