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

Li,Yu‑Guang; He,Jin‑Hua; Yu,Liu; Hang,Ze‑Ping; Li,Wang; Shun,Wen‑Hong; Huang,Guo Xian

doi:10.3892/mmr.2013.1845

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

Authors:
- Yu‑Guang Li
- Jin‑Hua He
- Liu Yu
- Ze‑Ping Hang
- Wang Li
- Wen‑Hong Shun
- Guo Xian Huang
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Affiliations: Department of Laboratory, Central Hospital of Panyu District, Guangzhou, Guangdong 511400, P.R. China, Department of Biochemistry, Medical College, Jinan University, Guangzhou, Guangdong 510630, P.R. China
Published online on: December 6, 2013 https://doi.org/10.3892/mmr.2013.1845
Pages: 541-546

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Abstract

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 5'cDNA ends (5'RACE) 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 5'RACE 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.

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