Systematic Validation of Predicted microRNAs for Cyclin D1

BMC Cancer. 2009 Jun 18;9:194. doi: 10.1186/1471-2407-9-194.

Abstract

Background: MicroRNAs are the endogenous small non-coding RNA molecules capable of silencing protein coding genes at the posttranscriptional level. Based on computer-aided predictions, a single microRNA could have over a hundred of targets. On the other hand, a single protein-coding gene could be targeted by many potential microRNAs. However, only a relatively small number of these predicted microRNA/mRNA interactions are experimentally validated, and no systematic validation has been carried out using a reporter system.

Methods: In this study, we used luciferease reporter assays to validate microRNAs that can silence cyclin D1 (CCND1) because CCND1 is a well known proto-oncogene implicated in a variety of types of cancers. We chose miRanda http://www.microRNA.org as a primary prediction method. We then cloned 51 of 58 predicted microRNA precursors into pCDH-CMV-MCS-EF1-copGFP and tested for their effect on the luciferase reporter carrying the 3'-untranslated region (UTR) of CCND1 gene.

Results: Real-time PCR revealed the 45 of 51 cloned microRNA precursors expressed a relatively high level of the exogenous microRNAs which were used in our validation experiments. By an arbitrary cutoff of 35% reduction, we identified 7 microRNAs that were able to suppress Luc-CCND1-UTR activity. Among them, 4 of them were previously validated targets and the rest 3 microRNAs were validated to be positive in this study. Of interest, we found that miR-503 not only suppressed the luciferase activity, but also suppressed the endogenous CCND1 both at protein and mRNA levels. Furthermore, we showed that miR-503 was able to reduce S phase cell populations and caused cell growth inhibition, suggesting that miR-503 may be a putative tumor suppressor.

Conclusion: This study provides a more comprehensive picture of microRNA/CCND1 interactions and it further demonstrates the importance of experimental target validation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3' Untranslated Regions
  • Carcinoma / genetics
  • Carcinoma / metabolism*
  • Cell Line, Tumor
  • Cyclin D1 / metabolism*
  • Gene Silencing
  • Genes, Reporter
  • Genes, Tumor Suppressor
  • Head and Neck Neoplasms / genetics
  • Head and Neck Neoplasms / metabolism*
  • Humans
  • MicroRNAs / genetics*
  • MicroRNAs / metabolism
  • RNA, Messenger / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • S Phase

Substances

  • 3' Untranslated Regions
  • MIRN16 microRNA, human
  • MIRN34 microRNA, human
  • MIRN503 microRNA, human
  • MicroRNAs
  • RNA, Messenger
  • Cyclin D1