Systematic validation of predicted microRNAs for cyclin D1

82Citations
Citations of this article
66Readers
Mendeley users who have this article in their library.

This article is free to access.

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 precursorsexpressed 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. © 2009 Jiang et al; licensee BioMed Central Ltd.

Cite

CITATION STYLE

APA

Jiang, Q., Feng, M. G., & Mo, Y. Y. (2009). Systematic validation of predicted microRNAs for cyclin D1. BMC Cancer, 9. https://doi.org/10.1186/1471-2407-9-194

Register to see more suggestions

Mendeley helps you to discover research relevant for your work.

Already have an account?

Save time finding and organizing research with Mendeley

Sign up for free