Depletion of human micro-RNA miR-125b reveals that it is critical for the proliferation of differentiated cells but not for the down-regulation of putative targets during differentiation.
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 and lin-41, 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.
Lee
Yong Sun
YS
Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia 22908, USA.
Kim
Hak Kyun
HK
Chung
Sangmi
S
Kim
Kwang-Soo
KS
Dutta
Anindya
A
D001483:Base Sequence; D015152:Blotting, Northern; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D015536:Down-Regulation; D006801:Humans; D035683:MicroRNAs / Q000737:chemistry*; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D016366:Open Reading Frames; D012313:RNA / Q000737:chemistry; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D035683:MicroRNAs; D009841:Oligonucleotides; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012313:RNA