MicroRNA fingerprints during human megakaryocytopoiesis

Abstract

microRNAs are a highly conserved class of noncoding RNAs with important regulatory functions in proliferation, apoptosis, development, and differentiation. To discover novel regulatory pathways during megakaryocytic differentiation, we performed microRNA expression profiling of in vitro-differentiated megakaryocytes derived from CD34(+) hematopoietic progenitors. The main finding was down-regulation of miR-10a, miR-126, miR-106, miR-10b, miR-17 and miR-20. Hypothetically, the down-regulation of microRNAs unblocks target genes involved in differentiation. We confirmed in vitro and in vivo that miR-130a targets the transcription factor MAFB, which is involved in the activation of the GPIIB promoter, a key protein for platelet physiology. In addition, we found that miR-10a expression in differentiated megakaryocytes is inverse to that of HOXA1, and we showed that HOXA1 is a direct target of miR-10a. Finally, we compared the microRNA expression of megakaryoblastic leukemic cell lines with that of in vitro differentiated megakaryocytes and CD34(+) progenitors. This analysis revealed up-regulation of miR-101, miR-126, miR-99a, miR-135, and miR-20. Our data delineate the expression of microRNAs during megakaryocytopoiesis and suggest a regulatory role of microRNAs in this process by targeting megakaryocytic transcription factors.

Fig. 1.

Northern blots and real-time miRNA-PCR validation of miRNA chip data in CD34 progenitor differentiation experiments. (A) Northern blots for miR-223, miR-130a, and miR-10a. Loading RNA control was performed with U6. (B) miRNA RT-PCR for miR-10a, miR-106, miR-126, and miR-130a. The miRNA expression is presented as fold difference with respect to CD34⁺ cells before culture. (C and D) Temporal array expression of miR-223, miR-15–A, and miR-16-1 by miRNA RT-PCR.

Fig. 2.

MAFB is a target of miR-130a. (A) MAFB mRNA and protein expression in CD34⁺ progenitors induced to megakaryocytic differentiation. β-Actin was used for RT-PCR and Western blot loading controls. (B) Relative repression of luciferase activity in MEG01 cells cotransfected with miR-130A and PGL3 3′UTR MAFB, PGL3 WT, and miR-130 seed match mutated. As a control scramble oligo sequences were cotransfected with PGL3 3′UTR MAFB. (C) Western blotting of MAFB total protein lysates in K562 cells transfected with miR-130a and scramble.

Fig. 3.

MiR-10a down-regulates HOXA1 by mediating RNA cleavage. (A) RT-PCR for HOXA1 gene expression in differentiated megakaryocytes (relative amount of transcript with respect to CD34⁺ progenitors at baseline). (B) hoxa1 protein expression in differentiated megakaryocytes. (C) Relative repression of luciferase activity of HOXA1 3′ UTR cloned in PGL3 reporter plasmid when cotransfected with miR-10a and control scramble. (D) Complementarity between miR-10a and the HOXA1 3′UTR as predicted by pictar. (E) Northern blotting for miR-10a gene expression in scramble and miR-10a precursor transfected K562 cells. (F) RT-PCR for HOXA1 gene expression in scramble and miR-10a precursor transfected K562 cells. (G) Western blotting for HOXA1 in K562 cells transfected with control scramble and precursor miR-10a.