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Silencing of MYCN by RNA interference in neuroblastoma

通过RNA干扰沉默神经母细胞瘤中的MYCN

基本信息

批准号：
17591861
负责人：
FUKUZAWA Masahiro
金额：
$ 2.05万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17591861/
关键词：
Neuroblastoma MYCN RNAi NB-1 アポトーシス分化

项目摘要

Although it has been suggested that the MYCN oncoprotein functions may influence tumorigenesis and patient survival in neuroblastoma (NB), 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. 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. TrkA, B, C and Ha-ras expression were analyzed by real time RT-PCR and morphological changes and differentiation appearance of the cells were evaluated before and after RNAi. After the MYCN-siRNA transfection, the expression level of the MYCN mRNA was significantly reduced to 30% of those of the cells before transfection and Western blotting revealed an obvious reduction in MYCN … More protein. On immunocytochemistry, intensity of nuclear staining of MYCN was weaker in the MYCN-siRNA transfected cells than in the cells before transfection. On WST-1 viability assay, cell proliferation after the MYCN-siRNA transfection was significantly suppressed. The TUNEL positive cells were frequently observed in the MYCN-siRMA transfected cells. Simultaneously multidirectional neurite extension and nuclear enlargement was observed. These morphological changes were consistent with neuronal differentiation in neuroblastoma. Moreover, TrkA and TrkC expression were significantly up-regulated after silencing MYCN. On the other hand, TrkB expression was down-regulated after silencing MYCN. Ha-ras expression did not change between before and after the transfection.In conclusion, using RNAi method, the knock-down of MYCN expression induced growth-inhibition, apoptotic activity and cell differentiation in MYCN-amplified NB-1 cell line. Thus, MYCN silencing by RNAi may provide a potential novel therapeutic option for aggressive neuroblastomas. Less

尽管有人认为 MYCN 癌蛋白功能可能影响神经母细胞瘤 (NB) 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表达的敲低。将MYCN siRNA（MYCN-siRNA）转染到MYCN扩增的细胞系NB-1中。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。通过实时RT-PCR分析TrkA、B、C和Ha-ras表达，并评估RNAi前后细胞的形态变化和分化外观。 MYCN-siRNA转染后，MYCN mRNA的表达水平显着降低至转染前细胞的30%，Western blotting显示MYCN蛋白明显减少。在免疫细胞化学上，MYCN-siRNA转染细胞中MYCN的核染色强度比转染前的细胞弱。在WST-1活力测定中，MYCN-siRNA转染后的细胞增殖被显着抑制。在MYCN-siRMA转染的细胞中经常观察到TUNEL阳性细胞。同时观察到多向神经突延伸和核增大。这些形态学变化与神经母细胞瘤中的神经元分化一致。此外，沉默 MYCN 后，TrkA 和 TrkC 表达显着上调。另一方面，沉默 MYCN 后 TrkB 表达下调。 Ha-ras表达在转染前后没有变化。总之，利用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。因此，RNAi 沉默 MYCN 可能为侵袭性神经母细胞瘤提供潜在的新治疗选择。较少的