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MYC stimulates EZH2 expression by repression of its negative regulator miR-26a

Sandrine Sander, Lars Bullinger, Kay Klapproth, Katja Fiedler, Hans A. Kestler, Thomas F. E. Barth, Peter Möller, Stephan Stilgenbauer, Jonathan R. Pollack and Thomas Wirth

Data supplements

Article Figures & Data

  • Figure 1

    miRNA expression profiling in MYC-dependent lymphoma. (A) Supervised analysis for MYC-regulated miRNAs (class comparison; P < .01, paired t test with random variance model paired samples) based on miRNAs displayed in panel C. Expression levels have been set to 0 in the untreated samples to display Dox-related changes. MYC-induced miRNAs are color coded in red, whereas repressed miRNAs are depicted in green. (B) Validation of microarray data by Northern blot analysis (doxycycline treatment for 18 hours). (C) Unsupervised average linkage cluster of MYC-induced lymphoma cell lines (n = 26) based on 154 differentially expressed miRNAs. Two major cell line clusters were identified, mainly characterized by a distinct miRNA pattern (blue bar).

  • Figure 2

    Proliferation changes after inducible miRNA expression in Namalwa. Namalwa cells transfected with an episomal Tet-inducible expression system were treated with Dox for 24 hours and 72 hours; subsequently, cell proliferation was determined and normalized to the empty control. Overexpression of miR-20a slightly increased cell counts, whereas induction of miR-489 and miR-494 caused no significant cell count alterations. In contrast, miR-181a, miR-16, and most prominently miR-26a expression resulted in reduced cell proliferation.

  • Figure 3

    miR-26a expression in MYC-induced lymphoma. (A,B) Northern blot analyses for miR-26a. (A) miR-26a expression in murine Myc-induced lymphoma cell lines after Dox treatment (18 hours). (B) miR-26a expression in human BL-derived cell lines and peripheral blood derived mononuclear cells (PBMNC). (C) Quantitative RT-PCR results evaluating miR-26a expression in primary human BL (n = 11), unselected B-CLL (n = 9), and CD19+-enriched B-CLL (n = 3) samples as well as in BL-derived cell lines (n = 3) and the CD19 fraction of the 3 enriched B-CLL cases. Normalized expression values are depicted (box plots indicate median expression and range of expression values). There are significant differences between BL and B-CLL samples with regard to miR-26a expression (P < .001, exact Wilcoxon rank sum test).

  • Figure 4

    miR-26a–induced phenotypic changes in a MYC expression background. (A) At indicated time points, cell count analysis was performed after induction of miR-26a expression in transfected Raji and Namalwa cells. After 72 hours of miR-26a overexpression, cell numbers were reduced in Raji and Namalwa cells (P = .031, exact Wilcoxon rank sum test). These effects were more pronounced after 96 hours of miRNA induction (P = .031, exact Wilcoxon rank sum test). As a control, empty vector- containing cells were treated with Dox for the same time periods. (B) Cell-cycle analysis in empty-vector and miR-26a-transfected Raji and Namalwa cells was determined by PI staining at indicated time points after Dox treatment. Compared with the empty-vector control, there was an increased percentage of cells in G1 phase within the miR-26a–expressing cell lines (P = .031, exact Wilcoxon rank sum test).

  • Figure 5

    EZH2 as a potential miR-26a target. (A) Protein expression analysis of selected miR-26a candidate targets in stably transfected Raji cells at indicated time points after miR-26a induction compared with an empty vector control. Actin/tubulin served as a loading control. (B) Transient transfection of miR-26a precursor and scrambled RNA into HEK-293 and Raji cells. Western blot results are shown for EZH2 and tubulin 48 hours after transfection. (C) Protein expression analysis of EZH2 after Dox treatment in 2 MYC-induced murine lymphoma cell lines. Starvation of Namalwa cells served as a positive control. (D) EZH2 protein expression in several human BL cell lines compared with endogenous miR-26a expression. (E) Sequence homology between miR-26a and human EZH2, site of miR-26a binding in the 3′-UTR of EZH2 is indicated. (F) Fold repression of luciferase activity as determined by reporter assay in HEK-293 cells. Luciferase expression was determined 24 hours after cotransfection of either the precursor miR-26a or a scrambled RNA. There was a significant reduction in luciferase activity in the miR-26a transfected cells compared with the controls (P = .004, exact Wilcoxon rank sum test). Error bars for 3 independent experiments (measured each in triplicate) are depicted.

  • Figure 6

    EZH2 knock-down in MYC-dependent BL cell lines. (A) At indicated time points, cell count analysis was performed after induction of EZH2 shRNA#1 or EZH2 shRNA #2 expression in transfected Raji and Namalwa cells. After 96 hours of shRNA expression, cell numbers were significantly reduced in Raji and Namalwa cells (P = .031, exact Wilcoxon rank sum test). As a control, empty vector containing cells were treated with Dox for the same time periods. (B) Protein expression analysis of EZH2 after Dox induced shRNA expression at indicated time points. (C) Cell cycle analysis in empty vector and shRNA-transfected Raji and Namalwa cells was determined by PI staining at indicated time points after Dox treatment. Within the shRNA-expressing cell lines, there was an increased percentage of cells in G1 phase compared with the empty vector control (P = .031, exact Wilcoxon rank sum test).

  • Table 1

    miRNAs associated with MYC-induced lymphoma

    Class comparisonDown-regulated miRNAsUp-regulated miRNAs
    High vs low MYC state
    miR-16miR-17-5p
    miR-26amiR-18
    miR-134miR-19b
    miR-207miR-20a
    miR-489miR-92
    miR-494miR-106a
    miR-130b
    B-cell phenotype (B- vs T-cell phenotype)
    let-7bmiR-30a-3p
    let-7cmiR-130b
    miR-15bmiR-155
    miR-30a-5pmiR-187
    miR-30bmiR-192
    miR-30dmiR-345
    miR-107miR-519e
    miR-128amiR-527
    miR-150
  • Table 2

    EZH2 target gene enrichment in miR-26a-induced gene expression pattern

    Gene symbolDescriptionProbe setGeometric mean of intensities (class empty/class miR-26a)Fold change after PcG knock-down*
    ARHGAP24Rho GTPase-activating protein 241566825_at0.70372921.5
    ATAD2ATPase family, AAA domain containing 2222740_at1.3870516−1.9
    CCNB1Cyclin B1214710_s_at1.3400383−1.5
    CCNE2Cyclin E2205034_at1.5680341−2.5
    CDC25ACell division cycle 25 homolog A (S. cerevisiae)1555772_a_at1.3642922−1.9
    CDC6Cell division cycle 6 homolog (S. cerevisiae)203968_s_at1.4108894−3.1
    CDT1Chromatin licensing and DNA replication factor 1228868_x_at1.5277866−2.2
    CHAF1AChromatin assembly factor 1, subunit A (p150)203976_s_at1.5635639−6.2
    CUGBP2CUG triplet repeat, RNA binding protein 2242268_at0.71931291.7
    DCC1defective in sister chromatid cohesion homolog 1 (S. cerevisiae)219000_s_at1.3318879−1.6
    GMNNGeminin, DNA replication inhibitor218350_s_at1.3320183−2
    GTSE1G2- and S-phase expressed 1215942_s_at1.4042593−3.4
    HELLSHelicase, lymphoid-specific220085_at1.3691602−2.3
    LRAPLeukocyte-derived arginine aminopeptidase240338_at0.6309561.4
    MCM10MCM10 minichromosome maintenance deficient 10 (S. cerevisiae)220651_s_at1.4244193−3.1
    MCM4MCM4 minichromosome maintenance deficient 4 (S. cerevisiae)222037_at1.2871303−4.8
    MKI67Antigen identified by monoclonal antibody Ki-67212022_s_at1.4520355−2.7
    MYBL1v-myb myeloblastosis viral oncogene homolog (avian)-like 1213906_at1.5254951−1.7
    ORC1LOrigin recognition complex, subunit 1-like (yeast)205085_at1.511178−2.9
    PDE4DIPPhosphodiesterase 4D interacting protein (myomegalin)232615_at0.76586411.1
    RFC4Replication factor C (activator 1) 4, 37kDa204023_at1.4484428−1.9
    SKP2S-phase kinase-associated protein 2 (p45)210567_s_at1.4732182−2.2
    SLC17A5Solute carrier family 17 (anion/sugar transporter), member 5221041_s_at0.77744431.3
    SOX5SRY (sex determining region Y)-box 5215768_at0.6858781.7
    TncRNATrophoblast-derived noncoding RNA238320_at0.54576271.9
    ZBTB1Zinc finger and BTB domain containing 11557036_at0.74101044.9
    ZBTB10zinc finger and BTB domain containing 10222863_at0.73214632.6
    • * Bracken et al.34

Supplementary Materials

  • Figure S1

    Supplementary PDF file available online.

  • Figure S2

    Supplementary PDF file available online.

  • Figure S3

    Supplementary PDF file available online.

  • Table S1

    Supplementary PDF file available online.

  • Table S2

    Supplementary PDF file available online.

  • Table S3

    Supplementary PDF file available online.