Tough decoy targeting of predominant let-7 miRNA species in adult human hematopoietic cells.
BACKGROUND
In humans, the heterochronic cascade composed of the RNA-binding protein LIN28 and its major target, the let-7 family of microRNAs (miRNAs), is highly regulated during human erythroid ontogeny. Additionally, down-regulation of the let-7 miRNAs in cultured adult CD34(+) cells or the over-expression of LIN28 in cultured erythrocytes from pediatric patients with HbSS genotype causes increased levels of fetal hemoglobin (HbF) in the range of 19-40% of the total. Therefore, we hypothesized that focused targeting of individual let-7 miRNA family members would exhibit regulatory effect on HbF expression in human adult erythroblasts.


METHODS
The expression levels of mature let-7 family members were measured by RT-qPCR in purified cell populations sorted from peripheral blood. To study the effects of let-7 miRNAs upon globin expression, a lentiviral construct that incorporated the tough decoy (TuD) design to target let-7a or let-7b was compared with empty vector controls. Transductions were performed in CD34(+) cells from adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Downstream analyses included RT-qPCR, Western blot and HPLC for the characterization of adult and fetal hemoglobins.


RESULTS
The expression of individual let-7 miRNA family members in adult peripheral blood cell populations demonstrated that let-7a and let-7b miRNAs are expressed at much higher levels than the other let-7 family members in purified adult human blood cell subsets with expression being predominantly in reticulocytes. Therefore, we focused this study upon the targeted inhibition of let-7a and let-7b with the TuD design to explore its effects upon developmentally-timed erythroid genes. Let-7a-TuD transductions significantly increased gamma-globin mRNA expression and HbF to an average of 38%. Let-7a-TuD also significantly decreased the mRNA expression of some ontogeny-regulated erythroid genes, namely CA1 and GCNT2. In addition, the erythroid-related transcription factors BCL11A and HMGA2 were down- and up-regulated, respectively, by let-7a-TuD, while ZBTB7A, KLF1 and SOX6 remained unchanged.


CONCLUSIONS
Overall, our data demonstrate that let-7 miRNAs are differentially expressed in human hematopoietic cells, and that targeted inhibition of the highly-expressed species of this family is sufficient for developmentally-specific changes in gamma-globin expression and HbF levels.
['de Vasconcellos|Jaira F|JF|', 'Byrnes|Colleen|C|', 'Lee|Y Terry|YT|', 'Allwardt|Joshua M|JM|', 'Kaushal|Megha|M|', 'Rabel|Antoinette|A|', 'Miller|Jeffery L|JL|']
D000328:Adult D001483:Base Sequence D002352:Carrier Proteins D002454:Cell Differentiation D049109:Cell Proliferation D002478:Cells, Cultured D005319:Fetal Hemoglobin D005786:Gene Expression Regulation D055785:Gene Knockdown Techniques D025743:HMGA2 Protein D006412:Hematopoietic Stem Cells D006801:Humans D035683:MicroRNAs D009687:Nuclear Proteins D012333:RNA, Messenger D012097:Repressor Proteins D012156:Reticulocytes D055545:gamma-Globins
let-7 miRNAs Fetal hemoglobin HbF let-7a let-7b Gamma-globin