The microRNA-26a target E2F7 sustains cell proliferation and inhibits monocytic differentiation of acute myeloid leukemia cells

Abstract

Blocks in genetic programs required for terminal myeloid differentiation and aberrant proliferation characterize acute myeloid leukemia (AML) cells. 1,25-Dihydroxy-vitamin D3 (VitD3) arrests proliferation of AML cells and induces their differentiation into mature monocytes. In a previous study, we showed that miR-26a was induced upon VitD3-mediated monocytic differentiation. Here, we identify E2F7 as a novel target of miR-26a. We show that E2F7 significantly promotes cell cycle progression and inhibits monocytic differentiation of AML cells. We also demonstrate that E2F7 binds the cyclin-dependent kinase inhibitor p21(CIP1/WAF1) (cyclin-dependent kinase inhibitor 1A) promoter repressing its expression. Moreover, interfering with E2F7 expression results in inhibition of c-Myc (v-myc myelocytomatosis viral oncogene homolog) transcriptional activity. This leads to the downregulation of c-Myc transcriptional target miR-17-92 cluster, whose expression has a well-defined role in contributing to block monocytic differentiation and sustain AML cell proliferation. Finally, we show that the expression of E2F7 is upregulated in primary blasts from AML patients. Thus, these findings indicate that the newly identified miR-26a target E2F7 might have an important role in monocytic differentiation and leukemogenesis.

Figure 1

E2F7 is a direct target of miR-26a. (a) Schematic representation of the constructs used in the luciferase assays. Positions and sequences of predicted miR-26a binding sites are shown. (b) Luciferase assays in HeLa cells transfected with the constructs indicated in panel a with an expression plasmid for miR-26a (+miR-26a) or control plasmid (mock). P-values are indicated

Figure 2

Expression analysis of miR-26a and E2F7. (a, left panel) qPCR analysis of miR-26a levels in HL-60 and U937 cells infected with a lentivirus for ectopic expression of miR-26a (Lenti-26a) or control (Mock inf.). Values were normalized for U6 expression. The histograms represent the means±S.E.M. from triplicates. (Right panel) Western blot analysis of E2F7 and p21 protein levels from cells transduced with control lentivirus (Mock inf.) or Lenti-26a. GAPDH was utilized as the loading control. (c) qPCR analysis of miR-26a levels during VitD3-induced monocytic differentiation of U937 cell line. Values were normalized for U6 expression. The histograms represent the means±S.E.M. from triplicates. (c) Western blot analysis of E2F7 and p21^CIP1/WAF1 protein levels during VitD3-induced monocytic differentiation of U937 cell line. GAPDH was utilized as the loading control. (c) qPCR analysis of E2F7 and miR-26a levels in primary leukemia cells derived from patients with M2, M4 and M5 AML subtypes (n=19) and monocytes (Mon.) isolated from healthy donors (n=5). Box plots graphically represent numerical data and the average of expression levels is indicated. Values were normalized with U6 snRNA. P-values are indicated

Figure 3

E2F7 counteracts monocytic differentiation of AML cells. (a) Western blot analysis of E2F7 levels from U937 cells infected with L-shE2F7 or L-shSCR. GAPDH was utilized as the loading control. (b) Morphological analysis of L-shE2F7- and L-shSCR-infected cells treated without (Ctr) or with 250 ng/ml of VitD3 for 72 h. (c) Representative FACS analysis of CD11b- and CD14-positive cells after VitD3 treatment. (d) The histogram represents the geometric mean±S.E.M. of CD11b/CD14-positive cells after VitD3 treatment and indicates the average intensity of CD11b/CD14 in a population. (e) qPCR analysis of monocytic specific markers. Data were normalized for GAPDH mRNA. The histograms represent the means±S.E.M. from three independent experiments

Figure 4

Interference against E2F7 inhibits AML cell proliferation. (a) Effect of L-shE2F7 and L-shSCR on cell proliferation. (b) Cell cycle distribution of cells transduced with L-shE2F7 and L-shSCR. The histograms represent the means±S.E.M. from three independent experiments. (c) Representative cell cycle analysis of cells transduced with L-shE2F7 and L-shSCR. (d) qPCR analysis of positive cell cycle regulators. Data were normalized for GAPDH mRNA. The histograms represent the means±S.E.M. from three independent experiments

Figure 5

Enforced E2F7 expression stimulates AML cell proliferation and counteracts monocytic differentiation of AML cells. (a) Western blot analysis of E2F7 protein levels in U937 cells transformed with EPB-PURO and EPB-PURO-E2F7. Data were normalized for GAPDH. (b) Effect of EPB-PURO and EPB-PURO-E2F7 on U937 proliferation upon Dox treatment (200 ng/ml). (c) Morphological analysis of EPB-PURO and EPB-PURO-E2F7 cells treated with Dox alone or in combination with VitD3 for 72 h. (d) Representative FACS analysis of CD11b- and CD14-positive cells after Dox and VitD3 treatment. (e) The histogram represents the geometric mean±S.E.M. of CD11b/CD14-positive cells after Dox and VitD3 treatment and indicates the average intensity of CD11b/CD14 in a population. (f) Cell cycle distribution of cells transduced with EPB-PURO and EPB-PURO-E2F7 and treated with Dox alone or in combination with VitD3 for 72 h. (g) Representative cell cycle analysis of (f)

Figure 6

E2F7 represses the expression of p21^CIP1/WAF1 in AML cells. (a) Western blot analysis of p21^CIP1/WAF1 protein levels in cells transduced with L-shE2F7 and L-shSCR. Data were normalized for GAPDH. (b) Localization of endogenous p21^CIP1/WAF1 protein in cells transduced with L-shE2F7 and L-shSCR. DNA staining with TO-PRO-3 and a merged view of green and blue channels of the same field is shown (merge). (c) ChIP analysis with anti-E2F7 antibody on p21^CIP1/WAF1 promoter in U937 cells before and after VitD3 treatment. (d) ChIP analysis with anti-E2F7 antibody on p21^CIP1/WAF1 promoter in U937 cells transduced with L-shE2F7 and L-shSCR. The recovered DNA in ChIP experiments was quantified by real-time PCR. Results are expressed as the relative level over control cells after correcting for differences in the amount of starting (input) chromatin materials. Experiments were performed in triplicate. P-values are indicated

Figure 7

(a) qPCR analysis of miR-17 and miR-20a in cells transduced with L-shE2F7 and L-shSCR. (b) qPCR analysis of miR-17 and miR-20a in cells transduced with Lenti-26a and mock. (c) ChIP analysis with anti-c-Myc antibody on miR-17-92 cluster promoter in cells treated with VitD3 for 72 h or transduced with L-shE2F7 and L-shSCR. The recovered DNA in ChIP experiments was quantified by real-time PCR. Results are expressed as the relative level over control cells after correcting for differences in the amount of starting (input) chromatin materials. Experiments were performed in triplicate. P-values are indicated