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Disruption of the C/EBPα—miR-182 balance impairs granulocytic differentiation

Wurm, Alexander Arthur, Zjablovskaja, Polina, Kardosova, Miroslava, Gerloff, Dennis, Bräuer-Hartmann, Daniela, Katzerke, Christiane, Hartmann, Jens-Uwe, Benoukraf, Touati, Fricke, Stephan, Hilger, Nadja, Müller, Anne-Marie, Bill, Marius, Schwind, Sebastian, Tenen, Daniel G., Niederwieser, Dietger, Alberich-Jorda, Meritxell, Behre, Gerhard·2017·Nature Communications
DOI 10.1038/s41467-017-00032-6PMC PMC5491528
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Abstract

Transcription factor C/EBPα is a master regulator of myelopoiesis and its inactivation is associated with acute myeloid leukemia. Deregulation of C/EBPα by microRNAs during granulopoiesis or acute myeloid leukemia development has not been studied. Here we show that oncogenic miR-182 is a strong regulator of C/EBPα. Moreover, we identify a regulatory loop between C/EBPα and miR-182. While C/EBPα blocks miR-182 expression by direct promoter binding during myeloid differentiation, enforced expression of miR-182 reduces C/EBPα protein level and impairs granulopoiesis in vitro and in vivo. In addition, miR-182 expression is highly elevated particularly in acute myeloid leukemia patients with C-terminal CEBPA mutations, thereby depicting a mechanism by which C/EBPα blocks miR-182 expression. Furthermore, we present miR-182 expression as a prognostic marker in cytogenetically high-risk acute myeloid leukemia patients. Our data demonstrate the importance of a controlled balance between C/EBPα and miR-182 for the maintenance of healthy granulopoiesis.

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Figures

Fig. 1 C/EBPα blocks miR-182 expression and both factors correlate inversely. a Next Generation Sequencing (NGS) for small RNAs in inducible K562-C/EBPα-ER cells revealed several miRNAs upregulated and downregulated by C/EBPα. MiR-182 is highlighted in bold (red arrow). Data summarize three independent experiments, while each colored box marks the relative expression of the indicated miRNA in comparison to the appropriate mean of each experiment. Red represents high fold change and green low fold change. b QPCR for miR-182 expression showed downregulation of miR-182 after induction of wild-type C/EBPα-p42 in K562-C/EBPα-ER cells. Induction of N-terminal mutant C/EBPα-p30, but not C-terminal mutant C/EBPα-BRM2 or control ER resulted in reduced miR-182 expression levels. Ethanol served as vehicle control. Cells were collected after treatment with β-estradiol for 6 h. Data represent the mean ± SD of three independent experiments (*p < 0.05). P values were calculated using unpaired Student’s t-test. c and d Relative expression levels of miR-182 were analyzed by qPCR c and correlate inversely to the protein amount of C/EBPα d in indicated leukemic cell lines, as analyzed by western blot. e Induced knockout of CEBPA by injection of pI:pC in CEBPA f/fMx1Cre+ mice led to reduced CEBPA mRNA levels compared to pI:pC injected CEBPA f/f fMx1Cre− mice and resulted in an upregulation of miR-182 in lineage−/c-Kit+ myeloid progenitor cells. Expression was measured using qPCR and quantified compared to the mean of all animals. Data represent the mean ± SD of four animals (**p < 0.01). P values were calculated using unpaired Student’s t-test. f Relative expression levels of miR-182 and CEBPA in sorted murine bone marrow subpopulations. LSK—Lineage−Sca-1+cKit+ myeloid progenitors, CMP—common myeloid progenitors, GMP—granulocyte-macrophage progenitor, NP—neutrophils. g Treatment of human CD34+ hematopoietic progenitor cells with G-CSF in vitro led to a rapid decrease of miR-182 expression and correlated inversely to C/EBPα protein levels. C/EBPα protein level was analyzed using western blot (left side, upper panel). The stage of differentiation was shown by flow cytometry after 14 days (left side, lower panel). MiR-182 expression was measured using qPCR (right panel). Normalization for miR-182 expression was done using U6 (human samples) or snoRNA-135 (mouse samples). For CEBPA expression normalization, GAPDH was used. Data represent the mean ± SD of three independent experiments (*p < 0.05). P-values were calculated using unpaired Student’s t-test
Fig. 1 C/EBPα blocks miR-182 expression and both factors correlate inversely. a Next Generation Sequencing (NGS) for small RNAs in inducible K562-C/EBPα-ER cells revealed several miRNAs upregulated and downregulated by C/EBPα. MiR-182 is highlighted in bold (red arrow). Data summarize three independent experiments, while each colored box marks the relative expression of the indicated miRNA in comparison to the appropriate mean of each experiment. Red represents high fold change and green low fold change. b QPCR for miR-182 expression showed downregulation of miR-182 after induction of wild-type C/EBPα-p42 in K562-C/EBPα-ER cells. Induction of N-terminal mutant C/EBPα-p30, but not C-terminal mutant C/EBPα-BRM2 or control ER resulted in reduced miR-182 expression levels. Ethanol served as vehicle control. Cells were collected after treatment with β-estradiol for 6 h. Data represent the mean ± SD of three independent experiments (*p < 0.05). P values were calculated using unpaired Student’s t-test. c and d Relative expression levels of miR-182 were analyzed by qPCR c and correlate inversely to the protein amount of C/EBPα d in indicated leukemic cell lines, as analyzed by western blot. e Induced knockout of CEBPA by injection of pI:pC in CEBPA f/fMx1Cre+ mice led to reduced CEBPA mRNA levels compared to pI:pC injected CEBPA f/f fMx1Cre− mice and resulted in an upregulation of miR-182 in lineage−/c-Kit+ myeloid progenitor cells. Expression was measured using qPCR and quantified compared to the mean of all animals. Data represent the mean ± SD of four animals (**p < 0.01). P values were calculated using unpaired Student’s t-test. f Relative expression levels of miR-182 and CEBPA in sorted murine bone marrow subpopulations. LSK—Lineage−Sca-1+cKit+ myeloid progenitors, CMP—common myeloid progenitors, GMP—granulocyte-macrophage progenitor, NP—neutrophils. g Treatment of human CD34+ hematopoietic progenitor cells with G-CSF in vitro led to a rapid decrease of miR-182 expression and correlated inversely to C/EBPα protein levels. C/EBPα protein level was analyzed using western blot (left side, upper panel). The stage of differentiation was shown by flow cytometry after 14 days (left side, lower panel). MiR-182 expression was measured using qPCR (right panel). Normalization for miR-182 expression was done using U6 (human samples) or snoRNA-135 (mouse samples). For CEBPA expression normalization, GAPDH was used. Data represent the mean ± SD of three independent experiments (*p < 0.05). P-values were calculated using unpaired Student’s t-testCC BY · PubMed Central
Fig. 2 MiR-182 expression is blocked by C/EBPα p42. a Schematic overview about the minimal MIR182 promoter region. b ChIP assay in K562-C/EBPα-ER cells revealed a binding of C/EBPα to both distal (#1) and proximal (#3) C/EBP binding sites. Analysis was calculated as a ratio between β-estradiol and ethanol treated cells. Data represent the mean ± SD of two independent experiments. c Transient overexpression of E2F1 in U937 cells leads to enhanced miR-182 expression levels at the indicated time points. Expression was measured using qPCR. Data represent the mean ± SD of three independent experiments (*p < 0.05). P values were calculated using unpaired Student’s t-test. d ChIP assay in K562-C/EBPα-ER cells showed a binding of E2F1 predominantly in the region of the predicted E2F binding site (#2). Data represent the mean ± SD of four independent experiments. (**p < 0.01). P values were calculated using unpaired Student’s t-test. e Schematic representation of different C/EBPα mutant forms used for luciferase assay. P42 represents the wild-type, p30 the N-terminal truncated mutant. BRM2 represents a point mutation in the E2F interaction domain at amino acids 294 and 297. The other mutants are insertions in the bZIP domain: bZIP-A (R300-D301 del—ins QN), bZIP-B (dupl. K313) and bZIP-C (dupl. K312). The asterisks mark the region of the point mutation. f Luciferase assay with full length MIR182 minimal promoter showed significantly reduced luciferase activity after C/EBPα-p42, C/EBPα-p30 and bZIP-mutant-A, but not C/EBPα-BRM2, bZIP-mutant-B and bZIP-mutant-C overexpression. g Effects of C/EBPα p42 and E2F on luciferase activity can be abolished using specific point mutations in the indicated binding sites. Luciferase activity was measured using different indicated MIR182 promoter constructs including mutations in the proximal C/EBP site (c2) and the E2F site (c4). Black circles highlight C/EBP binding sites and white circle the E2F site. Co-expression of E2F1 can partially rescue the effect of C/EBPα on promoter luciferase activity. Activity was normalized to pGL3-control. All luciferase experiments were normalized to transfection with equal amounts of pcDNA3.1 empty vector (backbone of all C/EBPα constructs and E2F). Renilla luciferase (pRL) served as internal normalization control. Data represent the mean ± SD of three independent experiments (*p < 0.05; **p < 0.01; ***p < 0.001). P values were calculated using unpaired Student’s t-test
Fig. 2 MiR-182 expression is blocked by C/EBPα p42. a Schematic overview about the minimal MIR182 promoter region. b ChIP assay in K562-C/EBPα-ER cells revealed a binding of C/EBPα to both distal (#1) and proximal (#3) C/EBP binding sites. Analysis was calculated as a ratio between β-estradiol and ethanol treated cells. Data represent the mean ± SD of two independent experiments. c Transient overexpression of E2F1 in U937 cells leads to enhanced miR-182 expression levels at the indicated time points. Expression was measured using qPCR. Data represent the mean ± SD of three independent experiments (*p < 0.05). P values were calculated using unpaired Student’s t-test. d ChIP assay in K562-C/EBPα-ER cells showed a binding of E2F1 predominantly in the region of the predicted E2F binding site (#2). Data represent the mean ± SD of four independent experiments. (**p < 0.01). P values were calculated using unpaired Student’s t-test. e Schematic representation of different C/EBPα mutant forms used for luciferase assay. P42 represents the wild-type, p30 the N-terminal truncated mutant. BRM2 represents a point mutation in the E2F interaction domain at amino acids 294 and 297. The other mutants are insertions in the bZIP domain: bZIP-A (R300-D301 del—ins QN), bZIP-B (dupl. K313) and bZIP-C (dupl. K312). The asterisks mark the region of the point mutation. f Luciferase assay with full length MIR182 minimal promoter showed significantly reduced luciferase activity after C/EBPα-p42, C/EBPα-p30 and bZIP-mutant-A, but not C/EBPα-BRM2, bZIP-mutant-B and bZIP-mutant-C overexpression. g Effects of C/EBPα p42 and E2F on luciferase activity can be abolished using specific point mutations in the indicated binding sites. Luciferase activity was measured using different indicated MIR182 promoter constructs including mutations in the proximal C/EBP site (c2) and the E2F site (c4). Black circles highlight C/EBP binding sites and white circle the E2F site. Co-expression of E2F1 can partially rescue the effect of C/EBPα on promoter luciferase activity. Activity was normalized to pGL3-control. All luciferase experiments were normalized to transfection with equal amounts of pcDNA3.1 empty vector (backbone of all C/EBPα constructs and E2F). Renilla luciferase (pRL) served as internal normalization control. Data represent the mean ± SD of three independent experiments (*p < 0.05; **p < 0.01; ***p < 0.001). P values were calculated using unpaired Student’s t-testCC BY · PubMed Central
Fig. 3 MiR-182 expression defines a subgroup of AML. a Schematic representation of different types of C/EBPα found in AML. b Relative expression of miR-182 in samples derived from bone marrow mononuclear cells (BMMNCs) of healthy donors (n = 3) or normal karyotype AML patients with either CEBPA wild-type (n = 12), CEBPA N-terminal mutation (n = 5) or CEBPA C-terminal mutation (n = 9). MiR-182 expression was elevated only in AML patients carrying a mutation in the C-terminal region of the CEBPA gene. Normalization was done using U6 and compared to the mean of all patient samples (** p< 0.01). P values were calculated using Mann-Whitney-U test. c Overall survival of high-risk AML patients according to the median divided miR-182 expression. P values were calculated using Log-rank test. d Overall survival of according to the CEBPA expression. Expression quartiles were created and the group with the lowest CEBPA expression (first quartile) was compared to the rest (second, third and fourth quartile). The only existing CEBPA mutated patient (ID2952) was excluded from the analysis. P values were calculated using Log-rank test. e Correlation between miR-182 and CEBPA expression in high-risk AML. Groups were divided into four quartiles according to the miR-182 expression. The low group represents the first quartile with the lowest expression levels of miR-182, while the high group combines the rest. Low miR-182 expressing patients showed significantly higher CEBPA levels (n = 41, *p < 0.05). P values were calculated using unpaired Student’s t-test. f Correlation between miR-182 and E2F2 expression in high-risk AML. Groups were divided according to the median miR-182 expression. High miR-182 expressing patients showed significantly higher E2F2 levels. (n = 42, *p < 0.05). P values were calculated using unpaired Student’s t-test. g Correlation between miR-182 expression and percentage of bone marrow blasts in the high-risk AML patient set. Groups were divided according to the median miR-182 expression. High miR-182 expressing patients showed significantly lower bone marrow blast counts. (n = 42, *p < 0.05). P-values were calculated using unpaired Student’s t-test. h Correlation between miR-182 and percentage of peripheral blood blasts in the high-risk AML patient set. Groups were divided according to the median miR-182 expression. High miR-182 expressing patients showed significantly lower peripheral blood blast counts. (n = 42, ***p < 0.001). P values were calculated using unpaired Student’s t-test
Fig. 3 MiR-182 expression defines a subgroup of AML. a Schematic representation of different types of C/EBPα found in AML. b Relative expression of miR-182 in samples derived from bone marrow mononuclear cells (BMMNCs) of healthy donors (n = 3) or normal karyotype AML patients with either CEBPA wild-type (n = 12), CEBPA N-terminal mutation (n = 5) or CEBPA C-terminal mutation (n = 9). MiR-182 expression was elevated only in AML patients carrying a mutation in the C-terminal region of the CEBPA gene. Normalization was done using U6 and compared to the mean of all patient samples (** p< 0.01). P values were calculated using Mann-Whitney-U test. c Overall survival of high-risk AML patients according to the median divided miR-182 expression. P values were calculated using Log-rank test. d Overall survival of according to the CEBPA expression. Expression quartiles were created and the group with the lowest CEBPA expression (first quartile) was compared to the rest (second, third and fourth quartile). The only existing CEBPA mutated patient (ID2952) was excluded from the analysis. P values were calculated using Log-rank test. e Correlation between miR-182 and CEBPA expression in high-risk AML. Groups were divided into four quartiles according to the miR-182 expression. The low group represents the first quartile with the lowest expression levels of miR-182, while the high group combines the rest. Low miR-182 expressing patients showed significantly higher CEBPA levels (n = 41, *p < 0.05). P values were calculated using unpaired Student’s t-test. f Correlation between miR-182 and E2F2 expression in high-risk AML. Groups were divided according to the median miR-182 expression. High miR-182 expressing patients showed significantly higher E2F2 levels. (n = 42, *p < 0.05). P values were calculated using unpaired Student’s t-test. g Correlation between miR-182 expression and percentage of bone marrow blasts in the high-risk AML patient set. Groups were divided according to the median miR-182 expression. High miR-182 expressing patients showed significantly lower bone marrow blast counts. (n = 42, *p < 0.05). P-values were calculated using unpaired Student’s t-test. h Correlation between miR-182 and percentage of peripheral blood blasts in the high-risk AML patient set. Groups were divided according to the median miR-182 expression. High miR-182 expressing patients showed significantly lower peripheral blood blast counts. (n = 42, ***p < 0.001). P values were calculated using unpaired Student’s t-testCC BY · PubMed Central
Fig. 4 C/EBPα is a direct target of miR-182. a Schematic overview about the highly conserved binding site for miR-182 in the 3′-UTR of the CEBPA mRNA. b–d Transient overexpression of human miR-182 led to a reduction of C/EBPα protein amount in b U937 cells, umbilical cord blood mononuclear cells (UCB-MNCs) c and primary AML-blasts d in vitro. e A block of miR-182 by either stable infection of U937 or Kasumi-1 cells with decoy-182 vector (left panels) or LNA treatment of primary AML blasts in vitro (right panel) led to elevated C/EBPα protein levels. f Schematic overview about the miR-182 binding site in the luciferase vector and the mutation of the indicated nucleotides. g Luciferase reporter and mutagenesis assay. U937 cells were co-transfected with either pGL3-control vector, pGL3-CEBPA-3′UTR or pGL3-CEBPA-mutant-3′UTR and pcDNA-6.2 empty (gray bars) or pcDNA-6.2-miR-182 (black bars). An overexpression of miR-182 inhibited the relative luciferase activity only when co-transfected with the pGL3-CEBPA-3′UTR. Normalization was done using Renilla luciferase construct (pRL). Data represent the mean ± SD of three independent experiments. (*p < 0.05) P values were calculated using unpaired Student’s t-test
Fig. 4 C/EBPα is a direct target of miR-182. a Schematic overview about the highly conserved binding site for miR-182 in the 3′-UTR of the CEBPA mRNA. b–d Transient overexpression of human miR-182 led to a reduction of C/EBPα protein amount in b U937 cells, umbilical cord blood mononuclear cells (UCB-MNCs) c and primary AML-blasts d in vitro. e A block of miR-182 by either stable infection of U937 or Kasumi-1 cells with decoy-182 vector (left panels) or LNA treatment of primary AML blasts in vitro (right panel) led to elevated C/EBPα protein levels. f Schematic overview about the miR-182 binding site in the luciferase vector and the mutation of the indicated nucleotides. g Luciferase reporter and mutagenesis assay. U937 cells were co-transfected with either pGL3-control vector, pGL3-CEBPA-3′UTR or pGL3-CEBPA-mutant-3′UTR and pcDNA-6.2 empty (gray bars) or pcDNA-6.2-miR-182 (black bars). An overexpression of miR-182 inhibited the relative luciferase activity only when co-transfected with the pGL3-CEBPA-3′UTR. Normalization was done using Renilla luciferase construct (pRL). Data represent the mean ± SD of three independent experiments. (*p < 0.05) P values were calculated using unpaired Student’s t-testCC BY · PubMed Central
Fig. 5 MiR-182 blocks granulocytic differentiation in vitro. a Overexpression of miR-182 reduced the amount of C/EBPα protein in both DMSO treated and ATRA treated U937 cells as analyzed by western blot. b and c Enforced expression of miR-182 led to a reduced percentage of CD11b-positive U937 cells after treatment with ATRA. b Representative FACS analysis for myeloid marker CD11b in miR-182 or scramble transfected U937 cells treated for 24 or 48 h with 1 µM ATRA to induce granulocytic differentiation and c summary of all experimental repetitions. Data represent the mean ± SD of three independent experiments. (*p < 0.05) P values were calculated using unpaired Student’s t-test. d Overexpression of miR-182 did not affect the amount of C/EBPα-ER fusionprotein in K562-C/EBPα-p42-ER cells as analyzed by western blot. e and f Enforced expression of miR-182 did not affect the percentage of CD11b-positive K562-C/EBPα-ER cells after treatment with β-estradiol. e Representative FACS analysis for myeloid marker CD11b in miR-182 or scramble transfected K562-C/EBPα-ER cells treated for 24 or 48 h with 5 µM β-estradiol to induce granulocytic differentiation and f summary of all experimental repetitions. Data represent the mean ± SD of three independent experiments. g Block of miR-182 by decoy lentiviral infection increased the amount of C/EBPα protein level in Kasumi-1 cells as analyzed by western blot. h and i A block of miR-182 accelerates myeloid differentiation of Kasumi-1 cells induced by 1 mM Sodium phenylbutyrate (SPB). h Representative FACS analysis for myeloid marker CD11b in decoy-miR-182 or control infected Kasumi-1 cells treated for 72 h with either vehicle control (left panel) or 1 mM SPB to induce granulocytic differentiation and i summary of all experimental repetitions. Data represent the mean ± SD of four independent experiments. (*p < 0.05) P values were calculated using unpaired Student’s t-test
Fig. 5 MiR-182 blocks granulocytic differentiation in vitro. a Overexpression of miR-182 reduced the amount of C/EBPα protein in both DMSO treated and ATRA treated U937 cells as analyzed by western blot. b and c Enforced expression of miR-182 led to a reduced percentage of CD11b-positive U937 cells after treatment with ATRA. b Representative FACS analysis for myeloid marker CD11b in miR-182 or scramble transfected U937 cells treated for 24 or 48 h with 1 µM ATRA to induce granulocytic differentiation and c summary of all experimental repetitions. Data represent the mean ± SD of three independent experiments. (*p < 0.05) P values were calculated using unpaired Student’s t-test. d Overexpression of miR-182 did not affect the amount of C/EBPα-ER fusionprotein in K562-C/EBPα-p42-ER cells as analyzed by western blot. e and f Enforced expression of miR-182 did not affect the percentage of CD11b-positive K562-C/EBPα-ER cells after treatment with β-estradiol. e Representative FACS analysis for myeloid marker CD11b in miR-182 or scramble transfected K562-C/EBPα-ER cells treated for 24 or 48 h with 5 µM β-estradiol to induce granulocytic differentiation and f summary of all experimental repetitions. Data represent the mean ± SD of three independent experiments. g Block of miR-182 by decoy lentiviral infection increased the amount of C/EBPα protein level in Kasumi-1 cells as analyzed by western blot. h and i A block of miR-182 accelerates myeloid differentiation of Kasumi-1 cells induced by 1 mM Sodium phenylbutyrate (SPB). h Representative FACS analysis for myeloid marker CD11b in decoy-miR-182 or control infected Kasumi-1 cells treated for 72 h with either vehicle control (left panel) or 1 mM SPB to induce granulocytic differentiation and i summary of all experimental repetitions. Data represent the mean ± SD of four independent experiments. (*p < 0.05) P values were calculated using unpaired Student’s t-testCC BY · PubMed Central
Fig. 6 Exogenous C/EBPα abrogates miR-182 mediated effects. a MiR-182 levels in 32D cells transduced with either miR-182 or scramble lentivirus. MiR-182 expression was measured by qPCR. b MiR-182 expressing 32D cells showed increased cell numbers under G-CSF conditions (black line) compared to control scramble transduced cells (gray line). 1 × 105 cells were initially seeded and cultured in medium without IL-3 and supplemented with 20 ng/ml G-CSF. Data represent the mean ± SD of three independent experiments. (*p < 0.05; **p < 0.01) P values were calculated using unpaired Student’s t-test. c Morphological analyses by cytospins followed by Wright-Giemsa-staining (top panel). MiR-182 expressing 32D cells exhibited less morphological signs of mature granulocytes and remained in an immature stage after treatment with G-CSF for 7 days. Representative plate from a CFU-assay of 32D-scramble or miR-182 expressing cells after 7 days under G-CSF conditions (bottom panel). Scale bar 5 µM. d Summary of 4 independent CFU-assays demonstrated enhanced colony forming unit ability of miR-182 expressing 32D cells after 7 days under G-CSF conditions. Data represent the mean ± SD. (**p < 0.01) P-values were calculated using unpaired Student’s t-test. e and f Stable overexpression of miR-182 led to a reduced percentage of Mac-1 positive 32D cells under both IL-3 and G-CSF conditions. e Representative flow cytometry analysis for myeloid marker Mac-1 in miR-182 or scramble expressing 32D cells under IL-3 conditions (left panel) or after treatment with 20 ng/ml G-CSF for 7 days (right panel) and f summary of all experimental repetitions. Y-axis indicate the percentage of Mac-1 positive cells. Data represent the mean ± SD of three independent experiments. (*p < 0.05; **p < 0.01; ***p < 0.001) P-values were calculated using unpaired Student’s t-test. g Western blot analysis of 32D cells stably expressing either scramble or miR-182 sequence revealed reduced C/EBPα protein levels under IL-3 conditions or after treatment for 7 days with 20 ng/ml G-CSF. h Transduction of 32D cells with exogenous murine CEBPA enhances the detectable amount of C/EBPα protein. Analysis was performed using western blot. i Exogenous C/EBPα lacking a natural 3′UTR can rescue the miR-182 induced phenotype in 32D cells. Representative flow cytometry analysis for Mac-1 in either parental 32D cells or 32D cells stably expressing miR-182 transduced with control or mCEBPA. Analysis was made 10 days after transduction
Fig. 6 Exogenous C/EBPα abrogates miR-182 mediated effects. a MiR-182 levels in 32D cells transduced with either miR-182 or scramble lentivirus. MiR-182 expression was measured by qPCR. b MiR-182 expressing 32D cells showed increased cell numbers under G-CSF conditions (black line) compared to control scramble transduced cells (gray line). 1 × 105 cells were initially seeded and cultured in medium without IL-3 and supplemented with 20 ng/ml G-CSF. Data represent the mean ± SD of three independent experiments. (*p < 0.05; **p < 0.01) P values were calculated using unpaired Student’s t-test. c Morphological analyses by cytospins followed by Wright-Giemsa-staining (top panel). MiR-182 expressing 32D cells exhibited less morphological signs of mature granulocytes and remained in an immature stage after treatment with G-CSF for 7 days. Representative plate from a CFU-assay of 32D-scramble or miR-182 expressing cells after 7 days under G-CSF conditions (bottom panel). Scale bar 5 µM. d Summary of 4 independent CFU-assays demonstrated enhanced colony forming unit ability of miR-182 expressing 32D cells after 7 days under G-CSF conditions. Data represent the mean ± SD. (**p < 0.01) P-values were calculated using unpaired Student’s t-test. e and f Stable overexpression of miR-182 led to a reduced percentage of Mac-1 positive 32D cells under both IL-3 and G-CSF conditions. e Representative flow cytometry analysis for myeloid marker Mac-1 in miR-182 or scramble expressing 32D cells under IL-3 conditions (left panel) or after treatment with 20 ng/ml G-CSF for 7 days (right panel) and f summary of all experimental repetitions. Y-axis indicate the percentage of Mac-1 positive cells. Data represent the mean ± SD of three independent experiments. (*p < 0.05; **p < 0.01; ***p < 0.001) P-values were calculated using unpaired Student’s t-test. g Western blot analysis of 32D cells stably expressing either scramble or miR-182 sequence revealed reduced C/EBPα protein levels under IL-3 conditions or after treatment for 7 days with 20 ng/ml G-CSF. h Transduction of 32D cells with exogenous murine CEBPA enhances the detectable amount of C/EBPα protein. Analysis was performed using western blot. i Exogenous C/EBPα lacking a natural 3′UTR can rescue the miR-182 induced phenotype in 32D cells. Representative flow cytometry analysis for Mac-1 in either parental 32D cells or 32D cells stably expressing miR-182 transduced with control or mCEBPA. Analysis was made 10 days after transductionCC BY · PubMed Central
Fig. 7 Enforced expression of miR-182 enhances replating capacity. a Schematic overview of the experimental strategy. Bone marrow cells were isolated from the tibia and femur and ficolled to obtain the mononuclear cells (BM-MNCs). Cells were infected with virus coding for murine miR-182 or scramble control and sorted for GFP + cells two days after infection. Cells were placed in semisolid medium and replated every 7–9 days. b Representative plates of CFU-assay after the fourth round of replating revealed more colonies after enforced miR-182 expression compared to control. c Summary of all replating rounds as a result of 4 independent wells of a 12 well plate. MiR-182 expressing mBM-MNCs showed significantly enhanced replating capacity. Data represent the mean ± SD of four independent wells. (***p < 0.001) P values were calculated using unpaired Student’s t-test. d Exogenous C/EBPα lacking a 3′UTR rescues the ability of miR-182 to enhance replating capacity: Representative plates of CFU-assay after third round of replating revealed more colonies after enforced miR-182 expression (182-ctr). This effect was rescued by additional infection with CEBPA coding lentiviral vectors (182-CEBPA). e Summary of all replating rounds as a result of 4 independent wells of a 12 well plate at the second and third replating round. MiR-182-CEBPA double expressing mBM-MNCs showed significantly reduced replating capacity compared to miR-182-control expression cells. Data represent the mean ± SD of four independent wells. (***p < 0.001) P values were calculated using unpaired Student’s t-test. scr = miR-scramble control vector, ctr = CEBPA empty control vector
Fig. 7 Enforced expression of miR-182 enhances replating capacity. a Schematic overview of the experimental strategy. Bone marrow cells were isolated from the tibia and femur and ficolled to obtain the mononuclear cells (BM-MNCs). Cells were infected with virus coding for murine miR-182 or scramble control and sorted for GFP + cells two days after infection. Cells were placed in semisolid medium and replated every 7–9 days. b Representative plates of CFU-assay after the fourth round of replating revealed more colonies after enforced miR-182 expression compared to control. c Summary of all replating rounds as a result of 4 independent wells of a 12 well plate. MiR-182 expressing mBM-MNCs showed significantly enhanced replating capacity. Data represent the mean ± SD of four independent wells. (***p < 0.001) P values were calculated using unpaired Student’s t-test. d Exogenous C/EBPα lacking a 3′UTR rescues the ability of miR-182 to enhance replating capacity: Representative plates of CFU-assay after third round of replating revealed more colonies after enforced miR-182 expression (182-ctr). This effect was rescued by additional infection with CEBPA coding lentiviral vectors (182-CEBPA). e Summary of all replating rounds as a result of 4 independent wells of a 12 well plate at the second and third replating round. MiR-182-CEBPA double expressing mBM-MNCs showed significantly reduced replating capacity compared to miR-182-control expression cells. Data represent the mean ± SD of four independent wells. (***p < 0.001) P values were calculated using unpaired Student’s t-test. scr = miR-scramble control vector, ctr = CEBPA empty control vectorCC BY · PubMed Central
Fig. 8 Enforced expression of miR-182 blocks granulocytic differentiation in vivo. a Schematic overview of the LSK based transplantation model. b MiR-182 levels in GFP+ LSK cells transduced with either miR-182 or scramble lentiviral particles. MiR-182 expression was measured by qPCR 48 h after transduction. c Representative flow cytometry analysis from blood (4 and 10 weeks) and bone marrow (10 weeks) after transplantation revealed a reduced percentage of Mac1+/Gr1+ granulocytes in the GFP+ fraction of mice transplanted with miR-182 transduced LSK cells compared to mice transplanted with scramble transduced cells. GFP− cells served as controls. d Summary of flow cytometry analysis from blood and bone marrow at indicated time points after transplantation. Percentage of mature Mac1+/Gr1+ granulocytes in the GFP+ fraction of mice transplanted with miR-182 transduced LSK cells was significantly reduced. No effect was observed in the GFP− fraction. Bars represent the summary of 3 independent bone marrow transplantations with the indicated total number of animals (4 weeks: scramble n = 9 and miR-182 n = 8; 10 weeks: scramble n = 8 and miR-182 n = 6; *p < 0.05; **p < 0.01; ns—not significant). P values were calculated using unpaired Student’s t-test. e MiR-182 expressing mice showed reduced CEBPA mRNA levels in the bone marrow. GFP + cells from the bone marrow 10 weeks after transplantation were sorted and analyzed by qPCR for CEBPA mRNA expression. Data represent the mean ± SD of four independent animals. (*p < 0.05) P values were calculated using unpaired Student’s t-test. f and g Cell morphology analysis from GFP+ sorted bone marrow cells 10 weeks after transplantation by Wright-Giemsa-staining showed reduced numbers of mature neutrophils in miR-182 transduced mice. f Representative photographs (Scale bar 5 µM) and g summary of four mice (scramble) and three mice (miR-182), respectively. (*p < 0.05) P values were calculated using unpaired Student’s t-test
Fig. 8 Enforced expression of miR-182 blocks granulocytic differentiation in vivo. a Schematic overview of the LSK based transplantation model. b MiR-182 levels in GFP+ LSK cells transduced with either miR-182 or scramble lentiviral particles. MiR-182 expression was measured by qPCR 48 h after transduction. c Representative flow cytometry analysis from blood (4 and 10 weeks) and bone marrow (10 weeks) after transplantation revealed a reduced percentage of Mac1+/Gr1+ granulocytes in the GFP+ fraction of mice transplanted with miR-182 transduced LSK cells compared to mice transplanted with scramble transduced cells. GFP− cells served as controls. d Summary of flow cytometry analysis from blood and bone marrow at indicated time points after transplantation. Percentage of mature Mac1+/Gr1+ granulocytes in the GFP+ fraction of mice transplanted with miR-182 transduced LSK cells was significantly reduced. No effect was observed in the GFP− fraction. Bars represent the summary of 3 independent bone marrow transplantations with the indicated total number of animals (4 weeks: scramble n = 9 and miR-182 n = 8; 10 weeks: scramble n = 8 and miR-182 n = 6; *p < 0.05; **p < 0.01; ns—not significant). P values were calculated using unpaired Student’s t-test. e MiR-182 expressing mice showed reduced CEBPA mRNA levels in the bone marrow. GFP + cells from the bone marrow 10 weeks after transplantation were sorted and analyzed by qPCR for CEBPA mRNA expression. Data represent the mean ± SD of four independent animals. (*p < 0.05) P values were calculated using unpaired Student’s t-test. f and g Cell morphology analysis from GFP+ sorted bone marrow cells 10 weeks after transplantation by Wright-Giemsa-staining showed reduced numbers of mature neutrophils in miR-182 transduced mice. f Representative photographs (Scale bar 5 µM) and g summary of four mice (scramble) and three mice (miR-182), respectively. (*p < 0.05) P values were calculated using unpaired Student’s t-testCC BY · PubMed Central
Fig. 9 Schematic model of the identified C/EBPα-miR-182 interaction. During myeloid differentiation (lower panel), C/EBPα is active, binds to the MIR182 promoter and reduces miR-182 expression. Elevated miR-182 levels, especially in AML with defective C/EBPα DNA binding capacity, reduce the amount of functional C/EBPα protein by direct binding to the CEBPA 3′UTR and block myeloid differentiation (upper panel). C/EBPα is a critical transcription factor involved in myelopoiesis and its inactivation is associated with acute myeloid leukemia (AML). Here the authors show a negative feedback loop between C/EBPα and miR-182 and identify this miRNA as a marker of high-risk AML
Fig. 9 Schematic model of the identified C/EBPα-miR-182 interaction. During myeloid differentiation (lower panel), C/EBPα is active, binds to the MIR182 promoter and reduces miR-182 expression. Elevated miR-182 levels, especially in AML with defective C/EBPα DNA binding capacity, reduce the amount of functional C/EBPα protein by direct binding to the CEBPA 3′UTR and block myeloid differentiation (upper panel). C/EBPα is a critical transcription factor involved in myelopoiesis and its inactivation is associated with acute myeloid leukemia (AML). Here the authors show a negative feedback loop between C/EBPα and miR-182 and identify this miRNA as a marker of high-risk AMLCC BY · PubMed Central

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Metadata

Authors
Wurm, A. A., Zjablovskaja, P., Kardosova, M., Gerloff, D., Bräuer-Hartmann, D., Katzerke, C., Hartmann, J.-U., Benoukraf, T., Fricke, S., Hilger, N., Müller, A.-M., Bill, M., Schwind, S., Tenen, D. G., Niederwieser, D., Alberich-Jorda, M., & Behre, G.
ORCID
Tenen, Daniel G.
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2017-06-29
Volume
8
Issue
1
ISSN
2041-1723
DOI
10.1038/s41467-017-00032-6
PMC
PMC5491528
Cited by
40
References
6
Language
en
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Wurm, A. A., Zjablovskaja, P., Kardosova, M., Gerloff, D., Bräuer-Hartmann, D., Katzerke, C., Hartmann, J.-U., Benoukraf, T., Fricke, S., Hilger, N., Müller, A.-M., Bill, M., Schwind, S., Tenen, D. G., Niederwieser, D., Alberich-Jorda, M., & Behre, G. (2017). Disruption of the C/EBPα—miR-182 balance impairs granulocytic differentiation. Nature Communications. https://doi.org/10.1038/s41467-017-00032-6