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 pGL3-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.

中文翻译：

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PCAN1 基因上游的两个功能性 NKX3.1 结合位点的表征，这些位点参与 PCAN1 基因转录的正调控。

背景 NKX3.1 和 PCAN1 都是与前列腺发育和前列腺癌相关的前列腺特异性基因。到目前为止，人们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 上调了 LNCaP 前列腺癌细胞中的 PCAN1 基因转录。为了理解调控机制，我们的工作重点是确定 PCAN1 基因上游的功能性 NKX3.1 结合位点，这可能参与 NKX3.1 对 PCAN1 表达的正调控。结果 我们克隆并表征了 PCAN1 基因上游的 2.6 kb 片段。使用 MatInspector 2.2 分析 2.6 kb 序列揭示了 NKX3.1 转录因子的五个潜在结合位点。荧光素酶报告基因检测、电泳迁移率变化检测、进行染色质免疫沉淀和RNA干扰以研究NKX3.1对前列腺癌细胞中PCAN1基因表达的影响。我们的结果表明，通过转染含有 NKX3.1 的质粒 (pcDNA3.1-NKX3.1)，PCAN1 启动子活性和 mRNA 表达增加，并且在 LNCaP 前列腺癌细胞中，靶向人 NKX3.1 的 RNA 干扰降低了 PCAN1 mRNA 表达. 电泳迁移率变化测定和染色质免疫沉淀的结果表明，NKX3.1 与 PCAN1 基因上游的 NBS1（-1848 至 -1836）和 NBS3（-803 至 -791）结合。荧光素酶报告基因检测表明，NBS1 和 NBS3 通过共转染含有 NKX3.1 的质粒增强了 pGL3 启动子载体中的启动子活性。此外，NBS1 或 NBS1 和 NBS3 的缺失降低了 PCAN1 启动子的活性，并消除了 NKX3.1 对 PCAN1 表达的正调控。结论 我们的结果表明，位于 PCAN1 基因上游 -1848 至 -1836 和 -803 至 -791 的两个功能性 NKX3.1 结合位点参与了 NKX3.1 对 PCAN1 基因转录的正调控。