Characterization of two functional NKX3.1 binding sites upstream of the PCAN1 gene that are involved in the positive regulation of PCAN1 gene transcription
Characterization of two functional NKX3.1 binding sites upstream of the PCAN1 gene that are involved in the positive regulation of PCAN1 gene transcription
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DOI：
10.1186/1471-2199-9-45
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发表时间：
2008-05-04
影响因子：
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通讯作者：
Jiang, Anli
中科院分区：
文献类型：
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作者：
Liu, Wenwen;Zhang, Pengju;Jiang, Anli
背景：NKX3.1和PCAN 1均为前列腺特异性基因，与前列腺发育和前列腺癌相关。到目前为止，对这两个基因表达的调控机制知之甚少。在本研究中，我们发现NKX3.1上调LNCaP前列腺癌细胞中的PCAN 1基因转录。为了解PCAN 1基因的调控机制，我们的工作重点是鉴定PCAN 1基因上游的NKX3.1结合位点，这些结合位点可能参与NKX3.1对PCAN 1基因表达的正调控。用MatInspector 2.2分析2.6 kb序列，发现NKX3.1转录因子的5个潜在结合位点。采用荧光素酶报告基因分析、电泳迁移率变动分析、染色质免疫沉淀和RNA干扰等方法研究NKX3.1对前列腺癌细胞PCAN 1基因表达的影响。我们的研究结果表明，转染含NKX3.1的质粒（pcDNA3.1- NKX3.1）可增加LNCaP前列腺癌细胞中PCAN 1启动子活性和mRNA表达，而针对人NKX3.1的RNA干扰可降低PCAN 1 mRNA表达。电泳迁移率变动分析和染色质免疫沉淀的结果表明，NKX3.1与PCAN 1基因上游的NBS 1（-1848至-1836）和NBS 3（-803至-791）结合。荧光素酶报告基因分析表明，NBS 1和NBS 3在共转染含NKX3.1质粒的pGL（3）-启动子载体中增强了启动子活性。此外，NBS 1缺失或NBS 1和NBS 3缺失均降低了PCAN 1启动子活性，并消除了NKX3.1对PCAN 1表达的正调控作用。我们的结果表明，两个功能性NKX3.1结合位点位于-1848至-1836和-803至-PCAN 1基因上游791个位点参与NKX3.1对PCAN 1基因转录的正调控。
Background: NKX3.1 and PCAN1 are both prostate-specific genes related to prostate development and prostate cancer. So far, little is known about the regulatory mechanisms of the expression of these two genes. In the present study, we found that NKX3.1 upregulated PCAN1 gene transcription in LNCaP prostate cancer cells. To understand the regulatory mechanisms, our work focused on identifying the functional NKX3.1 binding sites upstream of the PCAN1 gene, which might be involved in the positive regulation of PCAN1 expression by NKX3.1.Results: We cloned and characterized a 2.6 kb fragment upstream of the PCAN1 gene. Analysis of the 2.6 kb sequence with MatInspector 2.2 revealed five potential binding sites of NKX3.1 transcription factor. Luciferase reporter assays, electrophoretic mobility shift assays, chromatin immunoprecipitation and RNA interference were performed to study the effects of NKX3.1 on PCAN1 gene expression in prostate cancer cells. Our results showed that PCAN1 promoter activity and mRNA expression were increased by transfection with the NKX3.1 containing plasmid (pcDNA3.1- NKX3.1) and that PCAN1 mRNA expression was decreased by RNA interference targeting human NKX3.1 in LNCaP prostate cancer cells. The results of electrophoretic mobility shift assays and chromatin immunoprecipitation showed that NKX3.1 bound to NBS1 (-1848 to -1836) and NBS3 (-803 to -791) upstream of the PCAN1 gene. The luciferase reporter assays showed that NBS1 and NBS3 enhanced the promoter activity in pGL(3)-promoter vector with cotransfection of the NKX3.1 containing plasmid. Furthermore, the deletion of NBS1 or both NBS1 and NBS3 reduced PCAN1 promoter activity and abolished the positive regulation of PCAN1 expression by NKX3.1.Conclusion: Our results suggested that two functional NKX3.1 binding sites located at -1848 to -1836 and -803 to -791 upstream of the PCAN1 gene were involved in the positive regulation of PCAN1 gene transcription by NKX3.1.