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

Abstract

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-Omethyl 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 cotransfected 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. Micro-RNAs (miRNAs)1 are a class of small non-coding RNAs that are processed by Dicer from precursors with a characteristic hairpin secondary structure (1). It is now known that there are hundreds of evolutionarily conserved miRNAs in plants and animals, but their functions are not well understood The study of a miRNA function has been hampered by the lack of an efficient method to deplete a miRNA, especially in mammals where genetic experiments are hard to perform. Kawasaki and Taira (24) reported a knock-down of a miRNA by a siRNA against the loop region of the miRNA precursor, but the paper was retracted (25). Recently, 2Ј-O-methyl antisense oligonucleotide against a miRNA was reported to specifically knock-down the miRNA (26,27). Despite the successful knock-down of a miRNA in vitro and in vivo, this method has several limitations. First, a direct measurement of the depletion of a miRNA is difficult, because 2Ј-O-methyl antisense oligonucleotide binds to the miRNA and sequesters it from its target rather than induces its degradation. In addition, even a slight contamination of 2Ј-Omethyl antisense oligonucleotide in an RNA preparation interferes with assays to measure the miRNA level, such as Northern blot, primer extension assay, or RNase protection assay.2 Therefore, the only possible way to confirm the decrease of a miRNA is to employ a very indirect and cumbersome assay, measuring the level of expression of a reporter gene containing the ...

MicroRNA involvement in mammary gland development and breast cancer

“…Expression of some miRNA could be correlated with specific breast cancer biopathologic features, such as estrogen and progesterone receptor expression, tumour stage, vascular invasion, or proliferation index. High expression of human miR-125b seems to be present in differentiated cells or tissues [27]. It was observed that breast cancer primary Again, these data suggest an implication of miRNA in mammary gland biology.…”

Anti-miRNA oligonucleotides (AMOs): ammunition to target miRNAs implicated in human disease?

“…Given the vulnerability of unmodified dsRNA to nucleases in vivo, this class of compound could probably only be used in privileged local environments, for example in the central nervous system. 57 If a disease phenotype is derived from abnormal inhibition of mRNAs caused by, for example, excessive expression of miRNAs, AMOs complementary to either the mature miRNA or its precursors 58 can be designed (Figure 1). The earliest report of miRNA inhibition using AMOs describes the microinjection of DNA oligonucleotides of the same length and complementary to the target miRNA into Drosophila embryos.…”

“…Cheng et al 65 applied a library of Me-AMOs to human cancer cell lines to screen for miRNAs potentially involved in cell growth and apoptosis. As an alternative to direct blockage of a particular miRNA, Lee et al 58 recently suggested Me-AMOs directed against the loop region of a miRNA precursor as tools to determine miRNA function in vivo.…”

Implications of micro-RNA profiling for cancer diagnosis

“…However, upregulation of specific miRNAs, such as the mir-17-92 cluster described above, appears to be associated with oncogenesis (He et al, 2005b;Volinia et al, 2006). In addition, other miRNAs, including miR125b and miR-143, have been directly associated with the differentiation and proliferation of certain cell types (Esau et al, 2004;Lee et al, 2005). Finally, miRNA expression patterns may change when cells are treated with differentiation-promoting agents (Kasashima et al, 2004;Stegmaier et al, 2004), and recent studies have shown that undifferentiated human embryonic stem cells express certain miRNAs (Suh et al, 2004).…”

“…At least two examples already suggest that miRNA dysregulation may affect major oncogenes such as c-myc and RAS O'Donnell et al, 2005). Additionally, the recent successes of inhibition of miRNAs at the cellular level (Hutvagner et al, 2004;Meister et al, 2004;Lee et al, 2005) suggest the possibility of direct targeting of miRNAs that are amplified or upregulated in patient tumors.…”