Figure 1.

miR-34a inhibits the growth of neuroblastoma cells through apoptosis and suppressed DNA synthesis. (a) microRNAs in the 1p36 region. Five microRNAs were mapped between 0-10Mb corresponding to 1p36.22-1pter, which is commonly deleted in NB tumors, using the Sanger miRNA Registry (http://microrna.sanger.ac.uk/sequences/index.shtml, Release 10.0). The relative positions of them are depicted on the black bar which represents 0-10Mb of chromosome 1. Among these 5 microRNAs, three (miR-200b, 429 and 34a; marked with arrowheads) are predicted to target MYCN genes by the miRBase Targets available on the same website. Start and End mark the genomic coordinates for the stem-loop pre-microRNA sequences of these microRNAs (Human Genome Build 36.2). (b) miR-34a inhibits the growth of neuroblastoma cells. MYCN-amplified NB cell lines, IMR32 and LA-N-5, were transfected with synthetic miR-34a, miR-200b, miR-429, and mimic control, the growth was monitored using WST-1 assays for 4 days. The optical densities (OD) at 450nm corrected by OD_650nm were plotted over time after background subtraction of the readings from the media-only wells. miR-34a clearly suppressed the growth of both IMR32 and LA-N-5, but miR-200b and -429 had little or no effect. (c) A representative flow cytometry experiment shows miR-34a inducing apoptosis and suppression of DNA synthesis. Flow cytometry was used to study cell death (7-AAD staining), apoptosis (caspase 3 staining) and DNA synthesis (BrdU staining) in IMR32 cells at 48 hours after transfection. Blue: mimic control transfection; Red: miR-34a transfection. The bar graph summarizes the results of flow cytometry. Cell death and apoptosis were increased by 29% and 113% respectively, whereas DNA synthesis was suppressed by 27% in the miR-34a transfected cells.