Figure 1

Growth curves and cell cycle analysis. SK-N-AS^luc and NB1691^luc (1 × 10⁶) cells were reverse transfected with premiR-34a (30 μM) or a premiR-negative control molecule and cell pellets were analysed after 48 hours by qPCR for miR-34a, MYCN, BCL2, E2F1, E2F3 and CDC25A levels (Figure 1A and B, respectively). Additionally, cells were isolated at 24 hour intervals and nuclei were counted in triplicate for each sample using a Beckman Coulter Cell counter. Cells treated with synthetic miR-34a showed a marked reduction in cell growth relative to premiR-negative control-treated groups in both SK-N-AS^luc and NB1691^luc cells (Figure 1C and D, respectively * p = 0.004). In order to extend findings previously reported by Welch et al., [6] in SK-N-AS cells, premiR-34a- treated NB1691^luc (3 × 10⁵) cells were isolated 48 and 72 hours post-transfection and analysed by flow cytometry. Cell cycle and Annexin-V analysis was carried out on all samples (n = 3) and data was normalised to premiR-negative control treated cells (Figure 1E and 1). Findings indicate that over expression of miR-34a leads to significant reduction in S phase progression (*p < 0.01), an increase in G0/G1 initiation (**p < 0.001) and a corresponding increase in apoptosis (*p < 0.01).