p53过表达抑制同源盒基因NKX3.1启动子活性

NKX3.1是前列腺特异表达的同源盒基因，在前列腺癌的发生发展中起重要作用，而在前列腺癌进展中常会发生p53的基因突变.为研究两者之间的关系，构建NKX-3.1启动子(1 040bp)-荧光素酶报告基因重组质粒（pGL3-1040）及其缺失突变体,瞬时转染前列腺癌细胞LNCaP.通过荧光素酶表达活性分析,检测p53过表达对NKX3.1启动子活性的影响.结果表明：p53在LNCaP细胞中过表达可明显抑制NKX3.1启动子活性；RT-PCR及Western印迹检测p53过表达对NKX3.1表达的影响.结果表明，p53过表达可以明显抑制同源盒基因NKX3.1的表达.通过TRANSFAC软件分析，在NKX3.1基因上游-526至-507区存在一个p53反应元件的5′核心序列.缺失pGL3-1040中的p53反应元件核心序列并不能消除p53对NKX3.1启动子的抑制作用，表明p53不是通过p53反应元件直接抑制NKX3.1启动子活性.进一步通过5′缺失突变分析，发现NKX3.1启动子-140～+8 bp区仍受p53负调控.此148 bp区域中含有一个Sp1和一个CREB元件，瞬时共转染Sp1表达载体或CREB表达载体的结果表明，p53并不是通过与Sp1或CREB相互作用对NKX3.1启动子发挥抑制作用的.上述结果表明，p53过表达可以抑制同源盒基因NKX3.1启动子活性，下调NKX3.1基因的转录，其调控机制有待进一步研究.

Overexpression of p53 Inhibits Promoter Activity of NKX3.1 Gene

Homeogene NKX3.1is specially expressed in prostate and plays an important role in the development and growth of prostate cancer, while the mutation of p53 often occurs during the development of prostate cancer. In order to study the relationship between p53 and NKX3.1 in prostate cancer cells, the recombinant plasmid (pGL3-1040) of NKX3.1 promoter (1 040 bp)-luciferase reporter gene and its mutants were constructed and transiently transfected into prostate cancer cell LNCaP to analyze the effects of p53 overexpression on NKX3.1 promoter activity by using luciferase reporter assay. Our results showed that p53 overexpression inhibited NKX3.1 promoter activity and decreased NKX3.1expression in LNCaP cells. Analysis by TRANSFAC database indicated that a p53 responsive element (5′half site) was located in the region from -526 to -507 in the upstream of NKX3/1 gene. But the deletion of the core sequence of p53 responsive element from the NKX3.1 promoter did not abrogate the p53-mediated repression of the NKX3.1 promoter activity, suggesting that inhibition of p53 on the NKX3.1promoter was not achieved by binding to p53 responsive element. Furthermore, the results of 5′ deletion mutation analysis found that the region of the NKX3.1 promoter between the -140～+8 bp in the upstream of the NKX3.1 gene was negatively regulated by p53. The analysis showed that there were a Sp1 and a CREB elements within the region of -140～+8 bp. The results of transient transfection with Sp1 or CREB expression plasmid demonstrated that p53 also did not inhibit the NKX3.1 promoter by interaction with Sp1 or CREB. Although these results indicated that p53 overexpression was capable to inhibit the NKX3.1 promoter activity and downregulate the NKX3.1 gene expression, further studies would need to explore the mechanisms.