Potential mRNA degradation targets of hsa-miR-200c, identified using informatics and qRT-PCR

Gregory J. Hurteau, Simon D. Spivack, Graham J. Brock

Research output: Contribution to journalArticle

62 Citations (Scopus)

Abstract

Using an anchored oligo(dT) based RT-PCR approach we quantified endogenous expression of ten microRNAs in six cell lines. This identified a miRNA, miR-200c, with variable expression, ranging from undetectable in MDA-MB-231 and HT1080 to highly expressed in MCF7. The variable expression provided a model system to investigate endogenous interactions between miRNAs and their computationally predicted targets. As the expression level of the predicted mRNA targets and miR-200c in these lines should have an inverse relationship if cleavage or degradation results from the interaction. To select targets for analysis we used Affymetrix expression data and computational prediction programs. Affymetrix data indicated ∼3500 candidate mRNAs, absent in MCF7 and present in MDA-MB-231 or HT1080. These targets were cross-referenced against ∼600 computationally predicted miR-200c targets, identifying twenty potential mRNAs. Expression analysis by qRT-PCR of these targets and an additional ten mRNAs (selected using the prediction program ranking alone) revealed four mRNAs, BIN1, TCF8, RND3 and LHFP with an inverse relationship to miR-200c. Of the remainder, the majority did not appear to be degraded (and may be translational targets) or were undetectable in the cell lines examined. Finally, inhibition of miR-200c using an anti-miRNA 2′-0-Methyl oligonucleotide (AMO) resulted in an increase in expression of one of the targets, the transcription factor TCF8. These results indicate that a single miRNA could directly affect the mRNA levels of an important transcription factor, albeit in a manner specific to cell lines. Further investigation is required to confirm this in vivo and determine any translational effects.

Original languageEnglish (US)
Pages (from-to)1951-1956
Number of pages6
JournalCell Cycle
Volume5
Issue number17
StatePublished - Sep 1 2006
Externally publishedYes

Fingerprint

Informatics
RNA Stability
MicroRNAs
Degradation
Polymerase Chain Reaction
Messenger RNA
Cells
Cell Line
Transcription Factors
Oligonucleotides

Keywords

  • Cleavage
  • microRNA
  • miRNA
  • mRNA
  • Quantitative amplification

ASJC Scopus subject areas

  • Cell Biology
  • Biochemistry
  • Molecular Biology

Cite this

Potential mRNA degradation targets of hsa-miR-200c, identified using informatics and qRT-PCR. / Hurteau, Gregory J.; Spivack, Simon D.; Brock, Graham J.

In: Cell Cycle, Vol. 5, No. 17, 01.09.2006, p. 1951-1956.

Research output: Contribution to journalArticle

Hurteau, Gregory J. ; Spivack, Simon D. ; Brock, Graham J. / Potential mRNA degradation targets of hsa-miR-200c, identified using informatics and qRT-PCR. In: Cell Cycle. 2006 ; Vol. 5, No. 17. pp. 1951-1956.
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