目的:探索雷公藤内酯(triptolide, TP)治疗骨肉瘤(osteosarcoma, OS)的药理学机制和生物学途径。方法:通过全基因组测序,拓扑分析,多种公共数据库以及体外实验和临床组织等方法分析TP治疗OS的靶基因和介导的信号通路。结果:100 nM TP能有效抑制OS细胞系U2OS细胞增殖能力和活力,抑制细胞迁移和侵袭能力,促进U2OS细胞凋亡(P＜0.05)。使用RT-qPCR和蛋白免疫印迹分析OS和正常组织的NOC3样DNA复制调节因子(NOC3 like DNA replication regulator, NOC3L)、DEAH-box解旋酶15(DEAH-Box Helicase 15, DHX15)及WD重复域3(WD repeat domain 3, WDR3)mRNA和蛋白质表达,结果显示NOC3L、DHX15及WDR3 mRNA和蛋白质表达在OS组织中显著增高(P＜0.05);100 nM TP处理U2OS细胞24 h后,RT-qPCR和蛋白免疫印迹结果显示NOC3L、DHX15及WDR3 mRNA和蛋白质表达显著降低(P＜0.05);此外,细胞免疫荧光标记证实100 nM TP能有效抑制NOC3L、DHX15及WDR3表达;TP信号通路富集结果主要在Wnt信号通路,蛋白免疫印迹分析(以GAPDH为内参)结果显示,GSK-3β和p-GSK-3β表达增加,β-catenin和β-catenin(qSer)表达降低(P＜0.05)。结论:TP可通过抑制NOC3L、DHX15及WDR3表达和Wnt信号通路抑制OS进展。
Objective: To explore the pharmacological mechanism and biological pathway of triptolide (TP) in the treatment of osteosarcoma (OS). Methods: The target genes and mediated signaling pathways of TP for OS were analyzed by whole genome sequencing, topological analysis, multiple public databases as well as in vitro experiments and clinical tissues. Results: 100 nM TP could effectively inhibit the proliferation and viability of U2OS cells, inhibit cell migration and invasion, and promote apoptosis of U2OS cells (P＜0.05). The mRNA and protein expressions of NOC3 like DNA replication regulator(NOC3L)、DEAH-Box Helicase 15(DHX15)and WD repeat domain 3(WDR3) in OS and normal tissues were significantly increased by RT-qPCR and western blotting (P＜0.05), and the results showed that NOC3L, DHX15 and WDR3 were significantly increased in OS tissues (P＜0.05). After U2OS cells were treated with 100 nM TP for 24 h, RT-qPCR and Western blot results showed that the mRNA and protein expression of NOC3L, DHX15 and WDR3 were significantly decreased (P＜0.05). In addition, cell immunofluorescence labeling confirmed that 100 nM TP could effectively inhibit the expression of NOC3L, DHX15 and WDR3. The enrichment results of TP signaling pathway were mainly in Wnt signaling pathway. Western blot analysis (with GAPDH as internal reference) showed that the expression of GSK-3β and p-GSK-3β increased, and the expression of β-catenin and β-catenin (qSer) decreased (P＜0.05). Conclusion: TP inhibits OS progression by suppressing NOC3L, DHX15 and WDR3 expression and Wnt signaling pathway.
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