We used a murine spontaneous osteosarcoma cell line with high metastatic potential, the K7M2 cell line to study the role of Notch signaling in the biological manifestations of osteosarcoma, to understand its underlying mechanism in the regulation of cell proliferation and migration, and to improve patient prognosis in cases of osteosarcoma through the discovery of novel therapeutic targets, First, Notch expression in K7M2 was determined by immunostaining, and the γ-secretase inhibitor N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) was used to inhibit proteolytic cleavage of the Notch intracellular domain (NICD), resulting in the inhibition of Notch activation. By using the Sulforhodamine B assay, colony-forming units assay, Brdu and Ki67 staining, and flow cytometry assays of apoptosis and cell cycle stage, DAPT was found to inhibit K7M2 proliferation in a dose-dependent manner. By using wound healing and transwell migration assays, DAPT was found to inhibit K7M2 migration in a dose-dependent manner as well. By using a combination of micro-Raman spectroscopy and K-means clustering analysis, we found that DAPT inhibit a variety of important cell metabolism-related components in most K7M2 cell structures. Then, DAPT was found to inhibit Notch1ICD expression in a concentration-dependent manner, and this expression was directly correlated with Phospho-Erk1/2 (p-Erk) by using Western blotting. To confirm this finding, we used the Notch signaling ligand Jagged1 to activate the Notch signaling pathway, which in turn up-regulated p-Erk, resulting in increased proliferation and migration of K7M2. Using the Erk pathway inhibitor U0126, we showed that p-Erk was downregulated and the proliferation and migration of K7M2 decreased along with it. Finally, we constructed a K7M2 mouse para-tibial tumor model and lung metastatic model. We found DAPT inhibits p-Erk in vivo, effectively controls tumor growth, reduces angiogenesis, reduces metastasis to the lungs, and improves overall survival. In summary, Notch signaling plays an oncogene role and promotes metastasis in osteosarcoma through p-Erk. DAPT effectively inhibits osteosarcoma proliferation and metastasis in vivo and in vitro by inhibiting Erk phosphorylation. Therefore, the inhibition of Notch activation resulted the down-regulation of phosphorylation of Erk pathway can be used as potential therapeutic targets in clinical treatment to improve osteosarcoma prognosis.

中文翻译：

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Notch信号通过Erk磷酸化调节骨肉瘤的增殖和迁移。

我们使用了具有高转移潜能的小鼠自发性骨肉瘤细胞系，K7M2细胞系来研究Notch信号在骨肉瘤生物学表现中的作用，了解其在调节细胞增殖和迁移中的潜在机制，并改善患者的预后通过发现新的治疗靶点而在骨肉瘤患者中，首先，通过免疫染色确定K7M2中的Notch表达，并使用γ-分泌酶抑制剂N- [N-（3,5-二氟苯乙酰基）-L-丙氨酰] -S-苯基甘氨酸叔丁酯（DAPT）用于抑制Notch细胞内结构域（NICD）的蛋白水解裂解，从而抑制Notch激活。通过使用磺基罗丹明B检测，集落形成单位检测，Brdu和Ki67染色以及流式细胞术检测细胞凋亡和细胞周期阶段，发现DAPT以剂量依赖性方式抑制K7M2增殖。通过使用伤口愈合和transwell迁移分析，发现DAPT也以剂量依赖性方式抑制K7M2迁移。通过结合使用显微拉曼光谱和K-均值聚类分析，我们发现DAPT抑制了大多数K7M2细胞结构中多种重要的细胞代谢相关成分。然后，发现DAPT以浓度依赖的方式抑制Notch1ICD表达，并且通过Western印迹将该表达与Phospho-Erk1 / 2（p-Erk）直接相关。为了证实这一发现，我们使用了Notch信号配体Jagged1激活了Notch信号通路，从而上调了p-Erk，导致K7M2的增殖和迁移增加。使用Erk途径抑制剂U0126，我们发现p-Erk被下调，K7M2的增殖和迁移也随之减少。最后，我们构建了K7M2小鼠胫骨旁肿瘤模型和肺转移模型。我们发现DAPT可在体内抑制p-Erk，有效控制肿瘤的生长，减少血管生成，减少向肺的转移并提高总体生存率。总之，Notch信号传导起癌基因的作用，并通过p-Erk促进骨肉瘤的转移。DAPT通过抑制Erk磷酸化，在体内外有效地抑制骨肉瘤的增殖和转移。因此，抑制Notch激活导致Erk途径磷酸化的下调可作为临床治疗中潜在的治疗靶点，以改善骨肉瘤的预后。