The c-myb Protooncogene and microRNA (miR)-15a Form an Autoregulatory Loop That May Play a Role in Normal Human Erythropoiesis.

The c-myb proto-oncogene encodes an obligate hematopoietic cell transcription factor that contributes to lineage commitment, proliferation, and differentiation. Factors which regulate c-myb expression are of interest but remain incompletely defined. MicroRNAs (miRNAs) are being increasingly recognized as important regulators of cell development, and abnormalities in miRNA activity may also contribute to the pathogenesis of several hematologic malignancies. We speculated that miRNAs might also regulate c-Myb expression, a gene often aberrantly expressed in leukemia, lymphoma, and myeloma. Accordingly, we searched for potential miRNA binding sites in the 3′-UTR of the c-myb mRNA using the TargetScanS Target Database and identified 14 candidate miRNAs. Based on binding probability, three miRNAs (miR-15a, −107 and −150) were selected for further analysis using a luciferase reporter assay. 1191 bp of the human c-Myb 3′-UTR was subcloned downstream of the f-luc open reading frame to create the reporter construct (pBub1/Myb3U). This was co-transfected into HEK293T cell line with pRL-CMV (to normalize for transfection differences) and then either a control RNA oligonucleotide (ON) (miR-Control), miR-15a, miR-107, or miR-150 ONs. Relative luciferase activity of the pBub1/Myb3U construct was markedly diminished in cells co-transfected with miR-15a (74.1 ± 1.5%) or miR-107 (68.2 ± 5.3%) ONs, but only modestly with miR-150 ON (22.1 ± 4.2%). Functionality of miR-15a site was further tested by mutating the predicted miR-15a binding sites. This resulted in a 2 to 3 fold increase in luciferase activity, suggesting that miR-15a bound the predicated sites, and that they might be physiologically relevant. To test this possibility, miR-15a ONs were transfected into K562 human myeloid leukemia cells and the effects on c-Myb mRNA and MYB protein levels were determined. As expected with functional miRNA, c-myb mRNA levels did not change when compared to control treated cells, as measured by quantitative real-time PCR, but Myb protein levels were significantly decreased. Additionally, when analyzed by flow cytometry, miR-15a transfected cells were found to be arrested in G1 as might be expected in a Myb knockdown experiment. Conversely, when K562 cells were transfected with a methylphosphonate inhibitor (antisense) of miR-15a, endogenous c-myb expression increased. Moreover, exogenous expression in K562 cells of a c-Myb mRNA construct devoid of its 3′-UTR partially rescued the miR-15a induced cell cycle arrest. Interestingly, miR-15a levels were found to vary inversely with c-Myb mRNA expression levels in normal human CD34+ cells stimulated to develop along the erythroid, but not the myeloid lineage. These results suggested that miR-15a might play a specific, and potentially important role in regulating normal human erythropoiesis by modulating the expression of c-Myb, though we cannot exclude the possibility that additional miR-15a targets are also important. Finally, a siRNA mediated knockdown of c-Myb expression in K562 cells resulted in reduction of miR-15a expression, suggesting the possibility that Myb and miR-15a autoregulate their expression through a negative feed-back loop. We conclude that miR-15a regulates c-Myb expression in a physiologically significant, lineage specific manner in normal human hematopoietic cells. Since miR-15a localizes to ch13q14, a region often deleted in hematologic malignancies, we postulate that it might also play a role in leukemogenesis. This possibility is under active investigation in our laboratory.