Silencing of MYCN by RNA interference induces growth inhibition, apoptotic activity and cell differentiation in a neuroblastoma cell line with MYCN amplification.
Silencing of MYCN by RNA interference induces growth inhibition, apoptotic activity and cell differentiation in a neuroblastoma cell line with MYCN amplification.
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DOI：
10.3892/ijo.30.5.1189
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发表时间：
2007-05
影响因子：
5.2
通讯作者：
Keigo Nara;T. Kusafuka;A. Yoneda;T. Oue;S. Sangkhathat;M. Fukuzawa
中科院分区：
文献类型：
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作者：
Keigo Nara;T. Kusafuka;A. Yoneda;T. Oue;S. Sangkhathat;M. Fukuzawa
尽管有人认为 MYCN 癌蛋白功能可能影响神经母细胞瘤的肿瘤发生和患者生存，但这些功能的机制仍不清楚。为了阐明这种分子和生物学机制，我们使用 RNA 干扰 (RNAi) 方法进行了 MYCN 表达的敲除。将 MYCN-siRNA (MYCN-siRNA) 转染至 MYCN 扩增的细胞系 NB-1 中。为了验证siRNA的序列特异性，我们准备了三个对照组（siRNA对照组：与人类基因组中任何已知序列没有显着同源性的siRNA、模拟对照组：试剂和PBS、以及未转染对照组）。通过实时RT-PCR、蛋白质印迹、免疫细胞化学分析细胞的基因表达。通过WST-1测定来测量细胞增殖活性。进行TUNEL染色以评估细胞凋亡。 MYCN-siRNA转染后，MYCN mRNA的表达水平显着降低至三个对照组的30%（p<0.05）。 Western blotting结果显示，MYCN-siRNA组的MYCN蛋白水平明显降低。在免疫细胞化学上，MYCN-siRNA组的MYCN核染色强度弱于三个对照组。在 WST-1 活力测定中，与三个对照组相比，MYCN-siRNA 转染后的细胞增殖受到显着抑制（p<0.05）。 MYCN-siRNA组中经常观察到TUNEL阳性细胞。此外，MYCN-siRNA转染后，观察到提示神经元细胞分化的形态变化，并且TrkA和TrkC表达也显着上调。使用RNAi方法，MYCN表达的敲低诱导了MYCN扩增的NB-1细胞系的生长抑制、凋亡活性和细胞分化。
Although it has been suggested that the MYCN oncoprotein functions may influence tumorigenesis and patient survival in neuroblastoma, the mechanism of these functions remains unclear. To elucidate such molecular and biological mechanisms, we performed knock-down of MYCN expression using RNA interference (RNAi) method. MYCN-siRNAs (MYCN-siRNA) were transfected into the MYCN-amplified cell line NB-1. To verify the sequence specificity of the siRNA, we prepared three control groups (siRNA control group: siRNAs with no significant homology to any known sequences in human genome, mock control group: reagent and PBS, and the untransfected control group). The cells were analyzed by real-time RT-PCR, Western blotting, immunocytochemistry for gene expression. Cell proliferation activity was measured by WST-1 assay. TUNEL staining was performed to evaluate apoptosis. After the MYCN-siRNA transfection, the expression level of the MYCN mRNA was significantly reduced to 30% of those of the three control groups (p<0.05). Western blotting revealed an obvious reduction in MYCN protein level in the MYCN-siRNA group. On immunocytochemistry, intensity of nuclear staining of MYCN was weaker in the MYCN-siRNA group than in the three control groups. On WST-1 viability assay, cell proliferation after the MYCN-siRNA transfection was significantly suppressed compared to the three control groups (p<0.05). The TUNEL positive cells were frequently observed in the MYCN-siRNA group. Additionally, after the MYCN-siRNA transfection, the morphologic change which was suggestive of neuronal cell differentiation was observed and TrkA and TrkC expressions were also significantly up-regulated. Using RNAi method, the knock-down of MYCN expression induced growth-inhibition, apoptotic activity and cell differentiation in MYCN-amplified NB-1 cell line.