Combined EphB2 receptor knockdown with radiation decreases cell viability and invasion in medulloblastoma.
Combined EphB2 receptor knockdown with radiation decreases cell viability and invasion in medulloblastoma.
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DOI：
10.1186/s12935-017-0409-7
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发表时间：
2017
影响因子：
5.8
通讯作者：
Karam SD
中科院分区：
文献类型：
--
作者：
Bhatia S;Hirsch K;Bukkapatnam S;Baig NA;Oweida A;Griego A;Calame D;Sharma J;Donson A;Foreman N;Albanese C;Venkataraman S;Vibhakar R;Karam SD
髓母细胞瘤是最常见的小儿脑肿瘤类型之一，其特征在于细胞亚群表现出高侵袭潜力和耐辐射特性。此外，Eph受体家族的功能和信号转导失调已被证明在这种脑恶性肿瘤中赋予促肿瘤发生特征。在目前的研究中，我们研究了EphB 2敲低与辐射结合是否可以改变体外髓母细胞瘤的侵袭性和减少肿瘤的生长或存活率。免疫组化和Western blotting检测EphB 2受体的表达。对髓母细胞瘤患者数据集进行微阵列分析和mRNA分析，并与正常小脑进行比较。通过在人髓母细胞瘤细胞中的克隆形成测定来确定EphB 2敲低后的放射增敏作用。通过流式细胞术、MTT法、台盼蓝拒染法、xcelligence系统和Western印迹法分析EphB 2-siRNA在不存在或存在辐射的情况下对细胞周期分布、细胞活力和侵袭力的影响。我们观察到EphB 2在髓母细胞瘤细胞系和患者样品中表达，并且其下调使这些细胞对辐射敏感，如通过降低的克隆形成存活分数所证明的。与正常小脑相比，EphB 2在不同的髓母细胞瘤亚组中的表达也很高。EphB 2敲低后观察到的放射增敏作用部分由增强的G2/M细胞周期阻滞介导。我们还发现，与对照组相比，EphB 2敲低和辐射暴露的组合方法显著降低了髓母细胞瘤细胞的总体细胞活力。在基于xcelligence的侵袭测定中获得了类似的结果。蛋白质印迹分析还表明，与其他组相比，联合组中细胞增殖、细胞存活和侵袭分子的蛋白质表达发生了变化。总体而言，我们的研究结果表明，EphB 2受体的特异性靶向结合放射治疗可以作为髓母细胞瘤的有效治疗策略。未来的研究有必要在体内临床前模型中测试这种方法的有效性。本文的在线版本（doi：10.1186/s12935-017-0409-7）包含补充材料，可供授权用户使用。
Medulloblastoma is one of the most common types of pediatric brain tumor characterized by the subpopulation of cells that exhibit high invasive potential and radioresistant properties. In addition, dysregulated function and signaling by Eph family of receptors have been shown to impart pro-tumorigenic characteristics in this brain malignancy. In the current study, we investigated whether EphB2 knockdown in combination with radiation can alter invasiveness and decrease medulloblastoma tumor growth or viability in vitro. The expression of EphB2 receptor was analyzed by immunohistochemistry and Western blotting. Microarray analysis and mRNA analysis was performed on medulloblastoma patient datasets and compared to the normal cerebellum. The radiosensitization effect following EphB2 knockdown was determined by clonogenic assay in human medulloblastoma cells. Effects of EphB2-siRNA in absence or presence of radiation on cell cycle distribution, cell viability, and invasion were analyzed by flow cytometry, MTT assay, trypan blue exclusion assay, xcelligence system, and Western blotting. We observed that EphB2 is expressed in both medulloblastoma cell lines and patient samples and its downregulation sensitized these cells to radiation as evident by decreased clonogenic survival fractions. EphB2 expression was also high across different medulloblastoma subgroups compared to normal cerebellum. The radiosensitization effect observed following EphB2 knockdown was in part mediated by enhanced G2/M cell cycle arrest. We also found that the combined approach of EphB2 knockdown and radiation exposure significantly reduced overall cell viability in medulloblastoma cells compared to control groups. Similar results were obtained in the xcelligence-based invasion assay. Western blot analysis also demonstrated changes in the protein expression of cell proliferation, cell survival, and invasion molecules in the combination group versus others. Overall, our findings indicate that specific targeting of EphB2 receptor in combination with radiation may serve as an effective therapeutic strategy in medulloblastoma. Future studies are warranted to test the efficacy of this approach in in vivo preclinical models. The online version of this article (doi:10.1186/s12935-017-0409-7) contains supplementary material, which is available to authorized users.