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Open Access Highly Accessed Research article

Systematic validation of predicted microRNAs for cyclin D1

Qiong Jiang12, Ming-Guang Feng2* and Yin-Yuan Mo1*

Author Affiliations

1 Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, Springfield, IL, USA

2 Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou, PR China

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BMC Cancer 2009, 9:194  doi:10.1186/1471-2407-9-194

Published: 18 June 2009

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 webcite 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.