Department of Surgery, Division of Otolaryngology-Head and Neck Surgery, University of California-San Diego, 3350 La Jolla Village Drive, San Diego, CA 92161, U.S.A.
Otol Neurotol. 2010 Apr;31(3):460-6. doi: 10.1097/MAO.0b013e3181d2777f.
To investigate the early events in molecular progression toward schwannoma tumorigenesis, we developed an in vitro model of human Schwann cell tumorigenesis by merlin knockdown.
Neurofibromatosis 2 (NF2)-related and sporadic vestibular schwannoma (VS) exhibit loss of functional merlin (schwannomin). After loss of merlin expression in the Schwann cell, the initial steps toward VS tumorigenesis are unknown. Merlin, a putative tumor suppressor protein, interacts with many cellular proteins, regulating their function. Among these are receptor tyrosine kinases, including the epidermal growth factor receptor family B (ErbB) family receptors epidermal growth factor receptor and ErbB2. Functional merlin interacts with and internalizes these growth factor receptors, silencing their proliferation and survival signaling. Deregulation of CD44, the cell adhesion/signaling molecule and cancer stem cell marker, has also been implicated in VS tumorigenesis.
Merlin knockdown was performed using small interfering RNA transfection into human Schwann cell primary cultures. Knockdown was confirmed by real-time quantitative PCR, immunofluorescence, and Western analysis. Expression profiles of ErbB, merlin, and the stem cell markers nestin and CD44 were examined in knockdowns. Proliferation rate was assessed with bromodeoxyuridine incorporation, and radiation sensitivity was assessed using the Annexin assay in knockdowns versus controls.
Merlin knockdowns demonstrated increased proliferation rate, upregulation of epidermal growth factor receptor, ErbB2, and ErbB3, CD44, and nestin. Short-term merlin depletion had no effect on gamma irradiation sensitivity compared with controls.
Merlin depletion results in deregulation of ErbB receptor signaling, promotes a dedifferentiated state, and increases Schwann cell proliferation, suggesting critical steps toward schwannoma tumorigenesis.
为了研究向施万细胞瘤发生的分子进展的早期事件，我们通过 Merlin 敲低开发了一种人施万细胞肿瘤发生的体外模型。
神经纤维瘤病 2（NF2）相关和散发性前庭神经鞘瘤（VS）表现出 Merlin（施万omin）功能丧失。在施万细胞中 Merlin 表达丧失后，VS 肿瘤发生的最初步骤尚不清楚。 Merlin，一种假定的肿瘤抑制蛋白，与许多细胞蛋白相互作用，调节其功能。其中包括受体酪氨酸激酶，包括表皮生长因子受体家族 B（ErbB）家族受体表皮生长因子受体和 ErbB2。功能性 Merlin 与这些生长因子受体相互作用并内化它们，沉默其增殖和存活信号。细胞粘附/信号分子和癌症干细胞标志物 CD44 的失调也与 VS 肿瘤发生有关。
使用小干扰 RNA 转染到人施万细胞原代培养物中进行 Merlin 敲低。通过实时定量 PCR、免疫荧光和 Western 分析确认敲低。检查了敲低物中的 ErbB、 Merlin 和干细胞标志物巢蛋白和 CD44 的表达谱。通过溴脱氧尿苷掺入评估增殖率，并在敲低物与对照物之间使用 Annexin 测定评估辐射敏感性。
Merlin 敲低物显示增殖率增加，表皮生长因子受体、ErbB2 和 ErbB3、CD44 和巢蛋白上调。与对照物相比， Merlin 短期耗竭对γ辐射敏感性没有影响。
Merlin 耗竭导致 ErbB 受体信号转导失调，促进去分化状态并增加施万细胞增殖，提示向施万细胞瘤发生的关键步骤。
