Systematic validation of predicted microRNAs for cyclin D1.

  • Jiang Q
  • Feng M
  • Mo Y
 et al. 
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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 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.

Author-supplied keywords

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

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  • Qiong Jiang

  • Ming-Guang Feng

  • YY Yin-Yuan Mo

  • CM Croce

  • GA Calin

  • CZ Chen

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