NF-κB Mediates the Expression of TBX15 in Cancer Cells

TBX15 is a T-box transcription factor essential for development, also proposed as a marker in prostate cancer; and, recently, its antiapoptotic function indicates a role in carcinogenesis. Regulation of TBX15 is uncovered. In this study, we investigated the regulation of TBX15 expression in human cancer cells, by analyzing the regulatory function of a 5’-distal conserved region of TBX15. Bisulfite sequencing showed high methylation of the CpG island contained in this region that was not correlated with TBX15 mRNA levels, in the cancer cell lines analyzed; however, after 5-aza-dC treatment of TPC-1 cells an increase of TBX15 expression was observed. We also found a significant response of TBX15 to TNF-α activation of the NF-κB pathway using five cancer cell lines, and similar results were obtained when NF-κB was activated with PMA/ionomycin. Next, by luciferase reporter assays, we identified the TBX15 regulatory region containing two functional NF-κB binding sites with response to NF-κBp65, mapping on the -3302 and -3059 positions of the TBX15 gene. Moreover, a direct interaction of NF-κBp65 with one of the two NF-κB binding sites was indicated by ChIP assays. In summary, we provide novel data showing that NF-κB signaling up-regulates TBX15 expression in cancer cells. Furthermore, the link between TBX15 and NF-κB found in this study may be important to understand cancer and development processes.TBX15属于T-box转录因子（T-box transcription factor），在个体发育过程中发挥关键作用，同时被提议作为前列腺癌的标志物；近年来其抗凋亡功能提示其在肿瘤发生过程中具有一定功能。目前，TBX15的调控机制尚未被阐明。本研究中，我们通过分析TBX15基因5’端远端保守区域的调控功能，探究了该基因在人类癌细胞中的表达调控机制。亚硫酸氢盐测序（Bisulfite sequencing）结果显示，在所分析的癌细胞系中，该区域所包含的CpG岛甲基化程度较高，但该甲基化水平与TBX15的mRNA表达水平并无相关性；不过，在对TPC-1细胞施以5-氮杂-2’-脱氧胞苷（5-aza-dC）处理后，我们观察到TBX15的表达水平显著上调。我们还借助五种癌细胞系，发现TBX15可对肿瘤坏死因子-α（TNF-α）激活的核因子-κB（NF-κB）通路产生显著应答；而当用佛波酯/离子霉素（PMA/ionomycin）激活NF-κB时，也得到了相似的实验结果。随后，通过荧光素酶报告基因实验（luciferase reporter assays），我们鉴定出TBX15的调控区域包含两个可响应NF-κBp65的功能性NF-κB结合位点，这两个位点分别定位于TBX15基因的-3302和-3059位点处。此外，染色质免疫沉淀（ChIP, Chromatin Immunoprecipitation）实验结果证实，NF-κBp65可与这两个NF-κB结合位点中的其中一个发生直接相互作用。综上，本研究提供了全新的实验数据，证实NF-κB信号通路可在癌细胞中上调TBX15的表达。此外，本研究揭示的TBX15与NF-κB之间的调控关联，或许对解析肿瘤发生与个体发育过程具有重要的参考价值。