MicroRNA-96 Directly Inhibits γ-Globin Expression in Human Erythropoiesis

Fetal hemoglobin, HbF (alpha(2)gamma(2)), is the main hemoglobin synthesized up to birth, but it subsequently declines and adult hemoglobin, HbA (alpha(2)beta(2)), becomes predominant. Several studies have indicated that expression of the HbF subunit gamma-globin might be regulated post-transcriptionally. This could be confered by similar to 22-nucleotide long microRNAs that associate with argonaute proteins to specifically target gamma-globin mRNAs and inhibit protein expression. Indeed, applying immunopurifications, we found that gamma-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 gamma-globin expression by 50%, whereas the knock-down of endogenous microRNA-96 increased gamma-globin expression by 20%. Moreover, luciferase reporter assays showed that microRNA-96 negatively regulates expression of gamma-globin in HEK293 cells, which depends on a seedless but highly complementary target site located within the coding sequence of gamma-globin. Based on these results we conclude that microRNA-96 directly suppresses c-globin expression and thus contributes to HbF regulation.