| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2006: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2005: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Research Abstract |
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
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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
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