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Lincrnas MONC and MIR100HG Act As Oncogenes in Acute

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Lincrnas MONC and MIR100HG Act As Oncogenes in Acute Emmrich et al. Molecular Cancer 2014, 13:171 http://www.molecular-cancer.com/content/13/1/171 RESEARCH Open Access LincRNAs MONC and MIR100HG act as oncogenes in acute megakaryoblastic leukemia Stephan Emmrich†, Alexandra Streltsov†, Franziska Schmidt, Veera Raghavan Thangapandi, Dirk Reinhardt and Jan-Henning Klusmann* Abstract Background: Long non-coding RNAs (lncRNAs) are recognized as pivotal players during developmental ontogenesis and pathogenesis of cancer. The intronic microRNA (miRNA) clusters miR-99a ~ 125b-2 and miR-100 ~ 125b-1 promote progression of acute megakaryoblastic leukemia (AMKL), an aggressive form of hematologic cancers. The function of the lncRNA hostgenes MIR99AHG (alias MONC)andMIR100HG within this ncRNA ensemble remained elusive. Results: Here we report that lncRNAs MONC and MIR100HG are highly expressed in AMKL blasts. The transcripts were mainly localized in the nucleus and their expression correlated with the corresponding miRNA clusters. Knockdown of MONC or MIR100HG impeded leukemic growth of AMKL cell lines and primary patient samples. The development of a lentiviral lncRNA vector to ectopically express lncRNAs without perturbing their secondary structure due to improper termination of the viral transcript, allowed us to study the function of MONC independent of the miRNAs in cord blood hematopoietic stem and progenitor cells (HSPCs). We could show that MONC interfered with hematopoietic lineage decisions and enhanced the proliferation of immature erythroid progenitor cells. Conclusions: Our study reveals an unprecedented function of lncRNAs MONC and MIR100HG as regulators of hematopoiesis and oncogenes in the development of myeloid leukemia. Background myelodysplastic syndrome. In contrast, HOTAIR regu- It has become apparent that the vast majority of the lates expression of the HOXD gene family as well as other eukaryotic genome underlies prevalent transcription [1]. genes throughout the genome via re-targeting of Poly- Both DNA strands are pervasively transcribed, giving comb repressive complex 2 (PRC2) [6,7]. Enforced expres- rise to numerous different classes of non-coding RNAs sion of HOTAIR in epithelial cancer cells leads to altered (ncRNAs), including long intergenic RNAs (lincRNAs), histone H3 lysine 27 methylation, gene expression, and in- antisense RNAs and enhancer RNAs (eRNAs) [2]. This creased cancer invasiveness and metastasis. Similarly, abundant mixture of long (> 200 nt) and short (< 200 nt) HOTTIP affects expression of the HOXA gene family [8]. non-coding RNAs was misapprehended in the past as Recently, E2F1 transcription factor has been shown to ac- transcriptional noise or junk. However, accumulating tivate lncRNA ERIC, which restricts E2F-induced apop- evidence suggested that transcription factors and other tosis during cell cycle progression [9]. global regulators are prevalent targets of ncRNAs [3]. Acute myeloid leukemia (AML) is an aggressive form of Thereby, ncRNAs induce changes in histone marks and hematologic cancers with a 5-year overall survival between gene expression in cis and in trans. For example, XIST is 30 and 40% in adults [10]. While AML is generally less crucial for random inactivation of the X chromosome [4]. common in children, inherited molecular lesions can cause Beyond that, Xist RNA acts as a suppressor of hematologic a genetic background, which predisposes to malignant cancer [5]. Deletion of Xist results in the development transformation and AML. Particularly children with Down of a highly aggressive myeloproliferative neoplasm and syndrome (DS), i.e. trisomy 21, have a 400-fold increased risk [11] to develop acute megakaryoblastic leukemia * Correspondence: [email protected] (AMKL). Patients with DS-AMKL have an excellent prog- †Equal contributors nosis with 5-year overall survival rates of about 80%, while Pediatric Hematology and Oncology, Hannover Medical School, non-DS-AMKL patients have poor survival rates of only Carl-Neuberg-Straße 1, 30625 Hannover, Germany © 2014 Emmrich et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Emmrich et al. Molecular Cancer 2014, 13:171 Page 2 of 12 http://www.molecular-cancer.com/content/13/1/171 14% to 34% despite high intensity chemotherapy [12,13]. located in a phylogenetically conserved ncRNA ensemble, The molecular mechanisms underlying this AML subtype consisting of two other miRNAs (miR-99a and let-7c) remain incompletely understood. We recently reported the and the lncRNA hostgene MIR99AHG, which we termed characterization of an oncogenic microRNA (miRNA) on megakaryocytic oncogenic non-coding RNA (MONC) chromosome 21 (hsa21), miR-125b-2,whichishighly (Figure 1A). A homolog of the miR-99a ~ 125b-2 polycis- expressed in DS-AMKL and non-DS-AMKL. miR-125b-2 tron on hsa21 can be found in identical configuration in increased proliferation and self-renewal of human and the intron of the lincRNA MIR100HG on hsa11 (miR-100 ~ mouse megakaryocytic progenitors (MPs) and megakaryo- 125b-1). We could previously demonstrate that miR-100 ~ cytic/erythroid progenitors (MEPs) [14]. This small RNA is 125b-1 and miR-99a ~ 125b-2 protect megakaryoblasts A hsa21 MONC [MIR99AHG] TSS1 TSS2 TSS3 hsa11 MIR100HG TSS1 B 4.5 3.0 1.5 0.0 s s ll ll e 1 2 P e e 01 tes t cell ce 07 - B4 O- y ry y - CMK CMY N urkat SPC E -T-cell-T-c B- M- THP-1 J K56 H loc Meg yoc NK NOM 4- ar nu Kasumi-1 k a MonocytesCD4 CD8 Gr CD3 ega M C D 4 FC) R²=0.45; p=0.03 FC) 2 2 2 R²=0.27; p=0.12 R²=0.82; p=0.0003 -4 -2 2 4 -2 miR-99a let-7c -4 miR-125b miRNA expressionmiRNA (log miRNA expressionmiRNA (log MONC expression (log2 FC) Figure 1 MiR-99a/100 ~ 125b cluster host genes in hematopoietic cells and leukemia. A) Genomic architecture of the miR-99a/let-7c/miR-125b-2 (hsa21) and miR-100/let-7a-2/miR-125b-1 (hsa11) cluster. MIR99AHG (alias MONC)andMIR100HG represent the lincRNA host genes of the miRNA cluster, TSSs were determined by 5’RACE-PCR [15]. B) Transcript quantification of MONC and MIR100HG by qRT-PCR in sorted CD34+ HSPCs, CD36+/GlyA+ erythroid cells, CD41+/CD42b+ megakaryocytes, CD15+/CD66b+ neutrophil granulocytes, CD14+ monocytes, CD3+/CD4+/CD8− and CD3+/CD4−/CD8+ T-cells, respectively, CD56+/CD3− NK cells and CD19+/CD3−/CD56− B-cells (left panel;n=5each)aswellasindicatedcelllines(right panel). The B2M gene was used as reference; A.U., arbitrary units. C) Correlation plots and statistics of MONC and D) MIR100HG expression with their cluster miRNA expression in NB4, NOMO-1, THP-1, Kasumi-1, Jurkat, K562, M-07e, Meg-01, CMK and CMY cells measured by qRT-PCR. (B-D) Data are presented as mean ± s.d. Emmrich et al. Molecular Cancer 2014, 13:171 Page 3 of 12 http://www.molecular-cancer.com/content/13/1/171 and leukemic cells from TGFβ1-mediated proliferation K562 cells with low to absent endogenous MIR100HG arrest and apoptosis [15]. However, the role of the expression was mainly unaffected by sh-MIR100HG- lncRNA hostgenes in this ncRNA ensemble remained transduction (Additional file 1: Figure S1B-C). The colony- elusive. forming capacity of Meg-01 cells was decreased upon In the present study, we characterized the function of MIR100HG-knockdown (Figure 2C). This effect was even MONC and MIR100HG and demonstrate an unprece- aggravated in replating experiments for sh-MIR100HG #2, dented role of lncRNAs MONC and MIR100HG during the construct with the stronger knockdown efficacy hematopoiesis and the pathogenesis of AMKL. (Figure 2D). In BrdU cell cycle analyses of Meg-01 cells, we observed an increase in the apoptotic subG1 fraction Results accompanied by a decrease of cycling cells in S phase upon MiR-99a/100 ~ 125b cluster lincRNAs are overexpressed in MIR100HG knockdown (Figure 2E). Accordingly, we mon- AMKL itored a significant increase of Annexin+ apoptotic cells The miR-99a/100 ~ 125b clusters on hsa11 and hsa21 (Figure 2 F). Interestingly, MIR100HG knockdown changed are central regulators of stem cell homeostasis and the surface marker expression on the leukemic megakaryo- leukemogenesis and are hosted in introns of MIR100HG blasts (Figure 2G). While the percentage of CD36+ cells and MONC, respectively. We mapped the transcriptional increased from 11% in controls to 32%, the percentage of start sites (TSS) of both clusters by 5’RACE-PCR and CD41+ cells was ~1.8-fold reduced. demonstrated that the miRNAs are transcribed as one Taken together, knockdown of MIR100HG impaired cell polycistronic transcript together with their host genes viability and replating-efficiency of AMKL cells, while [15]. qRT-PCR expression profiling of spliced MONC changing lineage surface marker expression. and MIR100HG trancripts throughout hematopoietic line- ages demonstrated higher expression of MONC in mega- Knockdown of MONC reduces cell proliferation and viability karyocytes, HSPCs and B-cells and higher expression of MONC is encoded on hsa21 and highly upregulated in MIR100HG in erythroid cells, HSPCs and B-cells as com- both DS-AMKL (trisomy 21) and non-DS-AMKL cell pared to the other blood lineages (Figure 1B). Further- lines (Figure 1B). Therefore we sought to evaluate the more, MONC and MIR100HG are higher expressed in consequences of MONC knockdown in CMK and Meg-01 AMKL cell lines compared to various other leukemic cell cell lines, representing those two entities. As a control, lines (Figure 1B). Regression analysis confirmed positive we used K562 cells with low to absent MONC ex- correlation of MONC and MIR100HG with their respect- pression (Figure 1B). We designed a total of 8 different ive miRNA polycistrons (Figure 1C,D).
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