MicroRNA-96 Inhibits Fetal Hemoglobin Expression During Erythropoiesis

Fetal hemoglobin, HbF (α2γ2), is the main hemoglobin synthesized until birth; it subsequently declines and switches to adult hemoglobin, HbA (α2β2). Certain mutations within the globin gene locus can cause persistent HbF synthesis after birth, ameliorating symptoms in hemoglobinopathies. Analysis from e.g. Hb Corfu and δβ-thalassemia indicates that γ-globin gene expression is post-transcriptionally regulated (Efremov DG., Br J Haematol, Oct 1994; Chakalova L., Blood, 2005). The post-transcriptional regulation of HbF might involve regulatory non-coding RNAs. MicroRNAs (miRNAs) are a small non-coding family of 21 nucleotide RNAs that regulate gene expression post-transcriptionally by targeting mRNAs. After transcription and maturation the miRNA is incorporated into the miRNA-induced silencing complex (miRISC). The miRISC, which contains argonaute proteins (AGO), binds and silences the target mRNAs. Previous studies have reported a role of miRNAs in the developmental switch from HbF to HbA synthesis (Noh SJ, J Transl Med, 2009; Bianchi N., BMB Rep, 2009), however so far no direct interaction between miRNAs and the γ-globin mRNA has been reported. Moreover a function of miRISC in mature red blood cells (RBC) has not been shown yet. Therefore, we aimed (I) to characterize the miRISC composition in RBCs, (II) to identify miRNAs that are directly involved in the regulation of HbF and (III) to study their function during erythropoiesis.

To characterize miRISC in RBCs, we performed co-immunoprecipitations of the miRISC using antibodies directed against human AGO. The immunoprecipitated complexes were analyzed by Western-blot, mass spectrometry and qPCR. To identify miRNAs potentially involved in the regulation of HbF miRNA expression patterns in RBCs from adult and umbilical cord blood were analyzed by qPCR. To confirm the role of selected miRNAs, γ-globin protein levels were measured by ELISA in HEL cell cultures transfected with miRNA precursors or inhibitors as well as in primary erythroid cultures overexpressing miRNAs after lentiviral transduction. The direct interaction between miRNAs and the γ-globin mRNA was analyzed by luciferase reporter assay.

AGO1, 2, 3 and 4 and Importin 8 were detected in RBCs both from adult and cord blood. In adult RBC samples (n=3), expressing <1% HbF of total Hb, γ-globin mRNA was bound to AGO2 but not to AGO1 containing miRISC. However in cord blood (n=3), containing 90 % HbF and 30 times more γ-globin mRNA molecules than adult RBCs, the amount of γ-globin mRNA bound to AGO2 containing miRISC was 200 times less (p<0.05). With these indications, we started to analyze miRNA patterns in adult and cord blood RBCs. miR-96, miR-146a, miR-330-3p, let-7a and miR-888 were significantly downregulated in cord blood RBCs compared to adult RBCs. However, luciferase reporter assays demonstrated that only miR-96 was able to bind to the predicted target site within the γ-globin mRNA. Transfection of HEL cells with miR-96 inhibitor showed a twofold induction (p<0.01; n=3) of the γ-globin protein level compared to control cells transfected with scramble RNA. Consistently, transfection with miR-96 precursor in HEL cells showed a 15% reduction (p<0.05, n=3) of the γ-globin protein level. To study the miR-96 mediated inhibition of HbF during erythropoiesis, cord blood derived primary erythroid cultures were transduced with miR-96 lentiviruses. A 40% decrease (p<0.05, n=3) of γ-globin protein expression was observed in erythroid cells overexpressing miR-96 compared to cells transduced with the negative control virus. This γ-globin reduction was observed on 10 and 14 days old cultures, which contained 80% and 65% CD235+ CD71+ erythroblasts, respectively.

This study identifies miR-96 as direct inhibitor of the HbF expression during erythropoiesis. Specifically we observed that in cells with low HbF content, γ-globin mRNAs are bound to miRISC and are therefore directly inhibited by miRNAs. We demonstrated that miR-96 is binding to a seedless target site within the γ-globin open reading frame. Finally we showed that miR-96 overexpression reduces the expression of HbF in erythroleukemia cells and during late stages of cord blood derived erythropoiesis.

No relevant conflicts of interest to declare.