RESUMO
microRNAs (miRNAs) are small noncoding 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 miRNA202 (miR202) and that the promoter region of miR202 contains binding sites for the transcription factor E2F1. The aims of this study were to explore the regulation of MYCN expression by miR202 in the LAN5 human neuroblastoma cell line and to confirm the presence of binding sites for E2F1 in the miR202 promoter region. LAN5 cells were transfected with a synthetic miR202 mimic, an miRNA inhibitor or appropriate control miRNAs. miR202 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 miR202 and MYCN was examined using a luciferase reporter assay. The transcription start site of miR202 was determined by the rapid amplification of 5'cDNA ends (5'RACE) test and a chromatin immunoprecipitation (ChIP) assay was used to confirm binding sites for E2F1 in the miR202 promoter region. Overexpression of miR202 in LAN5 cells specifically inhibited MYCN protein expression. The 5'RACE test showed that the transcription start site of miR202 was at a thymidine, 312 bp upstream of the stemloop sequence. A ChIP assay demonstrated that E2F1 binds directly to the miR202 promoter region. miR202 is activated by E2F1 and in turn downregulates MYCN protein expression in the neuroblastoma cell line LAN5. Upregulation of miR202 may therefore be a novel strategy for neuroblastoma treatment.
