title: Systematic validation of predicted microRNAs for cyclin D1.
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.
  
  
  CONCLUSIONS
  This study provides a more comprehensive picture of microRNA/CCND1 interactions and it further demonstrates the importance of experimental target validation.
authors.affiliation: Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, Springfield, IL, USA. qjiang@siumed.edu
authors.forename: Qiong
authors.initials: Q
authors.lastname: Jiang
authors.forename: Ming-Guang
authors.initials: MG
authors.lastname: Feng
authors.forename: Yin-Yuan
authors.initials: YY
authors.lastname: Mo
mesh_terms: D020413:3' Untranslated Regions; D002277:Carcinoma / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D019938:Cyclin D1 / Q000378:metabolism*; D020868:Gene Silencing; D017930:Genes, Reporter; D016147:Genes, Tumor Suppressor; D006258:Head and Neck Neoplasms / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D000090063:Proto-Oncogene Mas; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016196:S Phase
chemical_list: D020413:3' Untranslated Regions; C000715470:MAS1 protein, human; C505362:MIRN16 microRNA, human; C522425:MIRN34 microRNA, human; C545473:MIRN503 microRNA, human; D035683:MicroRNAs; D000090063:Proto-Oncogene Mas; D012333:RNA, Messenger; D019938:Cyclin D1