MicroRNA29a regulates the expression of the nuclear oncogene Ski

Patients with acute myeloid leukemia (AML) with monosomy 7 (−7) or deletion of 7q (del7q) have a poor prognosis. We recently reported that the nucleoprotein Ski, the cellular homologue of the avian leukosis retrovirus oncogene v-SKI, is up-regulated in AML, especially in AML with −7/del7q, which presumably represses retinoic acid–induced myeloid differentiation.¹  In keeping with this, AML patients with low levels of SKI may benefit from combination therapy with retinoic acid.²  The molecular reason for up-regulation of SKI in AML with −7 is unclear. Therefore, we investigated whether a microRNA (miRNA) localized in the cancer fragile site of 7q could be a regulator of Ski. miRNAs are small RNA molecules that affect gene expression by posttranscriptional and translational mechanisms.³  Different miRNAs have been shown to regulate cell proliferation, differentiation, and apoptosis.^4-6  Aberrant miRNA expression has been linked to leukemogenesis in chronic lymphocytic leukemia,⁷  acute lymphoblastic leukemia,⁸  and AML.^9-11  miRNAs contribute to malignant transformation by genomic loss, because many miRNAs are tumor-suppressive and localized in cancer fragile sites.¹²  We describe here that expression of miR-29a localized on 7q32, is down-regulated in AML with −7/del7q and that miR-29a regulates Ski. These data suggest that loss of miR-29a may contribute to leukemogenesis by derepressing the nuclear oncogene SKI.

For detailed descriptions of patient cohorts, cell lines, and transient transfections, see supplemental Methods (available on the Blood Web site; see the Supplemental Materials link at the top of the online article).

We used an oligonucleotide array (mirVANA miRNA probe set; Ambion) and tested the miRNA expression pattern of 17 AML samples. The AML study was approved by the local ethics committee, and patients and donors gave written informed consent. For details, see supplemental Methods.

For a description of flow cytometry, see Ritter et al.¹ 

For details about the cell proliferation (MTT) assay, see supplemental Methods.

For Western blot, whole-cell proteins were extracted by use of a standard protocol for RIPA lysates. After SDS-PAGE, proteins were transferred to nitrocellulose membrane and incubated with antibody anti-Ski (sc-9140; Santa Cruz Biotechnology; 1:1000) or anti–β-actin (AC-74; Sigma-Aldrich; 1:5000).

For conditions and primers, see supplemental Methods.

For luciferase constructs (Figure 2A) and luciferase assay, see supplemental Methods.

For scientific graphing and statistical analysis, we used GraphPad Prism 5 software (GraphPad Prism Software Inc).

We performed an miRNA oligonucleotide array and compared the miRNA profile of normal bone marrow samples (n = 8) with that of AML samples with isolated −7/del7q or complex karyotype including −7/del7q (n = 17, cohort 1; supplemental Tables 1 and 2; blast count > 30%). A hierarchical cluster analysis demonstrated that AML samples had expression patterns different from normal bone marrow (supplemental Figure 1). Next, we looked for differential miRNA expression between normal bone marrow and AML with −7/del7q (supplemental Table 3). The only miRNA found to be down-regulated in AML samples and localized on 7q was miR-29a (Figure 1A). We then also analyzed 2 larger cohorts of patients for miR-29a expression using quantitative RT-PCR: cohort 2 (−7/del7q; n = 61; supplemental Table 4) and cohort 4 (normal karyotype; n = 54). Between these 2 cohorts, no significant different expression of miR-29a was found, which could be attributed to technical reasons; however, both cohorts showed a down-regulation compared with normal CD34-positive cells (supplemental Figure 2; supplemental Table 5). miR-29a is down-regulated in chronic lymphocytic leukemia¹³  and solid cancers.^14,15  Because our previous results indicated an overexpression of the nuclear oncogene SKI in AML with −7/del7q,¹  we examined whether miR-29a controls Ski expression.

We transfected HL60 cells with miRNA precursor 29a, 25, 183, or 335, localized on chromosome 7, and measured Ski using flow cytometry. Only miR-29a effectively lowered Ski in HL60 (supplemental Figure 3). Consistently, miR-29a reduced Ski compared with miR-scrambled in Western blot (Figure 1B). Flow cytometry revealed that overexpression of miR-29a (fold miR-29a expression compared with scrambled: 17) significantly increased expression of monocytic differentiation marker CD11b (P = .023). This could be enhanced by all-trans retinoic acid (ATRA; Figure 1C; P = .043). In keeping with this, overexpression of miR-29a decreased proliferation of HL60 cells (Figure 1D).

Ski may be a transcriptional corepressor¹⁶  or coactivator.¹⁷  In IFB melanoma cells, Ski acts as a coactivator of the neuronal cell adhesion molecule gene, Nr-CAM.¹⁸  We transfected IFB cells with miRNA precursor 29a (fold expression compared with scrambled: 25) and determined expression of Ski using Western blot and of Nr-CAM using PCR. Expression of miR-29a inhibited Ski protein and Nr-CAM gene expression, in keeping with our hypothesis (Figure 1E).

We next investigated whether inhibition of miR-29a would lead to increased Ski expression. NW1539 melanoma and HL60 cells express miR-29a (supplemental Figure 4). These cells were incubated with an inhibitor oligonucleotide against miR-29a or scrambled. Addition of anti-miR-29a induced Ski expression in NW1539 (Figure 1F) and HL60 (Figure 1F; fold down-regulation of miR-29a compared with scrambled: 10).

To analyze the function of miR-29a, we transfected HeLa cells with 3 different luciferase constructs of SKI wild-type 3′UTR (Figure 2A) and miRNA precursor 29a. miR-29a significantly reduced activity of the 5/6-UTR construct compared with other constructs (Figure 2B). Endogenous miR-29a also reduced activity of the 5/6-UTR construct (supplemental Figure 5), which indicates a binding site for miR-29a in this region (756-1252 bp). In silico analysis (www.targetscan.org) identified a putative binding site for miR-29a in the SKI 3′UTR at 1066-1088 bp (Figure 2A), which is conserved in Homo sapiens, mouse, rat, and dog. To confirm, we transfected HeLa cells with miRNA precursor 29a and luciferase constructs carrying wild-type or mutated predicted miR-29a binding site flanked by 130 bp of 5/6-UTR. The introduction of mutations of the putative binding region abolished the effect on luciferase activity, which supports the notion that miR-29a binds to this region (Figure 2C).

To address whether miR-29a is negatively associated with Ski expression in vivo, we analyzed 21 AML samples with different karyotypes (cohort 3; supplemental Table 6 and supplemental Figure 6). Samples with very high miR-29a expression revealed low Ski protein expression (P = .020; Figure 2D). Consistent with robust repression of Ski by miR-29a, no single patient sample showed high-level expression of Ski in the presence of miR-29a.

In conclusion, we show here that miR-29a regulates Ski expression in AML, thus suggesting a tumor-suppressive role of miR-29a, as has been described by others.¹⁹  In contrast, Han et al²⁰  observed that miR-29a is overexpressed and induces aberrant self-renewal in AML. We also observed a broad range of miR-29a expression in primary AML samples (Figure 2D). Because miRNAs do not regulate just one target, it is conceivable that in different AMLs, specific miRNAs may play different roles. Therefore, it is likely that the loss of miR-29a expression contributes to the up-regulation of the oncogene SKI in this subgroup of patients. This provides a mechanistic explanation for our original observation of high Ski expression in AML with −7/del7q. Because high-level expression of Ski is experimentally and clinically linked to retinoic acid resistance, this directly links drug resistance to a defined genetic aberration. Of note, it has been shown recently that miR-29a also regulates DNA methyltransferases.²¹  Together with our finding of miR-29a as a regulator of Ski, which is known to function in association with histone deacetylase in nuclear corepressor complexes,²²  this suggests that the tumor-suppressive role of miR-29a is based at least in part on deregulated epigenetic control of leukemogenic gene expression.

The authors thank the members of the AML-2003 study group for contributing patient samples and the participating patients for letting them study their tumor samples. They thank Professor Elke Jäger, Nord West Krankenhaus Frankfurt, Germany, for providing the cell line NW 1539. They thank Lukas Rycak and Lisa-Marie Weiss for technical support.

This work was supported in part by grants from the Bundesministerium für Bildung und Forschung (NGFN2: FKZ 01GS0449, NGFNplus: FKZ 01GS0880), the Deutsche Forschungsgemeinsschaft (Transregio 17, project C3; KFO 210, project 3), the Deutsche José Carreras Leukämie Stiftung, and a nonrestricted grant from Dr Reinfried Pohl.

Contribution: S.T. designed and performed research, collected data, analyzed and interpreted data, and wrote the manuscript; T.I. performed research, collected, analyzed, and interpreted data, performed statistical analysis, and wrote parts of the manuscript; J.R. performed research experiments and analyzed data from experiments; D.O. designed research and contributed analytical tools; T.S. designed research, interpreted data, and wrote parts of the manuscript; and A.N. designed research, interpreted and analyzed data, performed statistical analysis, and wrote the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Andreas Neubauer, Department of Hematology, Oncology and Immunology, Philipps University of Marburg, Medical Center of the University Giessen and Marburg, Baldingerstrasse, 35033 Marburg, Germany; e-mail: neubauer@ailer.ni-marburg.de.