PCP4/PEP19 downregulates neurite outgrowth via transcriptional regulation of Ascl1 and NeuroD1 expression in human neuroblastoma M17 cells
PCP4/PEP19 downregulates neurite outgrowth via transcriptional regulation of Ascl1 and NeuroD1 expression in human neuroblastoma M17 cells
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DOI：
10.1038/s41374-020-0462-z
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发表时间：
2020-07-08
影响因子：
5
通讯作者：
Tanimoto, Akihide
中科院分区：
文献类型：
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作者：
Kitazono, Ikumi;Hamada, Taiji;Tanimoto, Akihide
浦肯野细胞蛋白4/19(PCP4/PEP19)是最早发现于浦肯野细胞的7.6 kDa多肽。PCP4/PEP19是浦肯野细胞的分化标记物，在浦肯野细胞中起抗凋亡因子的作用。脑神经细胞也表达PCP4/PEP19，可能与神经细胞存活有关。然而，有证据表明，PCP4/PEP19也可能参与了神经元的分化。在此，我们通过分析培养的人神经母细胞瘤M17细胞的突起生长和神经元分化标志物的表达，探讨PCP4/PEP19的表达对神经元分化的影响。当PCP4/PEP19的表达被siRNA介导的基因敲除后，突起生长显著增加。在众多分化标志物中，PCP4/PEP19基因敲除后，NeuroD1的表达增加，而Ascl1的表达降低。此外，荧光素酶报告分析显示，PCP4/PEP19基因敲除后在转录水平上上调了NeuroD1的表达，下调了Ascl1的表达。这些结果提示了PCP4/PEP19的一种新功能，它通过调节NeuroD1和Ascl1的表达来抑制M17细胞的轴突生长和神经元分化。此外，免疫组织化学研究表明，PCP4/PEP19定位于人神经母细胞瘤细胞的细胞核。因此，PCP4/PEP19也可能是神经分化的核内负调控因子，因此可能成为促进神经母细胞瘤细胞分化的潜在治疗靶点。在神经母细胞瘤细胞中，PCP4/PEP19基因敲除后可诱导神经突起生长，上调NeuroD1的表达，下调Ascl1的表达，提示PCP4/PEP19可能通过调节NeuroD1和Ascl1的表达来抑制神经突起的生长和神经元的分化。免疫组织化学显示PCP4/PEP19在神经母细胞瘤细胞中有核定位。因此，PCP4/PEP19可能是神经元分化的核内负调控因子。
Purkinje cell protein 4/peptide 19 (PCP4/PEP19) is 7.6 kDa peptide originally found in Purkinje cells. PCP4/PEP19 is a differentiation maker of Purkinje cells, where it functions as an antiapoptotic factor. Cerebral neuronal cells also express PCP4/PEP19, which may be related to neuronal cell survival. However, evidence suggests that PCP4/PEP19 may also be involved in neuronal differentiation. Here, we investigated the effects of PCP4/PEP19 expression on neuronal differentiation by analyzing neurite outgrowth, and expression of neuronal differentiation markers in cultured human neuroblastoma M17 cells. When PCP4/PEP19 expression was reduced by siRNA-mediated knockdown, neurite outgrowth was significantly increased. Among many differentiation markers tested, expression of NeuroD1 was increased, while that of Ascl1 was decreased upon PCP4/PEP19 knockdown. Furthermore, luciferase reporter assays revealed that PCP4/PEP19 knockdown upregulated NeuroD1 and downregulated Ascl1 expression, at the transcriptional level. These results suggest a new function of PCP4/PEP19, which suppresses neurite outgrowth and neuronal differentiation through the regulation of NeuroD1 and Ascl1 expression in M17 cells. Furthermore, immunohistochemical studies showed that PCP4/PEP19 localizes in the nuclei of human neuroblastoma cells. Therefore, PCP4/PEP19 may also be an intranuclear negative regulator of neuronal differentiation and may thus be a potential therapeutic target to promote cellular differentiation in human neuroblastoma.PCP4/PEP19 knockdown in neuroblastoma cells induce neurite outgrowth, upregulation of NeuroD1 and downregulation of Ascl1 expression, suggesting that PCP4/PEP19 can suppress neurite outgrowth and neuronal differentiation through the regulation of NeuroD1 and Ascl1. Immunohistochemistry shows nuclear localization of PCP4/PEP19 in neuroblastoma cells. PCP4/PEP19 may therefore be an intranuclear negative regulator of neuronal differentiation.