Granulopoiesis is a tightly regulated process. A strictly regulated temporal expression of different transcription factors is crucial for proper neutrophil development. Dysregulation of transcription factors may result in impaired antimicrobial function of the neutrophils and may cause leukemia.
microRNAs (miRNAs) are small noncoding RNAs, that post-transcriptionally regulate protein expression. Dysregulation of miRNA expression has been shown to contribute to the development of neoplastic myeloid diseases such as myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML) and point to an important role of miRNAs in fine tuning of protein regulation in granulopoiesis.
We found that miR-130a is highly expressed in immature proliferating granulocytic precursors, and has a low expression in more mature cells in granulopoiesis. Using microRNA target-prediction software we found the transcription factor C/EBP-ε to be a putative target for miR-130a. C/EBP-ε is essential for expression of the genes encoding the proteins stored in the specific granules of the neutrophil. C/EBP-ε also play an important role for cessation of cell division at the myelocyte stage and the further progress of differentiation.
The biological effect of lacking C/EBP-ε can be observed in the C/EBP-ε knock-out mouse, with accumulation of immature granulocytic precursors in the bone marrow and a severely reduced number of terminally differentiated granulocytes in both bone marrow and peripheral blood. The anti-microbial defense is impaired in the C/EBP-ε knock-out mice which are significantly more susceptible to infection than wild-type mice.
We demonstrate that the mRNA for C/EBP-epsilon is present already in the late promyelocytes while the protein does not appear until the myelocyte stage. This indicates a posttranscriptional regulation of C/EBP-epsilon expression e.g. by microRNAs.
In-silico analysis has shown that the 3′-UTR of the C/EBP-epsilon mRNA contains one binding site for miR-130a. We have verified this by analysis of luciferase reporter constructs carrying the 3′-UTR of C/EBP-ε with the miR-130a binding-site. The repressive effect of miR-130a was abolished by point mutations in the miRNA–binding site.
Next we analyzed a murine granulocytic cell line (MPRO), which stably over expressed miR-130a, and found a reduction of the protein level of C/EBP-ε. We also found a significant decrease in the expression of different genes encoding proteins stored in specific granules such as lactoferrin and hCAP18. Interestingly, we also found elevated expression of MPO mRNA in these cells indicating a more immature phenotype of the cells.
Finally, we transiently transfected myelocytes and metamyelocytes from human bone marrow with pre-miR-130a and demonstrated that this has a significant effect on C/EBP-ε protein levels also in human cells. The reverse result was obtained when transfecting the cells with an anti-miR-130a oligonucleotide.
These results indicate that miR-130a plays an important regulatory role in granulopoiesis and if dysregulated, could lead to a compromised immune function and developmental progression through its regulation of C/EBP-ε.
Grant acknowledgment: The Danish Cancer Society, Lundbeck foundation, Brøchner Mortensen foundation, Medical Research Council.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.