Silencing of MYCN by RNA interference induces growth inhibition, apoptotic activity and cell differentiation in a neuroblastoma cell line with MYCN amplification

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.