靶向YAP协同强化selumetinib抑制神经纤维瘤相关施万细胞生长的体外研究

YAP targeting collaboratively enhanced the inhibition effects of selumetinib to neurofibroma-Schwann cells in vitro

目的:探讨selumetinib对神经纤维瘤相关施万细胞中YAP功能状态的影响,分析和评价联合靶向YAP及MEK1/2对施万细胞生长的体外抑制作用。方法:采用慢病毒转染构建NF1敲低的大鼠施万细胞模型,通过Western免疫印迹、RT-PCR方法验证敲低效率,结合免疫荧光染色,评估YAP蛋白的表达水平及功能状态。利用siRNA敲低YAP的表达。通过CCK-8实验检测不同药物(YAP抑制剂veteporfin、MEK1/2抑制剂selumetinib)处理条件下肿瘤细胞的增殖活性,平板克隆和微球形成实验观察不同处理条件下肿瘤细胞的体外肿瘤形成能力。根据Chou-Talalay法开展selumetinib和Veteporfin的联合指数分析。采用GraphPad Prism 8软件包对数据进行统计学分析。结果:成功构建了Nf1基因沉默的施万细胞模型(shNf1-RSC96),证实selumetinib促进神经纤维瘤相关施万细胞的YAP转录活性。进一步抑制YAP基因表达后,selumetinib对shNf1-RSC96细胞增殖的抑制作用得到显著增强。联合靶向抑制YAP及MEK1/2,Veteporfin增强selumetinib对肿瘤细胞活性的抑制并具有协同效应。结论:干预神经纤维瘤相关施万细胞中YAP的核易位及转录激活过程,可增强selumetinib对细胞增殖的抑制作用,联合靶向抑制YAP及MEK1/2可能是NF1相关神经纤维瘤潜在的治疗策略。

PURPOSE:To explore the functional status of YAP under selumetinib treatment,and to assess the efficacy of dual targeting YAP and MEK1/2 in inhibition of NF1-Schwann cells in vitro.METHODS:NF1-Schwann cell model was established by lentivirus transfection.RT-PCR and Western blotting were performed to identify the efficacy of gene knockdown.RT-PCR,Western blotting combined with immunofluorescence were carried out to detect the functional status of YAP.siRNA was adopted to knockdown YAP gene expression.CCK-8 was used to evaluate cell viability of Schwann cells under treatment of different drug combination(YAP inhibitor veteporfin and MEK1/2 inhibitor selumetinib).Colony and sphere formation were used to detect the ability of tumor formation in vitro.Combined index analysis was performed to predict the synergistic effect via Chou-Talalay method.GraphPad Prism 8 software package was used for data analysis.RESULTS:Herein,we established a NF1-neurofibroma Schwann cell model(shNf1-RSC96).Increased nuclear transloca-tion and transcriptional activation of YAP were confirmed under selumetinib intervening.Genetic inhibition of YAP signif-icantly enhanced the cytotoxicity of selumetinib to NF1-Schwann cells in vitro.Additionally,combined treatment with Verteporfin and selumetinib showed a synergistic effect in vitro.CONCLUSIONS:Blocking the nuclear translocation and transcriptional activation of YAP enhances inhibition effect of selumetinib to neurofibroma-Schwann cells.Dual targeting of YAP and MEK1/2 might be a promising therapeutic strategy for treating NF1-neurofibroma.