microRNA-202 suppresses MYCN expression under the control of E2F1 in the neuroblastoma cell line LAN-5

microRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by binding to the untranslated regions (UTRs) of target mRNAs. Bioinformatic software predicted that MYCN, a gene overexpressed in aggressive neuroblastoma cells, is a target gene of miRNA-202 (miR-202) and that the promoter region of miR-202 contains binding sites for the transcription factor E2F1. The aims of this study were to explore the regulation of MYCN expression by miR-202 in the LAN-5 human neuroblastoma cell line and to confirm the presence of binding sites for E2F1 in the miR-202 promoter region. LAN-5 cells were transfected with a synthetic miR-202 mimic, an miRNA inhibitor or appropriate control miRNAs. miR-202 expression levels prior to and following transfection were measured by quantitative polymerase chain reaction (PCR) and MYCN protein expression was measured by western blot analysis. The interaction between miR-202 and MYCN was examined using a luciferase reporter assay. The transcription start site of miR-202 was determined by the rapid amplification of 5cDNA ends (5RACE) test and a chromatin immunoprecipitation (ChIP) assay was used to confirm binding sites for E2F1 in the miR-202 promoter region. Overexpression of miR-202 in LAN-5 cells specifically inhibited MYCN protein expression. The 5RACE test showed that the transcription start site of miR-202 was at a thymidine, 312 bp upstream of the stem-loop sequence. A ChIP assay demonstrated that E2F1 binds directly to the miR-202 promoter region. miR-202 is activated by E2F1 and in turn downregulates MYCN protein expression in the neuroblastoma cell line LAN-5. Upregulation of miR-202 may therefore be a novel strategy for neuroblastoma treatment.