Tumour-suppressor microRNAs let-7 and mir-101 target the proto-oncogene MYCN and inhibit cell proliferation in MYCN-amplified neuroblastoma

Br J Cancer. 2011 Jul 12;105(2):296-303. doi: 10.1038/bjc.2011.220. Epub 2011 Jun 7.

Abstract

Background: MicroRNAs (miRNAs) regulate expression of many cancer-related genes through posttranscriptional repression of their mRNAs. In this study we investigate the proto-oncogene MYCN as a target for miRNA regulation.

Methods: A luciferase reporter assay was used to investigate software-predicted miRNA target sites in the 3'-untranslated region (3'UTR) of MYCN. The miRNAs were overexpressed in cell lines by transfection of miRNA mimics or miRNA-expressing plasmids. Mutation of the target sites was used to validate MYCN 3'UTR as a direct target of several miRNAs. To measure miRNA-mediated suppression of endogenous N-myc protein, inhibition of proliferation and inhibition of clonogenic growth, miRNAs were overexpressed in a MYCN-amplified neuroblastoma cell line.

Results: The results from this study show that MYCN is targeted by several miRNAs. In addition to the previously shown mir-34a/c, we experimentally validate mir-449, mir-19a/b, mir-29a/b/c, mir-101 and let-7e/mir-202 as direct MYCN-targeting miRNAs. These miRNAs were able to suppress endogenous N-myc protein in a MYCN-amplified neuroblastoma cell line. The let-7e and mir-202 were strong negative regulators of MYCN expression. The mir-101 and the let-7 family miRNAs let-7e and mir-202 inhibited proliferation and clonogenic growth when overexpressed in Kelly cells.

Conclusion: The tumour-suppressor miRNAs let-7 and mir-101 target MYCN and inhibit proliferation and clonogenic growth of MYCN-amplified neuroblastoma cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Brain Neoplasms / genetics
  • Brain Neoplasms / pathology*
  • Cell Line, Tumor
  • Cell Proliferation*
  • Cells, Cultured
  • Down-Regulation / genetics
  • Down-Regulation / physiology
  • Gene Amplification / physiology
  • Gene Expression Regulation, Neoplastic / genetics
  • Gene Targeting
  • Genes, Tumor Suppressor / physiology
  • Humans
  • MicroRNAs / genetics
  • MicroRNAs / physiology*
  • N-Myc Proto-Oncogene Protein
  • Neuroblastoma / genetics
  • Neuroblastoma / pathology*
  • Nuclear Proteins / antagonists & inhibitors*
  • Nuclear Proteins / genetics*
  • Oncogene Proteins / antagonists & inhibitors*
  • Oncogene Proteins / genetics*
  • Proto-Oncogenes / genetics

Substances

  • MIRN107 microRNA, human
  • MYCN protein, human
  • MicroRNAs
  • N-Myc Proto-Oncogene Protein
  • Nuclear Proteins
  • Oncogene Proteins
  • mirnlet7 microRNA, human