分泌蛋白Notum介导PI3K/AKT信号通路维持胃癌干细胞表型的分子机制及其分子标志物价值

The secretory protein Notum is up-regulated in gastrointestinal cancer, but its function and molecular mechanism remain to be elucidated. Up to date, there are little reports on the role of Notum in gastric cancer stem cells. Our previous work has found that Notum is highly expressed in gastric cancer, especially in tissues and serum of the early-stage gastric cancer. And Notum is significantly up-regulated in gastric cancer stem cells. Overexpression of Notum significantly enhances the tumor initiating ability of gastric cancer cells and their stemness. Second-generation sequencing combined with the results of PCR and WB suggest that PI3K/AKT signaling pathway targeting the stem cell-related gene ALDH1A2 may be involved in the role and molecular mechanism of Notum maintaining gastric cancer stem cells phenotype. Based on the above results, we hypothesize that Notum promotes the tumor initiating ability of gastric cancer cells through PI3K/AKT signaling pathway by upregulating ALDH1A2. This project intends to demonstrate the function and molecular mechanism of Notum in maintaining gastric cancer stem cells phenotype with a serious of experiments such as Crispr/Cas9 knockout or lentivirus overexpression, limiting dilution, Co-IP/MS, second-generation sequencing and luciferase report assay. And the clinical value of serum Notum as a molecular biomarker in early-stage gastric cancer will be also explored, which provides a new biomarker and target for the diagnosis and treatment of gastric cancer.

分泌蛋白Notum在消化系统肿瘤中表达上调，但功能机制不详，目前未见Notum与胃癌干细胞相关的报道。前期工作发现，Notum在胃癌患者尤其早期胃癌患者组织和血清中高表达，且Notum在胃癌干细胞中表达显著上调；过表达Notum显著增强胃癌细胞的肿瘤起始能力，并增强其干细胞特性；二代测序结合PCR和WB结果提示PI3K/AKT信号通路靶向调控干细胞相关基因ALDH1A2可能参与Notum维持胃癌干细胞特性。基于上述结果提出假说：Notum可通过PI3K/AKT信号通路上调ALDH1A2表达进而增强胃癌细胞的肿瘤起始能力。本项目拟通过Crispr/Cas9敲除或慢病毒过表达、有限稀释、Co-IP/质谱、二代测序、荧光素酶报告等实验证明Notum维持胃癌干细胞特性的功能及分子机制，并探讨血清中的Notum作为早期胃癌分子标志物的临床应用价值，以期为胃癌诊疗提供新标志物和新靶点。

胃癌干细胞是胃癌组织内真正驱动肿瘤发生和发展的“动力”，也是肿瘤复发和转移的根源，对肿瘤的发生和发展起着非常重要的作用。然而，人们对肿瘤干细胞的分子标志物及其发挥作用的分子调控机制仍不明确。分泌蛋白Notum在消化系统肿瘤中表达上调，但功能机制不详，目前未见Notum与胃癌干细胞相关的报道。.本课题前期工作发现，Notum在胃癌患者尤其早期胃癌患者组织和血清中高表达，且Notum在胃癌干细胞中表达显著上调。为验证上述实验猜想，本课题通过过表达和Crispr/Cas9敲除实验进一步证明，Notum过表达可显著增强胃癌细胞的肿瘤起始能力，并增强其干细胞特性；相反，敲除Notum的胃癌细胞其自我更新能力明显减弱且小鼠体内成瘤的能力也明显被削弱；二代测序结合PCR和WB结果提示PI3K/AKT信号通路靶向调控干细胞相关基因Sox2可能参与Notum维持胃癌干细胞特性。本课题采用PI3K/AKT信号通路的激活剂和抑制剂以及细胞回复实验结果进一步证实，Notum通过靶向Sox2调控PI3K/AKT信号通路，进而影响胃癌细胞的干细胞特性；此外，本课题通过筛选Notum的小分子抑制剂，试图挖掘可靶向作用Notum的药物，为临床治疗提供新思路。实验结果表明，Caffeine作为Notum的小分子抑制剂，可显著抑制胃癌细胞中Notum的表达水平，且能显著抑制胃癌细胞的成球能力。.因此，Notum可通过PI3K/AKT信号通路上调Sox2表达进而增强胃癌细胞的肿瘤起始能力。本项目证明Notum维持胃癌干细胞特性的功能及分子机制，并探讨血清中的Notum作为早期胃癌分子标志物的临床应用价值，以期为胃癌诊疗提供新标志物和新靶点。Caffeine作为一种Notum抑制剂，对胃癌的治疗可能发挥一定作用。