In myelodysplastic syndromes (MDS), functional defects of neutrophils result in high mortality because of infections; however, the molecular basis remains unclear. We recently found that miR-34a and miR-155 were significantly increased in MDS neutrophils. To clarify the effects of the aberrant microRNA expression on neutrophil functions, we introduced miR-34a, miR-155, or control microRNA into neutrophil-like differentiated HL60 cells. Ectopically introduced miR-34a and miR-155 significantly attenuated migration toward chemoattractants fMLF and IL-8, but enhanced degranulation. To clarify the mechanisms for inhibition of migration, we studied the effects of miR-34a and miR-155 on the migration-regulating Rho family members, Cdc42 and Rac1. The introduced miR-34a and miR-155 decreased the fMLF-induced active form of Cdc42 to 29.0 ± 15.9 and 39.7 ± 4.8% of that in the control cells, respectively, although Cdc42 protein levels were not altered. miR-34a decreased a Cdc42-specific guanine nucleotide exchange factor (GEF), dedicator of cytokinesis (DOCK) 8, whereas miR-155 reduced another Cdc42-specific GEF, FYVE, RhoGEF, and PH domain-containing (FGD) 4. The knockdown of DOCK8 and FGD4 by small interfering RNA suppressed Cdc42 activation and fMLF/IL-8–induced migration. miR-155, but not miR-34a, decreased Rac1 protein, and introduction of Rac1 small interfering RNA attenuated Rac1 activation and migration. Neutrophils from patients showed significant attenuation in migration compared with healthy cells, and protein levels of DOCK8, FGD4, and Rac1 were well correlated with migration toward fMLF (r = 0.642, 0.686, and 0.436, respectively) and IL-8 (r = 0.778, 0.659, and 0.606, respectively). Our results indicated that reduction of DOCK8, FGD4, and Rac1 contributes to impaired neutrophil migration in MDS.

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal disorders characterized by ineffective hematopoiesis resulting in numerical, morphological, and functional abnormalities in blood cells of multiple lineages (14). Most notably, quantitative and qualitative defects of neutrophilic granulocytes reduce bactericidal and fungicidal activities, resulting in life-threatening infections (57). MDS-derived neutrophils have demonstrated impairment in migration, production of reactive oxygen species, and phagocytosis. However, the molecular basis of the neutrophil dysfunction has yet to be clearly defined.

Neutrophil migration to infection sites is induced by chemoattractants, such as fMLF (8, 9) and IL-8 (IL-8/CXCL8) (10, 11). Both fMLF- and IL-8/CXCL8–induced migration have been shown to be affected in MDS-derived neutrophils (1214). A previous study suggested that disturbed activation of the Rac-ERK pathway and PI3K is responsible for the aberrant IL-8/CXCL8–induced migration in MDS (14). It has been reported that CD18 plays a critical role regarding fMLF-induced migration (15), and that expression of the CD11b–CD18 complex is decreased in MDS neutrophils (16). When stimulated with fMLF, activation of ERK1/2 and protein kinase B (PKB/Akt) was also attenuated in MDS (17). Although fMLF has been shown to activate various Rho family members that play essential roles in regulating cytoskeletal dynamics (18), there have yet to be studies on whether insufficient activation of Rho proteins is involved in aberrant fMLF-induced migration of MDS neutrophils.

Among the Rho family members, Cdc42 and Rac1 have been extensively studied as key regulators for cell migration. Cdc42 is required for actin polymerization and filopodial protrusion to maintain polarity, whereas Rac1 promotes actin assembly to regulate lamellipodia extension (1921). Both Rac1 and Cdc42 act as molecular switches by cycling between an inactive GDP-bound form and an active GTP-bound form. In response to stimuli, GDP-bound forms in the cytoplasm are recruited to the membrane, where guanine nucleotide exchange factors (GEFs) convert GDP to GTP (22, 23). GEFs are categorized into two distinct classes, dedicator of cytokinesis (DOCK) proteins and the diffuse B cell lymphoma (Dbl) family (23, 24). Of the DOCK proteins, DOCK8 is a Cdc42-specific GEF that critically regulates migration of dendritic cells (25), whereas DOCK2 and DOCK5 are identified as potent Rac regulators in neutrophils (26). Like FYVE, RhoGEF, and PH domain-containing 2 (FGD2) and FGD3, FGD4, also known as Frabin (FGD1-related F-actin binding protein), is a Cdc42-specific GEF belonging to the Dbl family (27).

We recently reported that miR-34a and miR-155 were significantly increased in neutrophils isolated from MDS patients compared with those from healthy cells (28). Aberrant expression of microRNAs (miRNAs) affects various cell functions. It has been shown that miR-34a, a target of p53 (29), not only inhibits proliferation by inducing apoptosis (30), but also suppresses migration and/or invasion of malignant cell lines via reduction of metalloproteinases and Fra-1 (3133). Regarding miR-155, it has been reported that its overexpression accelerates proliferation of cancer cells via repression of SHIP-1, a negative regulator of the Akt pathway (34), and inhibits migration of malignant and nonmalignant cells (3537). Because both miR-34a and miR-155 have been shown to affect migration of various cell types, the aberrantly increased miR-34a/miR-155 may be a cause of impaired migration of MDS neutrophils. Furthermore, a database (www.microRNA.org) predicts that miR-34a and miR-155 target Rac1 and several Cdc42-specific GEFs. Reduction of these molecules by the overexpressed miR-34a/miR-155 could impair activation of Cdc42 and Rac1, resulting in the inhibition of migration.

In this study, we examined whether miR-34a and miR-155 inhibited neutrophil migration by affecting activation of Cdc42 and Rac1. We demonstrated that overexpression of miR-34a and miR-155 attenuated migration of neutrophil-like differentiated HL60 (dHL60) cells toward fMLF/IL-8 via targeting different molecules in Cdc42/Rac-activating pathways. Furthermore, we studied the expression levels of the identified target molecules as well as their relationship with migratory activity in healthy and MDS neutrophils.

Peripheral blood was obtained from 12 healthy volunteers and 11 MDS patients consisting of 9 subjects with refractory cytopenia with multilineage dysplasia (RCMD), 1 with refractory cytopenia with unilineage dysplasia, and 1 with refractory anemia with excess blasts-2, according to the World Health Organization 2008 classification (38). Table I summarizes the clinical data of the patients and the genomic information obtained by target sequencing. Although none of the patients had experienced symptomatic infections for at least 1 y before the blood drawing, patient 8 experienced development of pneumonia several days after blood donation for this study. This study and the process of securing written informed consent from the patients and healthy control subjects were approved by the Ethics Committee of Fukushima Medical University (approval no. 1077), which is guided by local policy, national laws, and the World Medical Association Declaration of Helsinki.

Table I.
Hematological and clinical findings of patients
Patient No.Age (y)/SexSubtypeWBC (× 109/l)Neutrophils (× 109/l)Hb (g/dl)PLTs (× 1010/l)CytogeneticsTherapyGenetic Information
89/M RCMD 2.1 1.9 8.4 2.6 46,XY Azacitidine, transfusion (RBC) NE 
74/M RCMD 3.2 2.2 9.1 12.2 46,XY Transfusion (RBC) TET2(S1107P) 
89/F RCMD 2.0 1.5 7.2 30.5 46,XY,del (5)(q?); 46,XY Transfusion (RBC) TET2(Q810*)a 
70/F RCMD 3.5 1.5 10.3 13.7 46,XX,add (13)(q12); 46,XX Transfusion (RBC, PLTs), CyA NRAS(V9F), TET2(S1107P) NF1(T25651) 
72/F RCMD 2.1 0.7 9.9 24.3 46,XX CyA TET2(S1107P) GATA1 (H71P) 
71/F RCMD 3.1 1.7 10.1 11.6 46,XX None SF3B1(L1251F) SF3A1(N82I) 
82/M RCMD 3.1 2.2 8.4 1.2 46,XY Azacitidine, transfusion (RBC) TET2(S1290L) 
82/M RAEB2 1.9 1.1 9.4 2.9 47,XY,−2,−3,del (5)(q?),+8,−16,+mar1,+mar2, +mar3 48,XY,idem,+21 Cytarabine, aclarubicin, G-CSF NE 
64/M RCUD 6.1 3.8 15.0 13.7 46,XY,del (20)(q12q13); 46,XY None NRAS(V9F) TET2(S1107P) 
10 75/M RCMD 5.3 3.1 9.6 5.9 46,XY,del (20)(q11.2g13.3)46,XY None NE 
11 82/F RCMD 1.5 0.4 7.7 5.1 46,XX,del (12)(p?),46,XX Transfusion (RBC, PLTs) NE 
Patient No.Age (y)/SexSubtypeWBC (× 109/l)Neutrophils (× 109/l)Hb (g/dl)PLTs (× 1010/l)CytogeneticsTherapyGenetic Information
89/M RCMD 2.1 1.9 8.4 2.6 46,XY Azacitidine, transfusion (RBC) NE 
74/M RCMD 3.2 2.2 9.1 12.2 46,XY Transfusion (RBC) TET2(S1107P) 
89/F RCMD 2.0 1.5 7.2 30.5 46,XY,del (5)(q?); 46,XY Transfusion (RBC) TET2(Q810*)a 
70/F RCMD 3.5 1.5 10.3 13.7 46,XX,add (13)(q12); 46,XX Transfusion (RBC, PLTs), CyA NRAS(V9F), TET2(S1107P) NF1(T25651) 
72/F RCMD 2.1 0.7 9.9 24.3 46,XX CyA TET2(S1107P) GATA1 (H71P) 
71/F RCMD 3.1 1.7 10.1 11.6 46,XX None SF3B1(L1251F) SF3A1(N82I) 
82/M RCMD 3.1 2.2 8.4 1.2 46,XY Azacitidine, transfusion (RBC) TET2(S1290L) 
82/M RAEB2 1.9 1.1 9.4 2.9 47,XY,−2,−3,del (5)(q?),+8,−16,+mar1,+mar2, +mar3 48,XY,idem,+21 Cytarabine, aclarubicin, G-CSF NE 
64/M RCUD 6.1 3.8 15.0 13.7 46,XY,del (20)(q12q13); 46,XY None NRAS(V9F) TET2(S1107P) 
10 75/M RCMD 5.3 3.1 9.6 5.9 46,XY,del (20)(q11.2g13.3)46,XY None NE 
11 82/F RCMD 1.5 0.4 7.7 5.1 46,XX,del (12)(p?),46,XX Transfusion (RBC, PLTs) NE 
a

Asterisk represents stop codon.

CyA, cyclosporin A; F, female; Hb, hemoglobin concentration; M, male; NE, not examined; PLTs, platelets; RAEB2, refractory anemia with excess blasts 2; RCUD, refractory cytopenia with unilineage dysplasia.

As previously described (39), the granulocyte fraction was obtained by centrifugation through Lymphoprep (Axis-Shield, Oslo, Norway) followed by hypotonic lysis of erythrocytes. More than 92% of cells in the fraction were neutrophilic granulocytes, as confirmed by May-Grünwald and Giemsa staining.

The target sequence was carried out using the Human Myeloid Neoplasms Panel (catalog no. NGHS-003X; Qiagen, Hilden, Germany) for 50 genes (available at: https://www.qiagen.com/jp/shop/sample-technologies/dna/dna-preparation/generead-dnaseq-gene-panels-v2?catno=NGHS-003×#geneglobe). Exons of target genes were enriched by multiplex PCR according to the manufacturer’s instructions. After verifying quality and total amount of the samples by GeneRead Library Quantification System, next-generation sequencing was performed using MiSeq (Illumina, San Diego, CA). Sequence data were analyzed with Web-based software, QIAGEN NGS Data Analysis Web Portal (https://www.qiagen.com/jp/shop/genes-and-pathways/technology-portals/browse-ngs/next-generation-sequencing/?workflowstep%3dd7db5450-911c-44e6-9e30-6fb6d5e11eea). Silent mutations and known germline polymorphisms listed in public database (https://www.ncbi.nlm.nih.gov/SNP/) were excluded.

A human leukemic cell line HL60 was cultured in RPMI 1640 (Wako Laboratory Chemicals, Osaka, Japan) supplemented with 10% (v/v) heat-inactivated FBS (Nichirei Biosciences, Tokyo, Japan). Introduction of 50 nM of the following was carried out by square-pulse electroporation (280 V, 12 ms) using a Gene Pulser (Bio-Rad Laboratories, Hercules, CA): mirVana miRNA mimics (dsRNA oligonucleotides) of miR-34a (has-miR-34a-5p MC11030), miR-155 (has-miR-155-5p MC12601) and the negative control (4464058) (Life Technologies, Carlsbad, CA), mirVana miRNA inhibitors (single-stranded chemically modified oligonucleotides) against miR-34a (has-miR-34a-5p MH11030), miR-155 (has-miR-155-5p MH12601) and the negative control (4464076) (Life Technologies), small interfering RNAs (siRNAs; DOCK8: sense 5′-GAGACUUACUCUUCGAAGAtt-3′, antisense 5′-UCUUCGAAGAGUAAGUCUCca-3′, FGD4: sense 5′-GAAGGAGACUAAUGAGCAAtt-3′, antisense 5′-UUGCUCAUUAGUCUCCUUCat-3′, Rac1: sense 5′-CUACUGUCUUUGACAAUUAtt-3′, antisense 5′-UAAUUGUCAAAGACAGUAGgg-3′), and the negative control (AM4611) lacking homology for any known gene sequences (Life Technologies). It was confirmed that introduction of the siRNA control did not affect expression levels of DOCK8, FGD4, and Rac1. After 24 h, 500 μM dibutyryl cAMP (dbcAMP) (Sigma-Aldrich, St. Louis, MO) was added into the culture medium to induce differentiation toward a neutrophil-like phenotype for a further 48 h (Fig. 1A). For experiments using IL-8, HL60 cells were cultured with 1.25% DMSO for 4 d, and miRNAs or siRNAs were introduced by electroporation 3 d before migration assay.

FIGURE 1.

Differentiation of miRNA-overexpressing cells. (A) Experimental design. miR-34a, miR-155, or control miRNA was introduced into HL60 cells by electroporation. After 24 h, 500 μM dbcAMP was added into culture medium, and the cells were allowed to differentiate toward a neutrophil-like phenotype for 2 d. (B) Cell morphology and CD11b expression. To confirm cell morphology, we stained cells with May-Grünwald and Giemsa solutions. To measure cell surface expression of CD11b and CD18 by flow cytometry, we treated cells with mouse monoclonal anti-CD11b conjugated with PE or anti-CD18 with FITC (solid line) or mouse isotype IgG labeled with PE or FITC (broken line). Viable cells formed a single population according to forward and side scatters on flow cytometry, and this population was subjected to single-color analyses. (C) Amounts of MPO stored in dHL60 cells. (D) Amounts of elastase in dHL60 cells. Horizontal bars represent mean values of 9 to 15 experiments.

FIGURE 1.

Differentiation of miRNA-overexpressing cells. (A) Experimental design. miR-34a, miR-155, or control miRNA was introduced into HL60 cells by electroporation. After 24 h, 500 μM dbcAMP was added into culture medium, and the cells were allowed to differentiate toward a neutrophil-like phenotype for 2 d. (B) Cell morphology and CD11b expression. To confirm cell morphology, we stained cells with May-Grünwald and Giemsa solutions. To measure cell surface expression of CD11b and CD18 by flow cytometry, we treated cells with mouse monoclonal anti-CD11b conjugated with PE or anti-CD18 with FITC (solid line) or mouse isotype IgG labeled with PE or FITC (broken line). Viable cells formed a single population according to forward and side scatters on flow cytometry, and this population was subjected to single-color analyses. (C) Amounts of MPO stored in dHL60 cells. (D) Amounts of elastase in dHL60 cells. Horizontal bars represent mean values of 9 to 15 experiments.

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It was confirmed that the introduction of the controls for miRNA mimics and inhibitors did not alter miR-34a and miR-155 levels. In each experiment, the mimics increased miR-34a/miR-155 levels by >100-fold compared with those in the control miRNA-treated cells, and the decrease by inhibitors was more than 80%.

Total cellular RNA was isolated using Isogen (Nippon Gene, Toyama, Japan). First-strand cDNA was synthesized as described previously (40). Expression of three fMLF receptor (FPR) isoforms (FPR1-3) was quantified by real-time PCR in duplicate using SYBR Premix Ex Taq (Takara Bio, Otsu, Japan) and normalized by β-actin (40). The primers used for FPR isoforms were FPR1 sense 5′-GGCATCATCCGGTTCATCATT-3′, antisense 5′-AGGGCACTTGTCACATCCACT-3′; FPR2 sense 5′-GTCGGACCTTGGATTCTTGCT-3′, antisense 5′-CTTTTTGTGGATCTTGGCTGCA-3′; FPR3 sense 5′-CGCACAGTCAACACCATCTG-3′, antisense 5′-GTCATCACCCTCTTGGCCAGACTC-3′. Total cellular RNA was polyadenylated and reverse transcribed using a Mir-X miRNA First-strand Synthesis Kit (Clontech Laboratories, Mountain View, CA) to confirm the effects of miRNA introduction. The miRNAs were quantified by real-time PCR using miR-34a– or miR-155–specific primer and mRQ 3′ primer (Clontech Laboratories) and normalized by U6.

Cells were stained with mouse monoclonal anti-CD11b labeled with PE (eBioscience, San Diego, CA), mouse monoclonal CD18 conjugated with FITC (Sony Biotechnology, San Jose, CA), or isotype match control IgG labeled with PE or FITC (Beckman Coulter, Marseille, France). The cell-surface expressions of CD11b and CD18 were analyzed by FACSCanto II (BD Biosciences, San Jose, CA).

For the migration assay, 1 × 105 cells in the upper wells of a polycarbonate membrane chamber system with 3-μm pores (Cell Biolabs, San Diego, CA) were allowed to migrate into the lower wells containing 10 nM fMLF (Sigma-Aldrich) or 100 ng/ml IL-8 (PeproTech, Rocky Hill, NJ) for 90 min at 37°C. After aspirating out the contents remaining in the upper wells, the cells were collected from the lower chamber and the reverse side of the membrane, and treated with lysis buffer including CyQuant GR dye (Cell Biolabs) for 20 min at room temperature. The fluorescence of the cell lysate was immediately read at 480/520 nm.

The cells that were preincubated with 10 μg/ml cytochalasin B (Sigma-Aldrich) in assay buffer (PBS containing 0.9 mM CaCl2, 0.5 mM MgCl2, 10 mM HEPES, 10 mM glucose, and 0.1% BSA [pH 7.4]) at 37°C for 5 min were stimulated with 200 nM fMLF (Sigma-Aldrich), 300 nM human complement C5a (Sigma-Aldrich), or 5 ng/ml human GM-CSF (PeproTech) for 15 min and centrifuged. To measure myeloperoxidase (MPO) activity, we added 0.3 mM H2O2 (Wako Laboratory Chemicals) and 1 mM tetramethylbenzidine (Sigma-Aldrich) to both cell pellets lysed by 0.5% Triton X-100 and supernatant. After incubation for 5 min at room temperature, the enzyme reaction was terminated by adding 0.8 M acetic acid and 2 mM sodium azide to immediately read the absorbance at 630 nm. Elastase activity was analyzed by incubation with 0.45 mM of an elastase substrate N-t-BOC-l-alanine-p-nitrophenol ester (Sigma-Aldrich) for 25 min followed by absorbance measurement at 347 nm.

For Cdc42 and Rac1 activity analyses, 15 × 106 and 5 × 106 cells were incubated in HBSS containing 0.1% BSA with or without fMLF, respectively, at 37°C for 1 min. After immediate centrifugation, the cell pellets were treated with lysis buffer (25 mM Tris [pH 7.5], 50 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1% Nonidet P-40, 10% glycerol, inhibitors for proteinase and phosphatase) containing 5 mM diisopropyl fluorophosphates (Sigma-Aldrich) for 20 min on ice, and centrifuged at 10,000 × g for 10 min. While one tenth of the volume of the supernatant was collected for quantification of total Cdc42 or Rac1, the remaining volume was incubated with 15 μg Rac/Cdc42 (p21)-binding domain of human p21-activated protein kinase 1–bound agarose beads (Cell Biolabs) for 45 min at 4°C. The beads were washed three times with lysis buffer and subjected to immunoblotting of Cdc42 or Rac1.

For detection of DOCK8, Rac1, and ERK1/2 in dHL60, the cells were lysed by 1% Nonidet P-40 buffer. For analyses of DOCK8 and Rac1 in the neutrophils, Cdc42 and DOCK2 in dHL60, and FGD4 in both neutrophils and dHL60, total cell lysates were obtained from TCA-precipitated fraction, as previously described (41). RIPA buffer was used for detection of PKB/Akt in dHL60.

Proteins were separated on a 7.5, 10, or 15% NaDodSO4 polyacrylamide gel and transferred onto Immobilon-P transfer membrane (Millipore, Billerica, MA) for blotting with anti-DOCK8 (ab175208) (Abcam, Cambridge, U.K.), DOCK2 (ab124838; Abcam), FGD4 (ab97785; Abcam), ERK1/2 (ab184699; Abcam), pERK1/2 (Thr202/Thr204) (ab76299; Abcam), PKB/Akt (sc1618) (Santa Cruz Biotechnology, Santa Cruz, CA), phosphorylated PKB/Akt (Ser473) (04-736) (Millipore), or Cdc42/Rac1 (BD Biosciences), respectively. The membranes blotted with the first Abs were treated with a HRP-conjugated second Ab (Life Technologies). Anti-α-tubulin (sc-5286) and anti-GAPDH conjugated with HRP (sc-25778) were used as internal controls. Membranes were soaked in ImmunoStar Zeta (Wako), and signals were detected using the Molecular Image ChemiDoc XRS Plus System (Bio-Rad Laboratories). The reliability of the Abs was tested using positive and negative controls (Supplemental Fig. 1), and the linearity of quantification (Supplemental Fig. 2) was confirmed.

For three-group comparison, one-way ANOVA (IBM SPSS Statistics 17.0) was used, and the Mann–Whitney U test was used for the two-group comparison. Correlation coefficients were obtained by bivariate correlation. The p values <0.05 were considered significant. Regarding expression of miRNAs and proteins in individuals, the criteria of significant increase and reduction were set as higher and lower expressions than the two SDs from mean values of healthy control subjects, respectively.

We first examined whether the introduction of miR-34a and miR-155 affected dbcAMP-induced differentiation in HL60 cells. As shown in Fig. 1B, dbcAMP treatment induced segmented nuclei and cell-surface expression of the differentiation marker CD11b, which did not differ regardless of the ectopically introduced miRNAs. CD18 levels were also similar among the three differentiated cells. There were no significant differences in the amounts of MPO (Fig. 1C) and elastase (Fig. 1D) stored in the primary granules among the three cell types.

The effects of miR-34a and miR-155 on fMLF-induced migration were analyzed. In the absence of fMLF, 30.0–35.0% of the cells in the upper wells migrated through the 3-μm pores regardless of excessive miRNA expression. When fMLF was present in the lower chamber, 63.4 ± 13.4% of the control cells migrated, which was significantly reduced in the miR-34a– and miR-155–overexpressing cells (42.7 ± 14.6%, p < 0.05, and 40.3 ± 9.2%, p < 0.05, respectively) (Fig. 2A). In contrast, the treatment of inhibitors against miR-34a and miR-155 increased migration in the presence of fMLF to 72.9 ± 3.5% (p < 0.05) and 68.5 ± 8.2% (p < 0.05), respectively (Fig. 2B). The mRNA levels of three isoforms of the FPR, FPR1, FPR2, and FPR3, were not altered by overexpression of miR-34a or miR-155 (data not shown), suggesting that alteration of the FRP expression was not involved in attenuated migration of miRNA-overexpressing cells.

FIGURE 2.

Effects of miR-34a and miR-155 on degranulation and migration. (A) Effects of miRNA mimic introduction on fMLF-induced migration. (B) Effects of miRNA inhibitors on fMLF-induced migration. The cells treated with miRNA mimics or inhibitors were placed into the upper wells of a polycarbonate membrane chamber system and allowed to migrate through 3-μm pores toward the lower wells containing no chemoattractant (white column) or 10 nM fMLF (black column) at 37°C for 90 min. The ratios of the cells that migrated to the total cells plated in the upper wells are shown as mean ± SD of four to seven independent experiments performed in duplicate. (C) Released MPO. (D) Released elastase. Cells were incubated with indicated stimuli (200 nM fMLF, 300 nM C5a, and 5 ng/ml GM-CSF) at 37°C and centrifuged. The substrate was added to the supernatant to measure enzyme released into the assay buffer. The y-axes indicate the ratios of released enzyme to total enzymes initially stored in granules. *p < 0.05, **p < 0.01.

FIGURE 2.

Effects of miR-34a and miR-155 on degranulation and migration. (A) Effects of miRNA mimic introduction on fMLF-induced migration. (B) Effects of miRNA inhibitors on fMLF-induced migration. The cells treated with miRNA mimics or inhibitors were placed into the upper wells of a polycarbonate membrane chamber system and allowed to migrate through 3-μm pores toward the lower wells containing no chemoattractant (white column) or 10 nM fMLF (black column) at 37°C for 90 min. The ratios of the cells that migrated to the total cells plated in the upper wells are shown as mean ± SD of four to seven independent experiments performed in duplicate. (C) Released MPO. (D) Released elastase. Cells were incubated with indicated stimuli (200 nM fMLF, 300 nM C5a, and 5 ng/ml GM-CSF) at 37°C and centrifuged. The substrate was added to the supernatant to measure enzyme released into the assay buffer. The y-axes indicate the ratios of released enzyme to total enzymes initially stored in granules. *p < 0.05, **p < 0.01.

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We next examined the effects of miR-34a and miR-155 overexpression on release of MPO and elastase. In the absence of stimuli, 20–30% of total MPO and elastase were detected in the assay buffer, which was not altered by overexpression of miRNAs. In contrast, when stimulated with fMLF, C5a, and GM-CSF, the released MPO from the control cells was 81.1 ± 7.0, 74.5 ± 8.3, and 59.3 ± 14.1% of total MPO, respectively. The amounts of MPO released by these three stimuli were significantly greater in both cells with ectopic miR-34a (90.8 ± 4.6%, p < 0.05; 86.6 ± 4.8%, p < 0.01; 76.4 ± 10.1%, p < 0.05) and miR-155 (90.5 ± 5.5%, p < 0.05; 86.1 ± 5.9%, p < 0.01; 77.8 ± 8.5%, p < 0.01) than in the control cells (Fig. 2C). Elastase released in response to these three stimuli was also greater in the cells overexpressing miR-34a (59.4 ± 14.4%, p < 0.05; 57.8 ± 12.9%, p < 0.05; 55.1 ± 18.3%, p < 0.05) and miR-155 (59.2 ± 11.1%, p < 0.05; 57.1 ± 13.4%, p < 0.05; 55.4 ± 11.3%, p < 0.05) than those in the control cells (45.6 ± 9.8, 44.9 ± 11.0, and 41.4 ± 10.8%) (Fig. 2D).

Among migration-regulating Rho proteins, Rac2 and RhoA have been shown to critically regulate degranulation in neutrophils (4245). Therefore, we focused on other Rho proteins, namely Cdc42 and Rac1, to identify molecules responsible for attenuated migration. Neither mimics nor inhibitors of miR-34a/miR-155 altered the expression of Cdc42 protein (Fig. 3A, 3B). In the presence of 2.5 μM ML141, a Cdc42 inhibitor, fMLF-induced migration (Fig. 3C) and Cdc42 activation (data not shown) were completely blocked, confirming that Cdc42 activation was essential for fMLF-induced migration. The GTP-bound active form of Cdc42, which was hardly observed by pull-down assay without stimulation, became detectable with fMLF stimulation in the control cells. Introduction of miR-34a and miR-155 resulted in a decrease of active Cdc42 under stimulation with fMLF to 29.0 ± 15.9 and 39.7 ± 4.8% of that in the control cells, respectively (Fig. 3D). These data suggest that miR-34a and miR-155 did not target Cdc42 itself, but targeted some molecules involved in the activation of Cdc42.

FIGURE 3.

Effects of miR-34a and miR-155 on Cdc42. (A) Cdc42 protein levels in the cells with excessive miRNA. (B) Expression of Cdc42 protein in miRNA inhibitor-treated cells. Cdc42 protein levels were analyzed by immunoblotting. The ratios of Cdc42 band intensities to those of GAPDH are shown as mean ± SD of three to six independent experiments. (C) Effects of Cdc42 inhibition on fMLF-induced migration. A Cdc42 inhibitor ML141 was added to culture medium at the indicated concentrations, and the cells that migrated were quantified. The results shown are mean ± SD of four to five independent experiments. *p < 0.05. (D) Activation of Cdc42. The cells were incubated with or without 10 μM fMLF for 1 min, and the GTP-bound form of Cdc42 was pulled down by p21-binding domain of human p21-activated protein kinase 1 agarose beads. The bead-bound active Cdc42 and total Cdc42 in cell lysate were visualized by immunoblotting with anti-Cdc42. The ratios of active form to total Cdc42 in fMLF-stimulated cells were calculated. In the graph, the values from the fMLF-stimulated control cells are set as 1.0, and mean ± SD of three independent experiments are presented.

FIGURE 3.

Effects of miR-34a and miR-155 on Cdc42. (A) Cdc42 protein levels in the cells with excessive miRNA. (B) Expression of Cdc42 protein in miRNA inhibitor-treated cells. Cdc42 protein levels were analyzed by immunoblotting. The ratios of Cdc42 band intensities to those of GAPDH are shown as mean ± SD of three to six independent experiments. (C) Effects of Cdc42 inhibition on fMLF-induced migration. A Cdc42 inhibitor ML141 was added to culture medium at the indicated concentrations, and the cells that migrated were quantified. The results shown are mean ± SD of four to five independent experiments. *p < 0.05. (D) Activation of Cdc42. The cells were incubated with or without 10 μM fMLF for 1 min, and the GTP-bound form of Cdc42 was pulled down by p21-binding domain of human p21-activated protein kinase 1 agarose beads. The bead-bound active Cdc42 and total Cdc42 in cell lysate were visualized by immunoblotting with anti-Cdc42. The ratios of active form to total Cdc42 in fMLF-stimulated cells were calculated. In the graph, the values from the fMLF-stimulated control cells are set as 1.0, and mean ± SD of three independent experiments are presented.

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According to the gene target search program microRNA.org, of the DOCK family members highly expressed in neutrophils, only DOCK8 was a target of miR-34a. Although DOCK8 was not a target of miR-155, miR-155 was predicted to downregulate FGD4, another Cdc42-specific GEF belonging to the Dbl family. In miR-34a–introduced cells, the DOCK8 protein levels were 68.8 ± 11.1% of that in the control cells (p < 0.05), whereas miR-155 overexpression did not alter DOCK8 levels (Fig. 4A). When the miR-34a inhibitor was introduced to dHL60, the DOCK8 protein was increased 2.3 ± 0.9-fold (p < 0.05) (Fig. 4B). In contrast, the FGD4 protein, which was not altered by miR-34a, decreased to 50% of that in the control cells (p < 0.05) by introduction of miR-155 (Fig. 4C). The inhibitor of miR-155 increased FGD4 1.7 ± 0.3-fold (p < 0.05) (Fig. 4D).

FIGURE 4.

Modified expression of DOCK8 and FGD4. (A) DOCK8 protein levels in miRNA-introduced cells. (B) Expression of DOCK8 protein in miR-34a inhibitor-treated cells. (C) Effects of miRNA mimic introduction on FGD4 protein. (D) Expression of FGD4 in miR-155 inhibitor-introduced cells. (E) DOCK2 levels in miRNA mimic-induced cells. (F) Expression of DOCK2 in miRNA inhibitor-induced cells. Mean ± SD of four to six independent experiments are shown. *p < 0.05.

FIGURE 4.

Modified expression of DOCK8 and FGD4. (A) DOCK8 protein levels in miRNA-introduced cells. (B) Expression of DOCK8 protein in miR-34a inhibitor-treated cells. (C) Effects of miRNA mimic introduction on FGD4 protein. (D) Expression of FGD4 in miR-155 inhibitor-introduced cells. (E) DOCK2 levels in miRNA mimic-induced cells. (F) Expression of DOCK2 in miRNA inhibitor-induced cells. Mean ± SD of four to six independent experiments are shown. *p < 0.05.

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We also measured the protein levels of another GEF decisive for neutrophil migration, DOCK2, which is not a target of miR-34a or miR-155. Neither mimics (Fig. 4E) nor inhibitors (Fig. 4F) of miR-34a and miR-155 altered DOCK2 levels.

To clarify whether the reduction of DOCK8 and FGD4 led to the impairment of migration, we introduced siRNAs for DOCK8 and FGD4. In DOCK8 siRNA-treated cells (Fig. 5A), fMLF-induced migration was reduced by 84.6 ± 30.7% (Fig. 5B) and correlated with DOCK8 protein levels (r = 0.672, p < 0.05). Elevation of GTP-bound Cdc42 was also diminished by 94.3 ± 23.5% in DOCK8 siRNA-induced cells compared with that in the control cells (Fig. 5C). When endogenous FGD4 protein was decreased to 51.7 ± 24.7% by siRNA (Fig. 5D), fMLF did not increase migration (0.9 ± 0.1-fold that in unstimulated control cells) (Fig. 5E). Overall, the correlation coefficient between FGD4 levels and migration was 0.880 (p < 0.05). Although fMLF upregulated GTP-bound Cdc42 protein 3.9 ± 1.7-fold in the control cells, elevation of active Cdc42 was not observed in the FGD4 siRNA-treated cells stimulated with fMLF (0.7 ± 0.4-fold of the unstimulated control) (Fig. 5F).

FIGURE 5.

Roles of DOCK8 and FGD4 in fMLF-induced migration. (A) DOCK8 levels after siRNA introduction. DOCK8 siRNA or control siRNA was introduced to HL60 cells by electroporation. The cells were subsequently cultured in the presence of 500 μM dbcAMP, and DOCK8 protein levels were analyzed by immunoblotting 72 h after electroporation. The graph shows the mean ± SD of seven independent experiments. (B) Effects of DOCK8 siRNA on fMLF-induced migration. The ratios of migrating cells to total cells initially plated into the upper chamber are shown. The value from control siRNA is set as 1.0, and the results from three independent experiments are presented as mean ± SD. (C) Effects of DOCK8 siRNA on Cdc42 activation. The ratios of GTP-bound Cdc42 to total Cdc42 are presented. The values of unstimulated control cells are set as 1.0, and mean ± SD of four independent experiments are presented. (D) FGD4 protein levels after siRNA introduction. (E) Effects of FGD4 siRNA on fMLF-induced migration. The ratios of migrating cells to total cells from three independent experiments are shown as mean ± SD. (F) Modulation of fMLF-induced Cdc42 activation by FGD4 siRNA. The ratios of GTP-bound Cdc42 to total Cdc42 from four independent experiments are shown as mean ± SD. *p < 0.05.

FIGURE 5.

Roles of DOCK8 and FGD4 in fMLF-induced migration. (A) DOCK8 levels after siRNA introduction. DOCK8 siRNA or control siRNA was introduced to HL60 cells by electroporation. The cells were subsequently cultured in the presence of 500 μM dbcAMP, and DOCK8 protein levels were analyzed by immunoblotting 72 h after electroporation. The graph shows the mean ± SD of seven independent experiments. (B) Effects of DOCK8 siRNA on fMLF-induced migration. The ratios of migrating cells to total cells initially plated into the upper chamber are shown. The value from control siRNA is set as 1.0, and the results from three independent experiments are presented as mean ± SD. (C) Effects of DOCK8 siRNA on Cdc42 activation. The ratios of GTP-bound Cdc42 to total Cdc42 are presented. The values of unstimulated control cells are set as 1.0, and mean ± SD of four independent experiments are presented. (D) FGD4 protein levels after siRNA introduction. (E) Effects of FGD4 siRNA on fMLF-induced migration. The ratios of migrating cells to total cells from three independent experiments are shown as mean ± SD. (F) Modulation of fMLF-induced Cdc42 activation by FGD4 siRNA. The ratios of GTP-bound Cdc42 to total Cdc42 from four independent experiments are shown as mean ± SD. *p < 0.05.

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Another Rho protein Rac1 was identified as a target of miR-155 by the microRNA.org program. Endogenous Rac1 protein was significantly reduced by overexpression of miR-155 (56.2 ± 27.9% of that in the controls, p < 0.05), but not by miR-34a (113.6 ± 17.8%) (Fig. 6A). The miR-155 inhibitor significantly increased Rac1 (161.7 ± 17.6% of that in the controls) (p < 0.05) (Fig. 6B). When Rac1 was decreased to 55.0 ± 22.1% by siRNA (Fig. 6C), fMLF increased migrating cells only by 16.9 ± 22.0% (Fig. 6D), which was significantly smaller than that in the control siRNA-treated cells (78.0 ± 46.5%, p < 0.05). Rac1 levels were highly correlated with fMLF-induced migration (r = 0.840, p < 0.05). As shown in Fig. 6E, reduction of Rac1 concealed elevation of GTP-bound Rac1 by fMLF (0.7 ± 0.3-fold that in unstimulated control cells), whereas fMLF increased active Rac1 2.1 ± 0.2-fold in the control cells.

FIGURE 6.

Roles of Rac1 in fMLF-induced migration. (A) Effects of miR-34a and miR-155 mimics on Rac1 protein. Rac1 was immunoblotted and normalized by GAPDH. The graph shown is from four to six independent experiments. (B) Effects of miR-155 inhibitor on Rac1 expression. (C) Rac1 protein levels after siRNA introduction. The results were obtained from eight independent experiments. (D) Effects of Rac1 siRNA on migration toward fMLF. The ratios of migrating cells to total cells are shown. The value from unstimulated control cells is set as 1.0, and data from five independent experiments are shown as mean ± SD. (E) Modulation of fMLF-induced Rac1 activation by siRNA introduction. The ratios of GTP-bound Rac1 to total Rac1 in unstimulated control cells are set as 1.0, and data are shown as mean ± SD of three independent experiments. *p < 0.05.

FIGURE 6.

Roles of Rac1 in fMLF-induced migration. (A) Effects of miR-34a and miR-155 mimics on Rac1 protein. Rac1 was immunoblotted and normalized by GAPDH. The graph shown is from four to six independent experiments. (B) Effects of miR-155 inhibitor on Rac1 expression. (C) Rac1 protein levels after siRNA introduction. The results were obtained from eight independent experiments. (D) Effects of Rac1 siRNA on migration toward fMLF. The ratios of migrating cells to total cells are shown. The value from unstimulated control cells is set as 1.0, and data from five independent experiments are shown as mean ± SD. (E) Modulation of fMLF-induced Rac1 activation by siRNA introduction. The ratios of GTP-bound Rac1 to total Rac1 in unstimulated control cells are set as 1.0, and data are shown as mean ± SD of three independent experiments. *p < 0.05.

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The next question was whether reduction of DOCK8, FGD4, and Rac1 affects migration toward IL-8, which is known to be regulated by the ERK1/2 and PKB/Akt pathways (14). Because it had been reported that dbcAMP-differentiated cells do not respond chemotactically to IL-8 (46), we could not detect migration of dbcAMP-treated HL60 cells to IL-8. Therefore, DMSO was used as a differentiation inducer. It was confirmed that the cell-surface expressions of CD11b and CD18 did not differ among the DMSO-treated three cell types with ectopic miR-34a, miR-155, and control miRNA (Supplemental Fig. 3). IL-8–induced migration was significantly attenuated by both miR-34a (p < 0.05) and miR-155 (p < 0.05) (Fig. 7A) and enhanced by the inhibitors (Fig. 7B). In the presence of ML141, migration of dHL60 toward IL-8 was inhibited, suggesting the involvement of the Cdc42 pathway (Fig. 7C). When DOCK8 and FGD4 were reduced by siRNA, the dHL60 cells barely migrated toward IL-8 (Fig. 7D, 7E). The Rac1 inhibitor (Fig. 7F) and the introduction of Rac1 siRNA (Fig. 7G) also interfered with IL-8–induced migration (p < 0.05 and p < 0.05, respectively). The migratory response to IL-8 was positively correlated with expression levels of DOCK8 (r = 0.650, p < 0.05), FGD4 (r = 0.678, p < 0.05), and Rac1 (r = 0.893, p < 0.05) levels. Thus, DOCK8, FGD4, and Rac1 were involved in not only fMLF- but also IL-8–induced migration.

FIGURE 7.

Effects of DOCK8, FGD4, and Rac1 on IL-8–induced migration. (A) Effects of miR-34a and miR-155 mimics on IL-8–induced migration. (B) IL-8–induced migration under inhibition of miR-34a and miR-155. (C) Migration toward IL-8 in the presence of 2.5 μM ML141, a Cdc42 inhibitor. (D) Effects of DOCK8 siRNA and (E) FGD4 siRNA on migration. (F) IL-8–induced migration in the presence of 50 μM NSC23766, a Rac1 inhibitor. (G) Effects of Rac1 siRNA on migration. Data represent mean ± SD of three to five independent experiments. *p < 0.05, **p < 0.01.

FIGURE 7.

Effects of DOCK8, FGD4, and Rac1 on IL-8–induced migration. (A) Effects of miR-34a and miR-155 mimics on IL-8–induced migration. (B) IL-8–induced migration under inhibition of miR-34a and miR-155. (C) Migration toward IL-8 in the presence of 2.5 μM ML141, a Cdc42 inhibitor. (D) Effects of DOCK8 siRNA and (E) FGD4 siRNA on migration. (F) IL-8–induced migration in the presence of 50 μM NSC23766, a Rac1 inhibitor. (G) Effects of Rac1 siRNA on migration. Data represent mean ± SD of three to five independent experiments. *p < 0.05, **p < 0.01.

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We next investigated the effects of miR-34a/miR-155 on activation of ERK1/2 and PKB/Akt by fMLF, which was shown to be disturbed in MDS (14, 17, 47). Both miR-34a and miR-155 significantly inhibited the phosphorylation of ERK1/2 by fMLF (5.8 ± 3.6-fold, p < 0.05, and 5.6 ± 2.8-fold, p < 0.05, respectively, versus controls: 10.6 ± 5.3-fold) (Fig. 8A). The fMLF-induced ERK phosphorylation was enhanced by both miR-34a (p < 0.05) and miR-155 (p < 0.05) inhibitors (Fig. 8B). In contrast, the phosphorylation of PKB/Akt by fMLF was not significantly altered by either mimics (Fig. 8C) or inhibitors of miR-34a/miR-155 (Fig. 8D).

FIGURE 8.

Effects of miR-34a and miR-155 on phosphorylation of ERK1/2 and PKB/Akt. (A) Phosphorylation of ERK1/2 in response to fMLF in the cells with miRNA mimics. (B) ERK1/2 phosphorylation under miRNA inhibition. (C) Effects of miRNA mimics on fMLF-induced PKB/Akt phosphorylation. (D) PKB/Akt phosphorylation under miRNA inhibition. Effects of DOCK8, FGD4, and Rac1 siRNAs on phosphorylation of ERK1/2 (E) and PKB/Akt (F). The cells were stimulated with 1 μM fMLF for 2 min, and the reaction was terminated with ice-cold PBS. The cell lysates were subjected to immunoblotting using the indicated Abs. Mean ± SD of the ratios of phosphorylated form to total protein from 5 to 10 independent experiments are shown. *p < 0.05.

FIGURE 8.

Effects of miR-34a and miR-155 on phosphorylation of ERK1/2 and PKB/Akt. (A) Phosphorylation of ERK1/2 in response to fMLF in the cells with miRNA mimics. (B) ERK1/2 phosphorylation under miRNA inhibition. (C) Effects of miRNA mimics on fMLF-induced PKB/Akt phosphorylation. (D) PKB/Akt phosphorylation under miRNA inhibition. Effects of DOCK8, FGD4, and Rac1 siRNAs on phosphorylation of ERK1/2 (E) and PKB/Akt (F). The cells were stimulated with 1 μM fMLF for 2 min, and the reaction was terminated with ice-cold PBS. The cell lysates were subjected to immunoblotting using the indicated Abs. Mean ± SD of the ratios of phosphorylated form to total protein from 5 to 10 independent experiments are shown. *p < 0.05.

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To examine whether the reduction of the two GEFs and Rac1 interfered with activation of ERK1/2 and Akt, we quantified fMLF-induced phosphorylation of ERK and Akt under knockdown of DOCK8, FGD4, and Rac1. Compared with the control siRNA-treated cells (10.9 ± 5.6-fold), ERK phosphorylation was significantly attenuated by silencing Rac1 (6.5 ± 3.1-fold, p < 0.05), but not DOCK8 (11.2 ± 5.9-fold) or FGD4 (9.3 ± 4.0-fold) (Fig. 8E). In contrast, none of those siRNAs affected PKB/Akt phosphorylation (Fig. 8F).

To study whether the reduction of DOCK8, FGD4, and Rac1 was responsible for impaired neutrophil migration in MDS, we isolated peripheral neutrophils from 12 healthy volunteers and 11 MDS patients (Table I), including 7 with high-expression miR-34a (patients 1–5, 7, and 10 in Fig. 9B) and 2 (patients 3 and 5) with increased miR-155. As shown in Fig. 9A, fMLF-induced increase of migrating cells, which was 1.7 ± 0.2-fold in the healthy control subjects, was 1.1 ± 0.3-fold in MDS (p < 0.001). IL-8–induced migration was compared between six healthy control subjects and six patients (patients 6–11), and significant attenuation was found in MDS patients (1.2 ± 0.1-fold versus 1.6 ± 0.1-fold in the controls, p < 0.01).

FIGURE 9.

Migration and expression of DOCK8, FGD4, and Rac1 in neutrophils from MDS patients. (A) Comparison of neutrophil migration toward fMLF or IL-8 between healthy donors and MDS patients. Data of fMLF-induced migration was obtained from 11 healthy donors and 11 patients including 7 (1–5, 7, and 10) with significantly higher miR-34a expression and 2 (3 and 5) with overexpressed miR-155. Six patients (6–11) were subjected to comparison of IL-8–induced migration with six controls. The ratios of migrating cells to total cells in the absence of a stimulus are set as 1.0. **p < 0.01, ***p < 0.001. (B) Expression of DOCK8. The numbers above the photos indicate expression levels of miR-34a and miR-155, and ratios of band intensities of DOCK8 to those of α-tubulin in immunoblotting. The comparison of DOCK8 protein levels between the healthy control subjects and MDS patients (upper left), correlations between miR-34a and DOCK8 levels (upper right), and fMLF- (lower left) and IL-8–induced migration (lower right) are presented. Closed triangles and open squares represent healthy individuals and MDS patients, respectively. **p < 0.01. (C) Expression of FGD4. (D) Expression of Rac1. The detected FGD4 and Rac1 were normalized by GAPDH. Relationships between FGD4/Rac1 protein levels and miR-155 expression (left) and fMLF- (middle) and IL-8–induced (right) migration are shown. Closed triangles and open squares, respectively, represent healthy controls and MDS patients. NE, not examined.

FIGURE 9.

Migration and expression of DOCK8, FGD4, and Rac1 in neutrophils from MDS patients. (A) Comparison of neutrophil migration toward fMLF or IL-8 between healthy donors and MDS patients. Data of fMLF-induced migration was obtained from 11 healthy donors and 11 patients including 7 (1–5, 7, and 10) with significantly higher miR-34a expression and 2 (3 and 5) with overexpressed miR-155. Six patients (6–11) were subjected to comparison of IL-8–induced migration with six controls. The ratios of migrating cells to total cells in the absence of a stimulus are set as 1.0. **p < 0.01, ***p < 0.001. (B) Expression of DOCK8. The numbers above the photos indicate expression levels of miR-34a and miR-155, and ratios of band intensities of DOCK8 to those of α-tubulin in immunoblotting. The comparison of DOCK8 protein levels between the healthy control subjects and MDS patients (upper left), correlations between miR-34a and DOCK8 levels (upper right), and fMLF- (lower left) and IL-8–induced migration (lower right) are presented. Closed triangles and open squares represent healthy individuals and MDS patients, respectively. **p < 0.01. (C) Expression of FGD4. (D) Expression of Rac1. The detected FGD4 and Rac1 were normalized by GAPDH. Relationships between FGD4/Rac1 protein levels and miR-155 expression (left) and fMLF- (middle) and IL-8–induced (right) migration are shown. Closed triangles and open squares, respectively, represent healthy controls and MDS patients. NE, not examined.

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As depicted in Fig. 9B, MDS patients with high miR-34a levels showed significantly lower DOCK8 protein expression than the healthy volunteers (0.51 ± 0.41 versus 1.00 ± 0.41, p < 0.01), and inverse correlation was observed between miR-34a and DOCK8 levels (r = −0.464, p < 0.05). The DOCK8 levels positively correlated with both fMLF- and IL-8–induced migration (fMLF: r = 0.642, p < 0.01; IL-8: r = 0.778, p < 0.01). The FGD4 protein levels were not decreased in either patient 3 or 5 with miR-155 overexpression, whereas a significant reduction of FGD4 protein was observed in patients 1, 2, 4, 8, and 10 (0.47, 0.45, 0.48, 0.28, and 0.42 versus 1.00 ± 0.21 in the controls, respectively). Thus, the expression levels of FGD4 protein were not correlated with miR-155 levels. However, there was a strong correlation between FGD4 protein levels and fMLF/IL-8–induced migratory activities (fMLF: r = 0.686, p < 0.01; IL-8: r = 0.659, p < 0.05) (Fig. 9C). Although significantly low Rac1 expression was observed only in patient 4, who had a normal miR-155 level (0.45 versus 1.00 ± 0.20 in the controls), Rac1 protein levels positively correlated with fMLF/IL-8–induced migration (fMLF: r = 0.436, p < 0.05; IL-8: r = 0.606, p < 0.05) (Fig. 9D). Table II summarizes the migratory activities and protein levels in each patient.

Table II.
Protein levels and migration of patients
Patient No.DOCK8/α-TubulinFGD4/GADPHRac1/GADPHfMLF-Induced MigrationIL-8–Induced Migration
0.47 0.47a 0.96 1.25a NE 
0.41 0.45a 0.85 1.16a NE 
0.28 0.72 0.71 1.26a NE 
0.45 0.48a 0.45a 0.90a NE 
1.41 0.64 0.76 1.22a NE 
0.44 1.11 0.61 1.07a 1.06a 
0.13a 0.62 0.74 0.44a 0.98a 
0.32 0.28a 0.70 1.07a 1.13a 
0.07a 0.60 1.16 1.21a 1.31a 
10 0.41 0.42a 1.00 1.45 1.27a 
11 0.32 NE NE 1.33 1.25a 
Controls 1.00 ± 0.41 1.00 ± 0.21 1.00 ± 0.23 1.7 ± 0.2 1.6 ± 0.1 
Patient No.DOCK8/α-TubulinFGD4/GADPHRac1/GADPHfMLF-Induced MigrationIL-8–Induced Migration
0.47 0.47a 0.96 1.25a NE 
0.41 0.45a 0.85 1.16a NE 
0.28 0.72 0.71 1.26a NE 
0.45 0.48a 0.45a 0.90a NE 
1.41 0.64 0.76 1.22a NE 
0.44 1.11 0.61 1.07a 1.06a 
0.13a 0.62 0.74 0.44a 0.98a 
0.32 0.28a 0.70 1.07a 1.13a 
0.07a 0.60 1.16 1.21a 1.31a 
10 0.41 0.42a 1.00 1.45 1.27a 
11 0.32 NE NE 1.33 1.25a 
Controls 1.00 ± 0.41 1.00 ± 0.21 1.00 ± 0.23 1.7 ± 0.2 1.6 ± 0.1 
a

Significantly low protein levels or migratory activities, defined as a reduction of >2 SD from the mean values of the healthy controls.

NE, not examined.

Taken together, the reductions of DOCK8, FGD4, and Rac1, which did not always coincide with miR-34a or miR-155 overexpression in MDS neutrophils, correlated with attenuation of migratory activity in response to fMLF and IL-8.

Using neutrophil-like dHL60 cells, we demonstrated that cell migration toward fMLF and IL-8 was attenuated by miR-34a and miR-155, both of which were aberrantly increased in the neutrophils from MDS patients. The inhibitory effects of the miRNAs on migration were facilitated by reducing two Cdc42-specific GEFs, DOCK8 and FGD4, and Rac1. Further analyses of healthy and MDS neutrophils indicated that reduction of these molecules was correlated with the impaired neutrophil migration in MDS.

Overexpression of miR-34a and miR-155 did not seem to affect the differentiation of HL60 induced by dbcAMP, because no differences were observed in the morphology, expression of CD11b/CD18, and amounts of enzymes in primary granules among the cells with ectopic miR-34a, miR-155, or control miRNA. Therefore, the three cell types were considered to be in the same differentiation stage and available for comparison of functional interference by the miRNAs, and the impairment of migration was not attributable to CD11b/CD18 levels.

In miR-34a– and miR-155–overexpressing cells, inhibition of migration coincided with enhancement of degranulation, suggesting that Rac2 was unlikely to be involved in the inhibition of migration. Degranulation is predominantly regulated by Rac2, as shown by the lack of granule translocation to plasma membrane before docking and exocytosis in Rac2-deficient neutrophils (43, 48). In contrast, previous studies have reported cross talk between Rac2 and another Rho family member, Cdc42, which is known as a major regulator of neutrophil migration (42, 49). Activation of Cdc42 inhibited the translocation of Rac2 from the perinuclear zones to the plasma membrane, whereas inactivation of Cdc42 accelerated the production of reactive oxygen species (42, 49), which was dependent on Rac2 activity (50, 51). Therefore, the Cdc42 activating pathway was a candidate that caused suppression of migration. In addition to Cdc42, we speculated the involvement of Rac1. In human neutrophils, although Rac2 is more than 80% of the total Rac protein (52, 53), Rac1 is essential for directed migration (5456).

The impairment in fMLF– and IL-8–induced migration of neutrophils, which is essential for pathogen killing at infection sites (57), is a disadvantage to bactericidal functions. In contrast, enhanced enzyme release from granules could intensify bactericidal efforts and lead to tissue damage (48). We speculate that the coincidence of attenuated migration and enhanced degranulation increases susceptibility to local infection and deteriorates systemic inflammation, because MPO stimulates production of ROS and chemokines (58). However, according to a previous article by Dang et al. (59), MPO release from MDS neutrophils was similar to that from healthy control subjects. This may be because of neutralization of the miR-34a/miR-155–induced enhancement by additional factors that have inhibitory effects on degranulation in those patients.

To our knowledge, it is a novel finding that DOCK8 is involved in miR-34a–mediated inhibition of neutrophil migration induced by not only fMLF, but also IL-8. A decrease of DOCK8 by miR-34a mimic and an increase of DOCK8 by miR-34a inhibitor confirmed that miR-34a targeted DOCK8, as was suggested by the microRNA.org program. DOCK2, which lacked modulation by any of the miRNA mimics and inhibitors tested, was used as a negative control. A previous study showed that CCL21-induced dendritic cell migration was attenuated via impaired activation of Cdc42 at the leading edge membrane in DOCK8 knockdown mice (25). This report supports our findings, although we did not distinguish between local and global Cdc42 activation. In addition, miR-34a inhibited the activation of ERK1/2, which is known to play a crucial role in regulating cellular migration (14, 17). Because the database microRNA.org indicates that MAPK kinase 1, an upstream activator of the ERK1/2 pathway, is a target of miR-34a (60), we speculate that the attenuation of ERK1/2 phosphorylation by miR-34a was due to the reduction of MAPK kinase 1.

In MDS, DOCK8 reduction caused by miR-34a overexpression seems to be one of the causes of impaired neutrophil migration. Both fMLF/IL-8–induced migratory activities and DOCK8 protein levels were attenuated in MDS neutrophils, and correlation was detected between DOCK8 levels and migration. Overexpression of miR-34a has previously been demonstrated in CD34+ bone marrow cells derived from patients with early-stage MDS (61). If hematopoietic stem cells overexpressing miR-34a survive and differentiate, DOCK8 protein levels could be lowered in various types of blood cells. DOCK8 deficiency has been shown to affect migration of dendritic cells and CD4+ T cells, B cell activation, and CD8+ T cell survival and function (25, 6264). Thus, miR-34a–mediated DOCK8 reduction may interfere with acquired and natural immunity in MDS patients.

miR-155 inhibited migration via two different mechanisms from miR-34a: reduction of another Cdc42-specific GEF, FGD4; and a Rho protein Rac1, in dHL60. As the database predicted, ectopic miR-155 decreased FGD4 and Rac1, both of which were upregulated by the introduction of the miR-155 inhibitor. Knockdown of FGD4 and Rac1 by siRNA interfered with the activation of Cdc42 and Rac1, respectively, resulting in disturbed migration toward fMLF and IL-8. Rac1 was already shown to be required for neutrophil migration (54, 65) However, involvement of FGD4 in neutrophil migration had not been studied, whereas FGD4 had been shown to regulate motility of carcinoma cells and motor neurons (6668). This study demonstrated that FGD4 was indispensable for regulating migration of blood cells.

Overexpression of miR-155 did not affect PKB/Akt. Although attenuated activation of PBK/Akt has been reported in MDS-derived neutrophils and CD34+ cells (17, 47), our findings suggest that inhibition of PKB/Akt activation is not due to increased miR-155. miR-155 targets both SHIP-1, a phosphatase that negatively regulates PKB/Akt (34), and positive regulators of the Akt pathway, PI3Kγ subunits p84 and p101 (69). The effects of reduction of these molecules could have countervailed each other. In contrast, ERK1/2 activation was inhibited by miR-155. This effect could be attributed to silencing Rac1, which activates ERK1/2 (14, 70), because ERK1/2 itself has no miR-155 binding sites. As Rac1 siRNA suppressed ERK1/2 phosphorylation, miR-155 indirectly inhibited ERK1/2 activity via Rac1. Thus, miR-155 inhibited migration via suppression of FGD4 and Rac1, but not PKB/Akt.

In neutrophils, the elevation of miR-155 did not seem to be the only cause of suppression of FGD4 and Rac1, because expressions of FGD4 and Rac1 were preserved in the two patients with aberrantly high miR-155 levels, and miR-155–normal patients showed a decrease in FGD4 and Rac1. This may be attributed to the fact that the expression of these molecules is regulated by various factors, but not by a single miRNA. For example, FGD4 is also a target of miR-143 and miR-320a, which were recently found to be downregulated and upregulated in CD34+ cells from MDS patients, respectively (61, 71). Downregulation of miR-143 may mask the inhibitory effect of miR-155 on FGD4, whereas increased miR-320a could result in the reduction of FGD4 without aberrant expression of miR-155.

Instead of the ambiguous relationship between the expressions of miR-155 and its target molecules, the attenuated migration of MDS neutrophils seemed to contribute to the suppressed expression of FGD4 and Rac1, because expression levels of FGD4 and Rac1 were significantly correlated with fMLF- and IL-8–induced migration. Especially, FGD4, as well as DOCK8, showed a high correlation coefficient with migratory activity, suggesting that reduction of DOCK8 and FGD4 may be the major regulators of migration in neutrophils. In three patients (patients 3, 5, and 6), however, the reduction in FGD4, Rac1, and DOCK8 was not statistically significant, whereas migratory activities were significantly attenuated. In these patients, accumulation of a subtle decrease of multiple molecules, that is, DOCK8 and Rac1 in patient 6, may result in the impaired migration. Another possibility is that other mechanisms, such as inhibition of PKB/Akt, interfered with migratory response.

The clinical relevance of the reduction of the GEFs and Rac1 needs to be further studied. Not only DOCK8 (25, 6264), but also Rac1 (72), has been shown to affect immune cell functions. Our patients, except patient 8, however, experienced no symptomatic infections for at least 1 y. Migratory activities, expression of GEFs and Rac1, or any blood cell counts did not distinguish patient 8 from others. Regarding FGD4, of which the mutations are known to result in Charcot-Marie-Tooth disease (66, 73, 74), the effects of FGD4 loss on hematopoiesis are not clear. It is also of interest whether the expression of GEFs and Rac1 is connected to any somatic mutations frequently observed in MDS. Although Tet oncogene family member 2 (TET2) was mutated in at least 6 patients out of 11, more patients need to be studied to determine the roles of TET2 mutations in regulation of GEFs/Rac1 expression.

In this study, we identified mechanisms of attenuated migration by aberrantly increased miR-34a and miR-155, and demonstrated that the protein levels of DOCK8, FGD4, and Rac1 are downregulated in MDS neutrophils, which contributed to impaired migratory activity of MDS-derived neutrophils. These findings provide a new insight to unveil the pathophysiology behind the qualitative abnormalities in MDS neutrophils.

We thank Michiko Anzai and Sanae Sato (Fukushima Medical University) for their technical assistance, and Prof. Kenneth Nollet (Fukushima Medical University) for valuable advice in manuscript preparation.

This work was supported by Japanese Society for Promotion of Science Grants-in-Aid for Scientific Research (C) MO23591400 (to Y.S.), MO26461409 (to Y.S.), and MO24590325 (to J.K.).

The online version of this article contains supplemental material.

Abbreviations used in this article:

dbcAMP

dibutyryl cAMP

Dbl

diffuse B cell lymphoma

dHL60

differentiated HL60

DOCK

dedicator of cytokinesis

FGD

FYVE, RhoGEF, and PH domain-containing

FPR

fMLF receptor

GEF

guanine nucleotide exchange factor

MDS

myelodysplastic syndromes

miRNA

microRNA

MPO

myeloperoxidase

PKB

protein kinase B

RCMD

refractory cytopenia with multilineage dysplasia

siRNA

small interfering RNA

TET2

Tet oncogene family member 2.

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The authors have no financial conflicts of interest.

Supplementary data