Yong Sun Lee‡, Hak Kyun Kim‡§, Sangmi Chung¶, Kwang-Soo Kim¶ and Anindya Dutta‡∥
‡Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia 22908, §Institute of Molecular Biology and Genetics, School of Biological Sciences, Seoul National University, Seoul 151-742, Korea, and ¶Molecular Neurobiology Laboratory, McLean Hospital, Harvard Medical School, Belmont, Massachusetts 02478
∥ To whom correspondence should be addressed. Tel.: 434-924-1227; Fax: 434-924-5069.
Micro-RNAs are small non-coding RNAs that regulate target gene expression post-transcriptionally through base pairing with the target messenger RNA. Functional characterization of micro-RNAs awaits robust experimental methods to knock-down a micro-RNA as well as to assay its function in vivo
. In addition to the recently developed method to sequester micro-RNA with 2′-O
-methyl antisense oligonucleotide, we report that small interfering RNA against the loop region of a micro-RNA precursor can be used to deplete the micro-RNA. The depletion of miR-125b by this method had a profound effect on the proliferation of adult differentiated cancer cells, and this proliferation defect was rescued by co-transfected mature micro-RNA. This technique has unique advantages over the 2′-O
-methyl antisense oligonucleotide and can be used to determine micro-RNA function, assay micro-RNAs in vivo
, and identify the contribution of a predicted micro-RNA precursor to the pool of mature micro-RNA in a given cell. miR-125b and let-7
micro-RNAs are induced, whereas their putative targets, lin-28
, are decreased during in vitro
differentiation of Tera-2 or embryonic stem cells. Experimental increase or decrease of micro-RNA concentrations did not, however, affect the levels of the targets, a finding that is explained by the fact that the down-regulation of the targets appears to be mostly at the transcriptional level in these in vitro
differentiation systems. Collectively these results reveal the importance of micro-RNA depletion strategies for directly determining micro-RNA function in vivo
1 The abbreviations used are: miRNA, micro-RNA; siRNA, small interfering RNA; NP, neuronal precursor; EC, embryonic carcinoma; ES cell, embryonic stem cell; gapdh, glyceraldehyde-3-phosphate dehydrogenase gene; EB, embryoid bodies.
2 Y. S. Lee and A. Dutta, unpublished data.
* This work was partly supported by a post-doctoral fellowship program of the Korea Science and Engineering Foundation (to Y. S. L.) and National Institutes of Health Grant RO1 CA89406 (to A. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received October 28, 2004. Revision received February 15, 2005.