Impaired Neutrophil Migration Resulting from Mir-34a and Mir-155 Overexpressed in Neutrophils from Myelodysplastic Syndrome Patients

In myelodysplastic syndromes (MDS), functional defects of neutrophils result in high mortality due to infections. However, the molecular basis of neutrophil dysfunction remains unclear. We recently found that miR-34a and miR-155 were significantly increased in MDS neutrophils.

To determine the effects of the aberrant expression of the microRNAs on neutrophil functions, HL60 cells, in which miR-34a, miR-155 or control microRNA was ectopically introduced by electroporation, were cultured in the presence of 500 μM dibutyryl cyclic AMP for 2 days to induce differentiation toward a neutrophil-like phenotype. After electroporation, the introduced microRNA levels became 100-fold or higher than those in control microRNA-treated cells. In differentiated cells, the cell surface expression of the differentiation marker CD11b and total amounts of myeloperoxidase (MPO) and elastase in primary granules were not altered by excess of miR-34a or miR-155. When stimulated with 200 nM N-formylmethionyl-leucyl-phenylalanine (fMLP), the control cells released 81.1 ± 7.0 % of total MPO, which was significantly enhanced by overexpression of miR-34a (90.8 ± 4.6%; p<0.05) and miR-155 (90.5 ± 5.5%; p<0.05). Elastase release was also significantly increased by miR-34a (p<0.05) and miR-155 (p<0.05). In contrast, while 63.4 ± 13.4% of the control cells migrated toward 10 nM fMLP through 0.3 µm pores over 90 min, the migration of the cells was significantly reduced with excess of miR-34a and miR-155 (42.7 ± 14.6%; p<0.05 and 40.3 ± 9.2%; p<0.05, respectively). Thus, overexpression of miR-34a and miR-155 enhanced degranulation but attenuated fMLP-induced migration.

To clarify the mechanisms of repression of migration, we examined the effects of miR-34a and miR-155 on Cdc42 and Rac1, Rho family members that are known to regulate migration but not degranulation. Although neither microRNA altered the protein levels of Cdc42, a pull-down assay of the GTP-bound active form of Cdc42 generated by stimulation with 10 μM fMLP for 1 min showed that both miR-34a and miR-155 reduced the ratio of GTP-bound / total Cdc42 to 29.0 ± 15.9% and 39.7 ± 4.8% of those in the control cells, respectively. These results suggested that both microRNAs interfered with the activation process of Cdc42. According to the databases, DOCK8 and FGD4, guanine nucleotide exchange factors that activate Cdc42, are direct targets of miR-34a and miR-155, respectively. In the cells with excess of miR-34a and miR-155, the level of DOCK8 protein detected was 68.8 ± 11.1% and 95.8 ± 11.3% of those in the control cells, respectively. Knockdown of DOCK8 by siRNA decreased the amount of the fMLP-induced active form of Cdc42 by 94.0 ± 23.5% (p<0.05) and the migration rate by 84.6 ± 30.7% (p<0.05) compared with those in the control cells. In contrast, the FGD4 protein level was decreased by miR-155 (56.8 ± 28.6% of control) but not by miR-34a (108.6 ± 30.7% of control). When the FGD4 protein level was decreased to 58.3 ± 12.0% by siRNA, migration toward fMLP was completely blocked. These data showed that miR-34a and miR-155 inhibited migration toward fMLP via reduction of different Cdc42 activators, DOCK8 and FGD4, respectively. Another Rho family member Rac1, which is known as a target of miR-155, was decreased in miR-155-introduced cells (56.2 ± 27.9% of control) but not in miR-34a-overexpressing cells (113.6 ± 17.8% of control). Reduction of Rac1 by siRNA (55.0 ± 22.1% of control) significantly attenuated the migration toward fMLP by 78.0 ± 41.6% (p<0.05). Thus, overexpression of miR-34a attenuated migration via reduction of the Cdc42 activator DOCK8, while miR-155 inhibited migration via reduction of Rac1 and the Cdc42 activator FGD4. This study suggests that an increase of miR-34a and miR-155 contributes to impairment of migration via different mechanisms in MDS neutrophils.