LincRNAs MONC and MIR100HG act as oncogenes in acute megakaryoblastic leukemia - PubMed (original) (raw)

LincRNAs MONC and MIR100HG act as oncogenes in acute megakaryoblastic leukemia

Stephan Emmrich et al. Mol Cancer. 2014.

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) and MIR100HG 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.

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Figures

Figure 1

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) and MIR100HG 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 = 5 each) as well as indicated cell lines (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.

Figure 2

Figure 2

Knockdown of MIR100HG confers growth disadvantage to AMKL cells. A) Number of shRNA- or ctrl-transduced Meg-01 cells (n = 2). B) Fraction of Cerulean+ shRNA-transduced cells at indicated time points of culture is shown in relation to the ctrl construct (n = 2; Two-way ANOVA was performed to compare the mean of each construct at each time point to ctrl). C) Number of colonies from methylcellulose-based CFU assays of shRNA-transduced Meg-01 cells (n = 2). D) Cumulative number of CFUs after one round of replating of sh-MIR100HG #2 in Meg-01 cells (n = 2). E) Percentage of shRNA-transduced Meg-01 cells in subG1 (BrdU−/7-AAD−), G1 (BrdUlow/7-AADlow/high), S-phase (BrdU+/7-AADlow/high) and G2/M fraction (BrdUlow/7-AADhigh) (n = 2). Asterisks are indicated for subG1 and S phases. F) Percentage of apoptotic/dead (Annexin V+) shRNA-transduced Meg-01 cells after 5 days of culture (n = 2). G) Representative density plots of viable, Cerulean+ Meg-01 cells for indicated surface markers as measured by flow cytometry after 5 days of culture (n = 4). (A-G) Data are presented as mean ± s.d. *P < 0.05; **P < 0.01.

Figure 3

Figure 3

Knockdown of MONC reduces proliferation and viability of AMKL cells. A) Number of shRNA- or ctrl-transduced CMK and Meg-01 cells (n = 3). B) Fraction of Cerulean+ shRNA-transduced cells at indicated time points of culture is shown in relation to the ctrl construct (n = 3; Two-way ANOVA was performed to compare the mean of each construct at each time point to ctrl). C) Number of colonies from methylcellulose-based CFU assays of indicated shRNA-transduced CMK and Meg-01 cells (n = 3). D) Cumulative number of CFUs after one round of replating of sh-MONC in CMK and Meg-01 cells (n = 3). E) Percentage of shRNA-transduced Meg-01 cells in subG1 (BrdU−/7-AAD−), G1 (BrdUlow/7-AADlow/high), S-phase (BrdU+/7-AADlow/high) and G2/M fraction (BrdUlow/7-AADhigh) (n = 3). F) Percentage of apoptotic (Annexin V+/7-AAD−) and dead (7-AAD+) cells for shRNA-transduced CMK and Meg-01 cells measured by flow cytometry after 5 days of culture (n = 3). G-H) Merged density plots of viable, Cerulean+G) Meg-01 and H) CMK cells for indicated surface markers as measured by flow cytometry after 5 days of culture (n = 4); population frequencies with errors are displayed for respective framed gates. I) Number of colonies from methylcellulose-based CFU assays of indicated shRNA-transduced Down-Syndrome transient leukemia blasts (n = 2; error bars show variation). (A-H) Data are presented as mean ± s.d. *P < 0.05; **P < 0.01.

Figure 4

Figure 4

Design and evaluation of a lentiviral lncRNA overexpression vector. A) Schematic vector map. PCR primers used in B) are indicated. B) DNA electrophoresis gel of control PCRs validating the lncRNA expression and termination. Primer pair fwd1-rev1 demonstrates genomic integration of transduced cells, pair fwd2-rev1 indicates functional PolyA signal upon product absence in cDNA samples, and pair fwd2-rev2 detects specifically the MONC transcript. For all primer combinations plasmid DNA (vector plasmid, MONC plasmid) was used as a control for respective genomic or cDNA samples. Vector, empty LeGO-CeB/lnc. right graph: qRT-PCR quantification of MONC in transduced HT1080 cells (Data are presented as mean ± s.d. *P < 0.05; **P < 0.01).

Figure 5

Figure 5

MONC leads the expansion of immature erythroid precursors. A) qRT-PCR of _MONC_-transduced CD34+-HSPCs. B) Numbers of megakaryocytic (CD41+) and non-megakaryocytic (CD41−) CFUs from Megacult® assays of transduced HSPCs after 14 days (n = 2). C) Number of colonies in the methocellulose-based CFU-assay of transduced HSPCs after 12 days (n = 2). D) Representative flow cytometry dot plots of transduced HSPCs stained for CD71 and CD117 on day 4 of in vitro culture (n = 2). E) Representative flow cytometry histogram of CD13-stained transduced HSPCs on day 4 of in vitro culture (n = 2). Mean fluorescence intensities (MFI) are indicated. F-G) Representative flow cytometry dot plots of transduced HSPCs stained for F) CD36 and CD235a and G) stained for CD41 and CD42b after 7 days of mixed erythroid-megakaryocytic differentiation culture (n = 2); population frequencies with errors are displayed for respective framed gates. (A-G) Data are presented as mean ± s.d. *P < 0.05; **P < 0.01.

Figure 6

Figure 6

Subcellular localization of MONC and MIR100HG. A) RNA-FISH with LNA-probes for MONC and MIR100HG transcripts in CMK cells (n = 3). A scrambled oligo was used as negative control and a probe against poly-adenylated transcripts as a positive control (scale bars: 10 μm). B) qRT-PR for MONC and MIR100HG in fractionated RNA, showing a < 1 ratio. Cytoplasmic B2M RNA with a > 1 ratio is shown as control.

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