Figure 6.
miR-885-5p-induced growth arrest is p53 dependent in TP53 wild-type neuroblastoma cells. (a) IMR32, SH-EP and KELLY cells were transfected with siRNA against p53 or control siRNA using Lipofectamine 2000 and transfected with miRNA mimics after 24 h. p53 knockdown efficiency was evaluated at day 4 after transfection (insert below). Cell viability was measured by Alamar Blue assay for 24, 48 and 72 h after miRNA mimic transfection (upper panel). The data are represented as the means±S.D. of three experiments. The cell cycle profiles were performed by FACS analysis (middle panel). (a, insert) p53 knockdown efficiency is shown in western blots in IMR32, KELLY and SH-EP. (b) p53 knockdown and control siRNA transfected IMR32, KELLY and SH-EP cells were transfected with miRNA mimics and assayed for anchorage-independent growth in soft agar after 2 weeks in culture. Colonies were fixed and stained with crystal violet and counted using ImageJ software. The graphs show mean percentages of colonies number±S.D. of three experiments. (c) Protein expression of p21waf1, PUMA and p53 is shown in western blots of p53 knockdown and control siRNA transfected IMR32, KELLY and SH-EP cells at 3 days after transfection of miRNA mimics. (d) IMR32 and SK-N-BE(2)c were transfected with antimiR-885-5p or control antimiR (30 n) and assayed for anchorage-independent growth for 2 weeks. Colonies were counted using ImageJ software (mean percentages of colonies number±S.D. of three experiments are presented). Protein expression of p53, p21waf1, PUMA, CDK2 and MCM5 in antimiR-transfected IMR32 and SK-N-BE(2)c cells is shown in western blots. The relative densitometry values are shown on top of the western blot panels