Leukemia (2010) 24, 460–466 & 2010 Macmillan Publishers Limited All rights reserved 0887-6924/10 $32.00 www.nature.com/leu ‘ ORIGINAL ARTICLE Induction of microRNAs, mir-155, mir-222, mir-424 and mir-503, promotes monocytic differentiation through combinatorial regulation ARR Forrest1,2, M Kanamori-Katayama1, Y Tomaru1, T Lassmann1, N Ninomiya1, Y Takahashi1, MJL de Hoon1, A Kubosaki1, A Kaiho1, M Suzuki1, J Yasuda1, J Kawai1, Y Hayashizaki1, DA Hume3 and H Suzuki1 1LSA Technology Development Unit, Omics Science Center, RIKEN Yokohama Institute, Yokohama, Kanagawa, Japan; 2The Eskitis Institute for Cell and Molecular Therapies, Griffith University, Brisbane, Queensland, Australia and 3The Roslin Institute, Royal (Dick) School of Veterinary Studies, The University of Edinburgh, Roslin, UK Acute myeloid leukemia (AML) involves a block in terminal that might promote differentiation. MicroRNAs are short 21–22 differentiation of the myeloid lineage and uncontrolled prolif- nucleotide RNA molecules that cause translational repression eration of a progenitor state. Using phorbol myristate acetate and degradation of multiple mRNAs by binding target sequences (PMA), it is possible to overcome this block in THP-1 cells 0 5 (an M5-AML containing the MLL-MLLT3 fusion), resulting in in their 3 UTRs. Multiple microRNAs are implicated in differentiation to an adherent monocytic phenotype. As part of differentiation of cell lineages including skeletal muscle, FANTOM4, we used microarrays to identify 23 microRNAs that adipocytes and neurons6–8 and in mammals, the microRNA are regulated by PMA. We identify four PMA-induced micro- subsystem is essential for embryonic development. Dicer RNAs (mir-155, mir-222, mir-424 and mir-503) that when over- is required for differentiation of embryonic stem cells,9 Dicer expressed cause cell-cycle arrest and partial differentiation and (À/À) knockouts are embryonic lethal10 and Ago2 (À/À) when used in combination induce additional changes not seen 11 by any individual microRNA. We further characterize these pro- knockouts suffer severe hematopoietic defects. In addition differentiative microRNAs and show that mir-155 and mir-222 several microRNAs are deleted or amplified in cancer and have induce G2 arrest and apoptosis, respectively. We find been implicated as tumor suppressors12,13 or oncogenes.14,15 mir-424 and mir-503 are derived from a polycistronic precursor In this study we find that multiple microRNAs are induced mir-424-503 that is under repression by the MLL-MLLT3 upon PMA treatment and that four of these can promote both leukemogenic fusion. Both of these microRNAs directly target cell-cycle regulators and induce G1 cell-cycle arrest when partial monocytic differentiation and cell-cycle arrest in over- overexpressed in THP-1. We also find that the pro-differentia- expression experiments. Our data suggest a model whereby tive mir-424 and mir-503 downregulate the anti-differentiative multiple microRNAs are induced in conjunction with various mir-9 by targeting a site in its primary transcript. Our study transcription factors to cooperatively promote differentiation and highlights the combinatorial effects of multiple microRNAs inhibit cellular proliferation at multiple points in the cell cycle. within cellular systems. Leukemia (2010) 24, 460–466; doi:10.1038/leu.2009.246; published online 3 December 2009 Materials and methods Keywords: microRNA; acute myeloid leukemia; AML; monocyte; differentiation; systems biology The details of this section are available online as Supplementary Information. Results Introduction Multiple microRNAs are induced during PMA-induced THP-1 differentiation The FANTOM4 (Functional ANnoTation Of Mammals) project The expression patterns of microRNAs during PMA-induced used a combination of deep sequencing, microarrays, bioinfor- THP-1 differentiation were determined using Agilent microRNA matic predictions and siRNA perturbations to map a network of arrays (Palo Alto, CA, USA) on biological triplicate time– mammalian transcription factors and their targets.1 For this courses. The expression profile for each microRNA after PMA project we studied monocytic differentiation of the M5 acute 2 treatment is available through the FANTOM4 EdgeExpress myeloid leukemia (AML) cell line THP-1. This cell line contains 16 Database. Between undifferentiated (0 h) and differentiated the MLL-MLLT3 leukemogenic gene fusion3 and displays a (96 h) states we identified 21 microRNAs that were upregulated monoblastic phenotype; however, on phorbol myristate acetate and 2 that were downregulated with average expression (PMA) treatment it differentiates into an adherent monocytic changes of at least threefold (Table 1). Of the upregulated phenotype,4 closely approximating monoblast to monocyte microRNAs, several have been previously implicated in differentiation. hematopoiesis, including mir-424 that promotes monocytopoi- In this parallel study we monitored the expression dynamics 17 esis; mir-221, mir-222 and mir-24 that inhibit eryrthopoi- of microRNAs after PMA treatment and sought to identify those 18,19 esis and mir-155 that promotes B-cell proliferation and depletes myeloid and erythroid hematopoietic stem cell Correspondence: Dr ARR Forrest, Omics Science Center, RIKEN populations.20,21 Of the two downregulated microRNAs, Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, mir-210 promotes osteoblast differentiation22 and mir-9 blocks Kanagawa 230-0045, Japan. 23 E-mail: [email protected] B-cell differentiation by targeting PRDM1. We note that Received 12 May 2009; revised 8 September 2009; accepted 9 although detected in THP-1, the mir-17-92 polycistron, pre- October 2009; published online 3 December 2009 viously reported to prevent monocytic differentiation by Combinatorial effects of multiple microRNAs ARR Forrest et al 461 Table 1 MicroRNAs induced or repressed X3-fold in THP-1 after MicroRNAs were considered pro-differentiative (1) if there 96 h PMA-induced differentiation were more PMA-like changes than anti-PMA-like changes (at least 1.5 Â ) and (2) for the given microRNA the fraction of all Dynamically regulated MicroRNA Small RNA changes that were PMA-like was greater than the average microRNAs array sequencing fraction plus one standard deviation (Supplementary Table 2). None of the microRNAs completely reiterated the expression Fold change Fold Max change tpm changes observed with PMA, and no morphological differences were observed; however, using the above criteria, mir-155, Induced mir-222, mir-424 and mir-503 induced expression changes mir-221 5.7 10.9 106 251 suggesting partial differentiation including induction of known mir-24 3.2 2.2 65 581 markers of differentiation ITGAL (Cd11a), ITGB7 and ENG mir-222 3.3 6.4 55 722 mir-124 39.3 44 30 058 (Cd105) by mir-155; and ITGAL, ITGB2 and CSF1R by mir-503 mir-22 14.7 7.6 18 316 (Supplementary Figure 1). mir-155 and mir-503 had the greatest mir-29b 5.3 3.4 10 154 ratio of PMA-like to anti-PMA-like changes (2.9 Â ) and mir-503 mir-29a 5.2 2 6087 also had the greatest number of PMA-like changes in total (75 mir-503 6.8 8.3 4099 down, 35 up). As a comparison the additional negative control mir-424 5.1 1.9 1971 microRNA (mir-142, expressed in THP-1 but not regulated by mir-155 3 1.3 1445 mir-30a-5p 4.3 1.9 568 PMA) failed to induce pro-differentiative expression changes mir-132 12.3 11.5 294 and had a differentiation ratio of 0.3 Â . mir-542-3p 4.8 2.6 172 The combined effect of the four microRNAs was then tested mir-137 17.3 9.5 153 by co-overexpressing mir-155, mir-222, mir-424 and mir-503 in mir-151 10.2 1.8 105 equimolar amounts in THP-1 for 48 h. The mixture induced 69 mir-133a 7.7 ND 70 pro-differentiative changes, 39 of which were not observed in mir-542-5p 4.3 ND 67 mir-146b 5.6 ND 23 any of the four individual pre-miRNA transfections (including mir-193b 3.7 ND 19 modest induction of the key monocytic marker CD14, Supple- mir-133b 12 ND o1 mentary Figure 2). One-quarter of all changes induced by the mir-146a 17 ND ND mixture were PMA-like and the ratio of PMA-like to anti-PMA- like changes was 6.3 Â , far larger than observed for any of the Repressed individual miRNA transfections (Supplementary Table 2). mir-210 0.3 0.5 22 801 mi-9 0.3 0.04 17 871 Together this suggests that the mixture more closely mimics the PMA-induced expression changes by reducing the non- MicroRNAs are ranked by maximum tags per million in the small RNA 25 specific and anti-PMA-like changes and increasing the PMA-like sequencing that is described in Taft et al. changes. Finally, using this analysis, we also identify mir-9 as an repression of AML1,24 is not significantly downregulated anti-differentiative miRNA. 36% of all the genes downregu- suggesting our cells are at a different stage of differentiation. lated by mir-9 overexpression are normally upregulated Deep-sequencing of small RNAs from one of the three THP-1 with PMA and 26% of all the expression changes for mir-9 biological replicates time courses25 confirmed the differential were anti-differentiative, suggesting it helps maintain the expression of microRNAs detected by the arrays. Fold change undifferentiated monoblast state similar to its role in blocking 23 estimates by the two technologies varied (for example, mir-221 B-cell differentiation. was induced 5.7-fold on the arrays but 10.9-fold by sequencing) but repression or induction was consistent. We also estimated microRNA abundances from the sequencing and found mir-221 the most abundant inducible microRNA in THP-1 accounting mir-424-503 is a polycistronic microRNA cluster that for 11% of all tags at 96 h (106 251 tags per million (tpm)).