Box plots illustrating miR-542-5p expression in (A) MYCN amplified (MNA) versus non-MNA tumors; (B) different genetic subtypes of neuroblastoma; (C) different disease stages; and (D) different ages at diagnosis.

Number of invading cells detected per field at 200X magnification for Kelly (A), NB1691 (B) and SKNAS (C) cells transiently transfected with PremiR-542-5p, AntimiR-542-5p and a scrambled oligonucleotide negative control (NC). Graphs represent results from two biological replicate experiments with n=3 technical repeats. All p-values are relative to the negative control. Representative images of invading cells stained with crystal violet are displayed to the right of each set of graphs.

(A) Animals were sacrificed at moribundity and Mantle-Cox statistical analysis was used to compare overall survival in xenograft cohorts. (n = 4–7 animals per group; p < 0.003 for both NB1691 and SKNAS models). (B) The number of metastases counted post-mortem in Pre-miR 542-5p-treated groups compared to negative control (scrambled) groups. (C) qPCR validation of genes identified as differentially expressed in response to miR-542-5p by microarray gene expression profiling of Kelly cells (see Supplementary Table 1). Average fold change of genes is represented.

Kaplan-Meier survival plots confirm MYCN amplification as a significant risk factor in the 144 tumor dataset profiled for miRNA expression (A and B). Association between the lack of miR-542-5p expression (expression = 0) and lower EFS (p value = 0.0002) and OS (p value = 0.0013) (C and D).

Kelly (A), SKNAS (B), and NB1691 (C) were transiently transfected with PremiR-542-5p, AntimiR-542-5p, a scrambled oligonucleotide (negative control) or a kinesin siRNA (positive control for proliferation arrest). Relative cell number was determined over a 96hr period using an MTT assay. Graphs represent results from two biological replicate experiments with n=3 technical repeats.

NB1691 or SKNAS cells stably expressing firefly luciferase were transfected with either miR-542-5p mimics or negative control oligonucleotide and then introduced into the retroperitoneal cavity of CB17-SCID immune compromised mice (n = 4–7). Representative bioluminescent images of scanned mice obtained in negative control transfected animals compared to Pre-miR 542-5p-treated groups for NB1691 (A) and SKNAS (B) at day 21 post-inoculation. Note that scales of bioluminescence intensity differ for each image. Imaging using an IVIS Imaging System 100 Series was carried out at days 6, 10, 14 and 21 post-tumor cell inoculation for NB1691 (C) and SKNAS (D) inoculated mice and the data analysed and presented as the mean value for each cohort (photons/sec/cm²) ± SEM. P-values are for day 21 post tumor cell inoculation.