血根碱靶向Notum调控Wnt/β-catenin通路抑制肺癌干细胞自我更新的分子机制

The self-renewal characteristic of lung cancer stem cells is one of the critical factors of leading to the failure of lung cancer treatment. Notum as a specific negative feedback protein in Wnt/β-catenin self-renewal pathway, the mechanism in lung cancer is unclear. Previous studies have found that sanguinarine is the main anti-cancer component of Chinese Herb celandine, which can significantly inhibit the self-renewal of lung cancer stem cells. Sanguinarine can can up-regulate the expression of notum at transcription level and inhibit the corn protein β-catenin and its downstream target gene in Wnt-pathway through gene-chip analysis and rescue experiments. When notum was suppressed, the sanguinarine’s ability to inhibit the self-renewal of lung cancer stem cells were weakened. According to this, we postulated that sanguinarine can inhibit the self-renewal of lung cancer stem cells by regulating Wnt/β-catenin signaling pathway through notum. This project will further elucidated the the methylation site of sanguinarine activated notum and the regulation mechanism of sanguinarine to notum’s upstream transcription factors by taking advantage of molecular cloning, virus transfection, biological information prediction and living imaging technology and so on. The project will reveal the molecular mechanism of sanguinarine to inhibit lung cancer stem cells and clarify the effect of notum in lung cancer, providing the theoretical foundation for sanguinarine application in clinical.

肺癌干细胞自我更新是导致肺癌治疗失败的关键因素之一，Notum作为Wnt/β-catenin自我更新通路中的特异性负反馈调节蛋白，目前在肺癌中的机制尚不清楚。前期在“金水相生”抗肺癌干细胞理论指导下，已发现中药白屈菜中的主要抗癌成分血根碱能显著抑制肺癌干细胞的自我更新；通过芯片分析及回复实验表明，血根碱可以在转录水平上调Notum表达，抑制Wnt通路中β-catenin及其下游靶基因；当Notum抑制时，血根碱对肺癌干细胞的自我更新能力减弱。据此提出“血根碱是否通过靶向Notum调控Wnt/β-catenin通路进而抑制肺癌干细胞自我更新”的假说；本课题将综合运用分子克隆、病毒转染、生物信息预测、动物活体成像等技术，进一步从甲基化及转录调控水平，明确Notum在肺癌干细胞自我更新中的作用，深入阐明血根碱激活Notum的具体位点与分子机制，为血根碱在临床的应用提供理论基础和实验依据。

肺癌干细胞自我更新是导致肺癌治疗失败的关键因素之一，Notum作为Wnt/β-catenin自我更新通路中的特异性负反馈调节蛋白。前期在“金水相生”抗肺癌干细胞理论指导下，已发现中药白屈菜中的主要抗癌成分血根碱能显著抑制肺癌干细胞的自我更新，血根碱可以在转录水平上调Notum表达，抑制Wnt通路中β-catenin及其下游靶基因；当Notum抑制时，血根碱对肺癌干细胞的自我更新能力减弱。本课题在前期研究基础上，进一步从表观遗传学角度，分析Notum启动子区的甲基化CpG岛和CpG位点，并通过甲基化特异性PCR法（MSP）检测未发现Notum甲基化程度受血根碱调控；利用转录因子数据库JASPAR、TRANSFAC预测Notum启动子区潜在转录因子及其结合位点，筛选并构建KLF9和EGR2的结合位点突变体质粒，通过Luciferase荧光酶报告基因表达量及Truncation分段实验验证，发现血根碱上游发挥转录调控作用机制与KLF9转录因子有关；为血根碱在临床的进一步应用提供理论基础和实验依据，创新中医药复方及单体治疗肺癌的研究思维和方法，提高肺癌治疗的有效性。