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. 2020 Aug;30(4):237-248.
doi: 10.1089/nat.2019.0831. Epub 2020 Apr 2.

MYCN Silencing by RNAi Induces Neurogenesis and Suppresses Proliferation in Models of Neuroblastoma with Resistance to Retinoic Acid

Ruhina Maeshima  1 , Dale Moulding  2 , Andrew W Stoker  3 , Stephen L Hart  1
Affiliations

MYCN Silencing by RNAi Induces Neurogenesis and Suppresses Proliferation in Models of Neuroblastoma with Resistance to Retinoic Acid

Ruhina Maeshima et al. Nucleic Acid Ther. 2020 Aug.

Abstract

Neuroblastoma (NB) is the most common solid tumor in childhood. Twenty percent of patients display MYCN amplification, which indicates a very poor prognosis. MYCN is a highly specific _target for an NB tumor therapy as MYCN expression is absent or very low in most normal cells, while, as a transcription factor, it regulates many essential cell activities in tumor cells. We aim to develop a therapy for NB based on MYCN silencing by short interfering RNA (siRNA) molecules, which can silence _target genes by RNA interference (RNAi), a naturally occurring method of gene silencing. It has been shown previously that MYCN silencing can induce apoptosis and differentiation in MYCN amplified NB. In this article, we have demonstrated that siRNA-mediated silencing of MYCN in MYCN-amplified NB cells induced neurogenesis in NB cells, whereas retinoic acid (RA) treatment did not. RA can differentiate NB cells and is used for treatment of residual disease after surgery or chemotherapy, but resistance can develop. In addition, MYCN siRNA treatment suppressed growth in a MYCN-amplified NB cell line more than that by RA. Our result suggests that gene therapy using RNAi _targeting MYCN can be a novel therapy toward MYCN-amplified NB that have complete or partial resistance toward RA.

Keywords: MYCN; neuroblastoma; retinoic acid treatment; siRNA.

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Conflict of interest statement

No competing financial interests exist.

Figures

FIG. 1.
FIG. 1.
Cytotoxicity assay. Cell viability of SK-N-BE(2) cells treated with 50 or 10 nM siMYCN/siNeg was assessed using MTS assay reagent. SiRNA transfections at 50 nM using RNAiMAX were notably toxic, while the cell viability of the cells treated with 10 nM siRNAs was almost the same or slightly lower (statistically nonsignificant) to untransfected negative control cells (P < 0.001 between 50 nM siMYCN and 10 nM siMYCN, and P < 0.001 between 50 nM siMYCN and untransfected control cells). In addition, the values between 50 nM siNeg and untransfected control cells were also statistically different, with P < 0.01 indicating that toxicity was due to the formulation itself rather than MYCN silencing. The intensity was normalized to untransfected negative control cells (n = 5). In the graph, each bar represents the mean ± SD, ***P < 0.001. SD, standard deviation; siRNA, short interfering RNA.
FIG. 2.
FIG. 2.
Relative expression of MYCN mRNA and NTRK1 mRNA in NB cell lines 48 h after siMYCN transfection quantified by qRT-PCR. (a) SK-N-BE(2), Kelly, LAN-5, and SK-N-SH cells were treated with siMYCN and siNeg for 48 h, and the MYCN mRNA expression level was quantified using qRT-PCR. The values were normalized by the value of siNeg at each concentration. siMYCN silenced MYCN up to 45.9% in SK-N-BE(2), 51.2% in Kelly, 12.5 in LAN-5, and 67.5% in SK-N-SH cells. All the concentrations of siMYCN significantly reduced MYCN mRNA in the three cell lines, except LAN-5 (n = 3). (b) After the transfection, mRNA of NTRK1 was quantified by qRT-PCR. NTRK1 was significantly upregulated 2-3-fold in SK-N-BE(2) cells and 1.3- to 4.4-fold in Kelly cells, compared to cells treated with siNeg (n = 3). In all the graphs, each bar represents the mean ± SD, **P < 0.01, ***P < 0.001. NB, neuroblastoma; qRT-PCR, quantitative real-time RT-PCR.
FIG. 3.
FIG. 3.
Immunoblotting of N-Myc and Trk following siMYCN transfections. The number under the band is the relative expression level calculated from the intensity of the band. (a) SK-N-BE(2) (n = 3) and (b) Kelly cells (n = 2) were transfected with siMYCN/siNeg for 3 days, and the samples were probed with anti-N-Myc antibody and anti-Pan-Trk antibody. N-Myc bands appeared between 64 and 51 kDa, and Pan-Trk bands above 97 kDa. The bands were quantified and normalized to untransfected control cells and plotted in charts for N-Myc and Trk, and compared by a Student's t-test (**P < 0.01, *P < 0.05). siMYCN induced N-Myc reduction and Trk upregulation. (c, d) Comparison of the effects on N-Myc and Trk expressions of treatment with RA in (c) SK-N-BE(2) (n = 2) and (d) Kelly (n = 2) cells shown in immunoblots and quantified in charts. Each bar represents the mean, and the error bar is SD. Each filled square, triangle or circle represents data points from the same individual experiment (n = 2 or 3).
FIG. 3.
FIG. 3.
Immunoblotting of N-Myc and Trk following siMYCN transfections. The number under the band is the relative expression level calculated from the intensity of the band. (a) SK-N-BE(2) (n = 3) and (b) Kelly cells (n = 2) were transfected with siMYCN/siNeg for 3 days, and the samples were probed with anti-N-Myc antibody and anti-Pan-Trk antibody. N-Myc bands appeared between 64 and 51 kDa, and Pan-Trk bands above 97 kDa. The bands were quantified and normalized to untransfected control cells and plotted in charts for N-Myc and Trk, and compared by a Student's t-test (**P < 0.01, *P < 0.05). siMYCN induced N-Myc reduction and Trk upregulation. (c, d) Comparison of the effects on N-Myc and Trk expressions of treatment with RA in (c) SK-N-BE(2) (n = 2) and (d) Kelly (n = 2) cells shown in immunoblots and quantified in charts. Each bar represents the mean, and the error bar is SD. Each filled square, triangle or circle represents data points from the same individual experiment (n = 2 or 3).
FIG. 4.
FIG. 4.
Cell morphology changes after siMYCN transfection or RA treatment. (a) SK-N-BE(2) cells were transfected with siMYCN/siNeg and incubated for 6 days. The morphology was altered by transfection relative to controls, with the formation of elongated neurites (arrows in 10 nM siMYCN) and the presence of rounder and smaller cell bodies. The scale bar represents 100 μm. (b) SK-N-SH, LAN-5, SK-N-BE(2), and Kelly cells were treated with 5 or 10 μM RA for 48 h. RA induced neurite elongation in SK-N-SH and LAN-5 cells, while SK-N-BE(2) and Kelly cells did not respond significantly with respect to their morphology. The scale bar represents 100 μm.
FIG. 5.
FIG. 5.
Quantification of neurites after siMYCN transfection of SK-N-BE(2) cells. siMYCN significantly induced differentiation in SK-N-BE(2) cells (n = 10), and there were significant differences in (a) the neurite length and (b) the number of the neurites between siMYCN and siNeg. The neurite length on day 6 was longer than that in day 2, while the number of the neurite was almost the same during the 4 days. In all the graphs, each bar represents the mean ± SD, ***P < 0.001.
FIG. 6.
FIG. 6.
Analysis of cell viability after siMYCN treatment in SK-N-BE(2) cells. (a) SK-N-BE(2) cells were treated with 10 nM siMYCN, and the cell growth rates were measured using the CCK-8 assay reagent at four time points. Ten nanomolars siMYCN remarkably reduced the growth rate during the 3 days (n = 3). Each dot represents the mean, and error bar is SD.*P < 0.05, **P < 0.01, ***P < 0.001. (b) SK-K-BE(2) cells transfected with 10 nM siMYCN and siNeg with/without DMSO or treated with 5 μM RA. The proliferation rate was measured using resazurin in PBS on day 6 time point. Each bar represents the mean, and the error bar is SD. Two-way ANOVA Bonferroni's multiple comparisons test was performed (Table 1), *P < 0.05, **P < 0.01, ***P < 0.001. DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline.
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