食管癌重要功能基因LCN2过表达正反馈调控环分子机制及临床意义研究

Our previous studies have shown the clinical significance of LCN2 overexpression in esophageal cancer, which is positively correlated with poor cancer cell differentiation, invasion and metastasis, as well as a shortened survival time of patients with esophageal cancer, suggesting LCN2 was an important functional gene for esophageal cancer. Our recent studies further revealed that the secreted LCN2 protein binds to its specific receptor in cell membrane and activates the intracellular ERK signaling pathway, which in turn leads to the overexpression of LCN2 gene and the increased LCN2 protein, thus forming a positive feedback regulation loop. However, one deeper important scientific issue remains unsolved: which transcriptional activator protein in this positive feedback regulation loop participates in the up-regulation of LCN2 gene. To answer this question, our project intends to apply the joint experiment methods including the luciferase reporter gene system, the key promoter sequence location and site-directed mutagenesis, Electrophoretic Mobility Shift Assay (EMSA), chromatin immunoprecipitation, Chip-PCR and protein mass spectrometry and other experimental techniques, combined with bioinformatics, to systematically identify transcriptional activator protein and its binding transcriptional activation elements for LCN2 in esophageal cancer. Our work may lead to deep insights into the detailed interaction between transcriptional activator and its binding elements and may uncover the molecular mechanisms of positive feedback regulation loop for the overexpression of LCN2 in esophageal cancer, as well as its clinical significance.

我们以往研究发现，LCN2基因在食管癌中显著异常过表达，与癌细胞的不良分化、移动侵袭，以及患者生存期缩短等高度相关，是食管癌重要功能基因。近年，我们进一步研究发现，在食管癌细胞中，分泌至胞外的LCN2蛋白与胞膜上的特异受体结合，跨膜激活胞内ERK信号通路，使LCN2基因更大量过表达，LCN2蛋白分泌增加，构成正反馈调控环。然而，此正反馈调控环中间，究竟由何转录激活蛋白激活LCN2基因过表达，这一更深层次重要科学问题尚不清楚。为此，本项目拟通过联合运用荧光素酶报告基因系统、启动子关键序列定位定点突变、凝胶滞留与超滞留、染色质免疫共沉淀、Chip-PCR以及蛋白质谱等系列实验技术，结合生物信息学，系统鉴定食管癌中LCN2基因的转录激活蛋白及其所结合的转录激活元件，深入研究激活蛋白与激活元件间的精细相互作用情况，理顺重要功能基因LCN2在食管癌中过表达正反馈调控环分子机制，同时探讨其临床意义。

我们以往研究发现，LCN2基因在食管癌中显著异常过表达，与癌细胞的不良分化、移动侵袭，以及患者的生存期缩短等高度相关，表明是十分重要的食管癌功能基因。最近，我们的研究结果进一步提示，在食管癌中存在着LCN2基因表达正反馈环。然而，在上述正反馈环中，最终通过何种转录激活蛋白，上调LCN2基因表达的分子机制尚不清楚。为此，本项目拟通过联合运用基因克隆、荧光素酶报告基因检测系统、基因转染、染色质ChIP、生物信息学分析等系列实验技术，鉴定LCN2基因的转录激活蛋白及其转录激活元件，并深入探讨激活蛋白与激活元件之间的精细相互作用情况，理顺食管癌中LCN2基因表达正反馈环调控分子机制。 .研究结果表明，生物信息学预测LCN2基因启动子区存在着这些转录激活蛋白的有效结合位点分别位于-710~-699及-629~-616区段潜在的EGR1结合位点、-273~-260及-222~-209区段的潜在TCF7L2结合位点、还有可能位于-158~-148及-69~-59区段的潜在STAT3结合位点。进一步的双荧光素酶报告基因检测及染色质CHIP检测发现，EGR1及TCF7L2可能参与到食管癌LCN2正反馈表达调控环中，是LCN2正反馈表达调控环关键转录因子。蛋白免疫印迹的检测显示：在不同浓度的rhLCN2作用下，内源性EGR1及TCF7L2的表达呈递增趋势。同时在食管癌细胞中过表达ERG1或TCF7L2可以显著提高食管癌中LCN2的表达，且随转染质粒浓度的提高LCN2的表达渐强，LCN2表达亦增强；而且EGR1或TCF7L2上调LCN2表达受MEK/ERK调控。体外功能实验显示：食管癌细胞中EGR1或TCF7L2的过表达，通过细胞微丝骨架系统重排，可显著提高食管癌细胞的移动及侵袭能力。.综上所述，TCF7L2与EGR1协同参与到食管癌LCN2的正反馈调控中，是LCN2正反馈调控的关键转录因子。