MicroRNA-96 directly inhibits γ-globin expression in human erythropoiesis

Abstract

Fetal hemoglobin, HbF (α(2)γ(2)), is the main hemoglobin synthesized up to birth, but it subsequently declines and adult hemoglobin, HbA (α(2)β(2)), becomes predominant. Several studies have indicated that expression of the HbF subunit γ-globin might be regulated post-transcriptionally. This could be confered by ∼22-nucleotide long microRNAs that associate with argonaute proteins to specifically target γ-globin mRNAs and inhibit protein expression. Indeed, applying immunopurifications, we found that γ-globin mRNA was associated with argonaute 2 isolated from reticulocytes that contain low levels of HbF (<1%), whereas association was significantly lower in reticulocytes with high levels of HbF (90%). Comparing microRNA expression in reticulocytes from cord blood and adult blood, we identified several miRNAs that were preferentially expressed in adults, among them miRNA-96. The overexpression of microRNA-96 in human ex vivo erythropoiesis decreased γ-globin expression by 50%, whereas the knock-down of endogenous microRNA-96 increased γ-globin expression by 20%. Moreover, luciferase reporter assays showed that microRNA-96 negatively regulates expression of γ-globin in HEK293 cells, which depends on a seedless but highly complementary target site located within the coding sequence of γ-globin. Based on these results we conclude that microRNA-96 directly suppresses γ-globin expression and thus contributes to HbF regulation.

Figure 1. γ-globin mRNA is bound by AGO2 in reticulocytes with low HbF content.

(A) Western blot analysis of AGO1, 2, 3, 4, γ-globin and β-globin in reticulocytes purified from umbilical cord blood (CB) and blood from adults (AB). Actin was included as loading control. (B) AGO2-containing RNA-protein complexes were immunoprecipitated from equal amounts of cord blood reticulocyte (CB) and adult blood reticulocyte (AB) lysates using rat monoclonal anti-AGO2 antibodies; as isotype control, non-specific rat immuno-globulin (IgG) as well as rat monoclonal anti-AGO1 antibodies were used. Immunoprecipitations were compared to lysate (input) and subjected to immunoblot analysis using anti-AGO2 antibodies. (C) The amounts of α-globin (HBA), β-globin (HBB), γ-globin (HBG) and δ-globin (HBD) mRNA immunoprecipitated together with AGO2 from CB and AB were compared to amounts of globin mRNAs that were non-specifically immunoprecipitated with control IgG. As control GAPDH and DNMT3a mRNAs were analyzed. For every sample the same amount of precipitated RNA was analyzed. The values are expressed as fold-enrichment over IgG immunoprecipitations, and represent mean±SEM (n = 3).

Figure 2. miRNA expression-patterns in cells expressing high HbF-levels differ from cells with low HbF-levels.

Total RNA isolated from reticulocytes from cord blood (CB) (n = 3), adult blood (AB) (n = 3), K562 cells treated with hemin (n = 3) and untreated K562 cells (n = 3) was used to generate miRNA expression profiles. Red color indicates higher expression and blue color lower expression compared to the global mean. miRNAs were classified into three groups: miRNAs significantly less expressed in cells with high HbF content, i.e. CB compared to AB and K562 cells treated with hemin compared to untreated K562 cells (down), miRNAs significantly less expressed in CB compared to AB (downregulated in CB), miRNAs less expressed in cells with high HbF content (upregulated in CB). The relative quantification (RQ) values, representing the fold enrichment, and the corresponding p values (determined by two tailed Student's t-test) are presented.

Figure 3. miR-96, miR-146a, let-7a are predicted to target the open reading frame of the γ-globin mRNA.

(A) Represented are the 5′ untranslated region (5′UTR, light blue), open reading frame (ORF, dark blue) and 3′UTR (gray) of γ-globin mRNA (HBG). By using RNA hybrid , the free energy of the individual miRNA:mRNA hybridization was determined and the corresponding base pairing shown. The hybridizations of γ-globin mRNA with miR-96, miR-146a and let-7a (black squares) showed either well-defined mRNA:miRNA pairing (miR-146a and let-7a) consisting of a seed region containing eight base pairs, followed by a four-base pair bulge region and a 3′ complementary region as described or seedless base pairing (miR-96), all within the open reading frame (ORF) of HBG. (B) The amounts of miR-96, miR-146a and let-7a immunoprecipitated together with AGO2 from CB and AB were compared to amounts of miR-96, miR-146a and let-7a that were non-specifically immunoprecipitated with control IgG. For every sample the same amount of precipitated RNA was analyzed. The values are expressed as 1000-fold-enrichment over IgG immunoprecipitations, and represent mean±SEM (n = 3). P values were determined by the Student's t-test.

Figure 4. miR-96 inhibits γ-globin expression in human erythropoiesis.

(A,C,E) Cord blood-derived erythroid cultures (CB) were transduced with miRNA precursors; (B,D,F) Adult bone marrow-derived erythroid cultures (BM) were transduced with anti-miRNAs. Cells were harvested and analyzed at day 8 (d8), 11 (d11) and 14 (d14). (A–B) Specific amounts of γ-globin per total protein concentration and (C–D) relative amounts of γ-globin compared to negative control, which was set to 100%, were measured by ELISA. Values represent mean ± SEM of 3 experiments (n = 3). P values were determined by the Student's t-test. * p<0.05. (E–F) Representative cytospins of erythroid cells on d8, d11 and d14. The cytospins were stained with May Grünwald Giemsa and images were acquired with a Zeiss Axioskop2 microscope equipped with a Zeiss Plan-Apochromat 63×/1.4 oil immersion objective lens and a Zeiss AxioCam MRc digital camera. Images were recorded using Zeiss AxioVision AC release 4.5.0 software.

Figure 5. miR-96 targets the γ-globin ORF.

HEK239T cells were co-transfected with the psiCHECK-2 reporter vector expressing Renilla luciferase fused to γ-globin cDNA and the control mimic (neg ctrl), miR-96 mimic, miR-146a mimic or small interfering RNA against γ-globin (siRNA). (A) Relative Renilla luciferase activity in cell lysates normalized to firefly luciferase are shown. (B) Relative Renilla luciferase activities after co-transfection of the control mimic (black) or miR-96 mimic (grey) with wild-type (wt) γ-globin cDNA or mutated γ-globin cDNA (mut) at the predicted miR-96 target site are represented. Values represent mean±SEM of six experiments. P values were determined by the Student's t-test. (C) Predicted miR-96 binding site within the ORF of γ-globin and binding site mutations tested (indicated in bold italics).