Notch信号通路通过抑制Eph通路调控骨肉瘤干细胞样特性

Notch signaling pathway regulates osteosarcoma stem cell characteristics by inhibiting Eph pathway

目的研究Notch信号通路维持骨肉瘤干细胞样特性的作用及其机制。方法体外细胞实验中，取对数生长期的人骨肉瘤细胞MG63细胞系，利用慢病毒技术转导NICD1、Numb．shRNA上调Notch信号通路活性，通过肿瘤成球实验，流式细胞术检测细胞表面标记物Stro-1、CD117，Westernblot和qRT，PCR检测干细胞相关基因Sox2和Oct4，观察Notch信号通路对骨肉瘤干细胞样特性的影响。在动物实验中，根据注射成瘤细胞不同，将裸鼠随机分NICDl过表达组、DAPT抑制剂组和对照组。连续观察8周，通过记录每周肿瘤的体积和重量，比较各组成瘤后体积重量变化，观察Notch信号通路对动物成瘤的影响。进一步探索Notch信号通路调控Eph信号通路的体外细胞实验中，利用Westernblot检测Eph通路相关蛋白EphB，p-EphB评价Notch信号通路对于Eph通路的调控作用。在体外实验MG63骨肉瘤细胞培养基中添加具有活性的ephrinB1后，利用Westernblot、肿瘤成球率、Sox2、Oct4表达量变化，观察Eph通路激活后对肿瘤细胞干性的影响。结果体外细胞实验中NICDl、Numb—shRNA成功上调Notch信号通路活性，Notch信号通路激活的骨肉瘤体外肿瘤成球率上调、流式细胞检测Stro-1／CD117双阳性比率增高，于细胞相关基因Sox2、Oct4表达量上调。动物实验发现Notch信号通路激活可促进体内成瘤，而抑制Notch信号通路会抑制体内成瘤。将DAPT处理后的骨肉瘤组织做原代培养，细胞成球显著降低。探索Notch信号通路调控Eph信号通路的体外细胞实验中，Westernblot结果显示Notch通路激活后，磷酸化的EphB水平降低，Eph通路被抑制。而DAPT处理组p-EphB水平升高，但总EphB的表达量无明显变化。MG63骨肉瘤细胞培养基中添加具有活性的ephrinB1后，Westernblot结果显示EphB磷酸化显著上调，证实Eph通路被激活，骨肉瘤细胞的成球率下降和Sox2、Oct4表达量下降。结论Notch通路的激活在促骨肉瘤细胞的干细胞样特性发挥重要作用，并可能通过Notch信号通路对EPH通路的下调有关。

Objective To investigate the role of Notch signaling pathway to maintain the stem cell-like characteristics of osteosarcoma and its underlying mechanism. Methods Lentiviral NICD1 or Numb-shRNA was transduced into MG63 osteosarcoma cells to activate Notch activity in vitro. The impact of Notch on osteosarcoma stem cells were assessed by the tumor sphere for- mation assay and flow cytometry analysis of cell surface markers STRO-1/CD117. The expression of stem cell related genes （Sox2, Oct4） were evaluated by Western blot and qPCR. The nude mice were randomly divided into 3 groups： the NICD1 overexpression （NICD-OE） group, the DAPT group and the control （CON） group. The tumor growth was monitored for 8 weeks and the tumor vol- ume and weight were recorded weekly. To investigate whether Notch regulates Eph pathway, Eph pathway related protein EphB, p- EphB was measured by Western blot. The impact of ephrinB 1 on NICD overexpression cell were assessed by tumor sphere forma- tion assay. The expression of Sox2 and Oct4 was evaluated by Western blot. Results NICD1 overexpression or Numb-shRNA in- creased the activity of Notch pathway. The Notch-activated osteosarcoma showed enhanced in vitro tumor spheroid formation capacity, increased Stro-1/CD117double positive ratio, and upregulated expression of Sox2 and Oct4 in vitro. In animal experiments, it was found that activation of Notch pathway promoted tumor formation in vivo and Notch inhibition decreased it. The primary os- teosarcoma cells were obtained from mice xenograft treated with DAPT and its tumor sphere formation capacity was significantly reduced. Finally, The Notch pathway inhibits the phosphorylation of EphB, as well as the downstream signal pathway of EphB, but there is no significant change in total EphB. The activation of Eph pathway inhibited Notch induced up-regulation of tumor sphere formation and Sox2 and Oct4 expression. Conclusion Notch signaling pathway maintains the stem cell-like characteristics of osteosarcoma probably by inhibiting the Eph pathway.