ORIGINAL ARTICLE Glucocorticoid-regulated microRNAs and mirtrons in acute lymphoblastic leukemia J Rainer 1,6 , C Ploner 2,6 , S Jesacher 1 , A Ploner 3 , M Eduardoff 1 , M Mansha 1,2 , M Wasim 1,2 , R Panzer-Gru ¨ mayer 4 , Z Trajanoski 5 , H Niederegger 1 and R Kofler 1,2 1 Tyrolean Cancer Research Institute, Innsbruck, Austria; 2 Division of Molecular Pathophysiology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria; 3 Division of Genomics and RNomics, Biocenter, Medical University of Innsbruck, Innsbruck, Austria; 4 Children’s Cancer Research Institute and St Anna Kinderspital, Vienna, Austria and 5 Institute for Genomics and Bioinformatics, Graz University of Technology, Graz, Austria Glucocorticoids (GCs) induce apoptosis in lymphoid lineage cells and are therefore used in the therapy of acute lympho- blastic leukemia (ALL) and related malignancies. MicroRNAs (miRNAs) and the related mirtrons are B22 nucleotide RNAs derived from polymerase-II transcripts and implicated in the control of essential biological functions, including apoptosis. Whether GCs regulate miRNA-encoding transcription units is unknown. We investigated miRNA/mirtron expression and GC regulation in 8 leukemia/lymphoma in vitro models and 13 ALL children undergoing systemic GC monotherapy using a combination of expression profiling techniques, real time reverse transcription (RT)-PCR and northern blotting to detect mature miRNAs and/or their precursors. We found that mature miRNA regulations can be inferred from expression data of their host genes. Although a simple miRNA-initiated canonical pathway to GC-induced apoptosis or cell cycle arrest did not emerge, we identified several miRNAs/mirtrons that were regulated by GC in patients and cell lines, including the myeloid-specific miR-223 and the apoptosis and cell cycle arrest-inducing miR15B16 clusters. In an in vitro model, overexpression of miR15bB16 mimics increased and silencing by miR15bB16 inhibitors decreased GC sensitivity. Thus, the observed complex changes in miRNA/mirtron expression during GC treatment might contribute to the anti-leukemic GC effects in a cell context-dependent manner. Leukemia (2009) 23, 746–752; doi:10.1038/leu.2008.370; published online 15 January 2009 Keywords: acute lymphoblastic leukemia; microRNA; glucocorticoid; apoptosis; expression profiling Introduction Glucocorticoids (GCs) have pronounced effects on metabolism, differentiation, proliferation and cell survival in many tissues. In the lymphoid system, they affect cell cycle progression, influence immunoglobulin and lymphokine production and, most notably, induce apoptosis in immature lymphoblasts. The latter has been implicated in the generation of the immune repertoire and the regulation of immune responses, 1–3 and is used clinically in the treatment of childhood acute lympho- blastic leukemia (ALL) and other lymphoid malignancies. 4 GCs mediate their effects through the GC receptor (GR), a ligand- activated transcription factor of the nuclear receptor super- family that resides in the cytoplasm and, upon ligand binding, translocates into the nucleus, where it modulates gene expression by binding to specific DNA response elements or by protein–protein interactions with other transcription factors. 5 A large number of protein-encoding genes have been identified that are regulated by GCs in lymphoid lineage cells in experimental systems 6 and related clinical samples, 7,8 but the genes responsible for cell death induction are not well under- stood (refer recent reviews by Schaaf and Cidlowski, 2 Schmidt et al., 6 Distelhorst, 9 Haarman et al. 10 ). MicroRNAs (miRNAs) are tiny non-coding RNAs that induce post-transcriptional gene silencing through base pairing with their target mRNAs (for recent reviews and a mammalian miRNA expression atlas refer Kim and Nam, 11 Filipowicz et al., 12 Grosshans and Filipowicz, 13 Cullen, 14 Bartel, 15 Landgraf 16 ). They are transcribed by RNA polymerase II as long primary transcripts referred to as ‘pri-miRNAs’. miRNAs are encoded by one arm of a stem-loop structure embedded in introns or, less frequently, exons of protein-coding or non-coding transcripts. In many cases, the corresponding host genes and transcripts have been defined. 17 Subsequent to transcription, the pri-miRNAs stem-loop is cleaved by Drosha, an RNase III family member, to generate B70 nucleotide (nt) precursors called pre-miRNAs. In some instances, an entire intron consists of such a stem-loop structure, which is released by the splicing machinery in a Drosha-independent manner. miRNAs generated by this mechanism are referred to as ‘mirtrons’. 18–20 Pre-miRNAs are exported by Exportin-5 to the cytoplasm, where they are further processed by Dicer, another RNase III enzyme, to generate B22 base pair intermediates that enter effector complexes called miRISC (miRNA-containing RNA- induced silencing complex). Here, they are rapidly converted into single-stranded ‘mature miRNAs’ that target mRNAs and thereby affect their translation and/or stability. 21,22 Expression and regulation of miRNAs/mirtrons and their host genes can be studied at the level of the primary transcripts (pri-miRNAs), as polyadenylated and spliced mRNAs, as B70 nucleotide pre-miRNAs or as B22 nucleotide mature miRNAs using different techniques (Figure 1). A strong correlation between the tissue-specific expression patterns of the flanking mRNA and the embedded miRNA has been observed for some miRNAs, 14,17 suggesting that reliable mature miRNA expression patterns might be derived from host gene expression data. This raises the attractive possibility of exploiting conventional whole genome expression profiles not only for defining expression and regulation of miRNA-containing host genes, but also for obtaining relevant information regarding the corresponding mature miRNAs (provided miRNA and host gene are transcribed in the same orientation). Such a strategy is particularly relevant in the case of existing expression profiling data derived from clinical samples that are often unique and/or difficult to obtain. Received 26 August 2008; revised 5 November 2008; accepted 28 November 2008; published online 15 January 2009 Correspondence: Dr Professor R Kofler, Division of Molecular Pathophysiology, Biocenter, Medical University of Innsbruck, Fritz- Pregl-Strae 3, Innsbruck, Tyrol A-6020, Austria. E-mail: Reinhard.Kofler@i-med.ac.at 6 These authors contributed equally to this work. Leukemia (2009) 23, 746–752 & 2009 Macmillan Publishers Limited All rights reserved 0887-6924/09 $32.00 www.nature.com/leu