MYCN silencing induces differentiation and apoptosis in human neuroblastoma cells

Abstract

MYCN amplification strongly correlates with unfavorable outcomes in patients with neuroblastoma. The aim of this study was to investigate the role of MYCN in neuroblastoma cell differentiation and apoptosis. We used the technique of RNA interference to inhibit MYCN gene expression in neuroblastoma cells with variable expression of MYCN. Our results showed that inhibition of MYCN gene expression in MYCN amplified cells induced apoptosis and suppressed cell growth; neuronal differentiation also occurred after MYCN gene silencing. Moreover, N-myc downregulation was associated with decreased Bcl-xL protein levels and caspase-3 activation. These data show that small interfering RNA directed to MYCN, which plays a crucial role in neuroblastoma cell survival, may provide a potential novel therapeutic option for aggressive neuroblastomas.

Figure 1. Knockdown of N-myc expression using small interfering RNA in MYCN amplified and non-amplified neuroblastoma cell lines

(A) Western blot analysis of N-myc protein expression in MYCN amplified cell lines; LAN-1, IMR-32, JF and MYCN non-amplified SK-N-SH cells at 24 h post-transfection with siMYCN. (B) Real time RT-PCR analysis of MYCN mRNA expression in LAN-1, IMR-32, JF and SK-N-SH at 6 h post-transfection.

Figure 2. siRNA-mediated inhibition of N-myc expression and expression in aggressive neuroblastoma cell line BE(2)-C

(A) N-myc protein in BE(2)-C cells transfected with siRNA against MYCN or NTC at 8, 12, 24, 48 and 72 h. β-actin was used to check for equal protein loading of cell lysates. (B) WST-8 assay was used to compare the growth of siMYCN and siNTC transfected BE(2)-C cells. (C) DNA fragmentation was measured at 48, 72 h post-transfection with either siMYCN or siNTC. Data represent mean ± SEM. *P < 0.05. (D) BE(2)-C cells were transfected with siRNA against MYCN and protein levels for N-myc, Bcl-xL and cleaved caspase-3 were detected at 48 h post-transfection. β-actin was used to check for equal protein loading.

Figure 3. MYCN downregulation by RNAi induces apoptosis in MYCN amplified neuroblastoma cells

(A) Cells were transfected with siMYCN or siNTC and DNA fragmentation was analyzed at 48, 72 h post-transfection. Data represent mean ± SE. *P < 0.05. (B) SK-N-SH and SH-SY5Y cells were transfected with siMYCN and siNTC. After 72 h, the cells were analyzed by Western blot for caspase-3 and cleaved caspase-3. β-actin was used to check for equal protein loading.

Figure 4. MYCN downregulation by RNAi induces differentiation in MYCN amplified neuroblastoma cells

(A) BE(2)-C, LAN-1 and IMR-32 cells were transfected with siMYCN or siNTC. Cell morphology was analyzed by microscopy with representative images shown. (B) IMR-32 cells were transfected with siMYCN or siNTC and the protein was analyzed by Western blot for N-myc and NSE at 8 and 12 h post-transfection. β-actin was used to check for equal protein loading.