MiR-382-5p controls hematopoietic stem cell differentiation through the downregulation of MXD1

MicroRNAs are key regulators of gene expression that control stem cell fate by post-transcriptional down-regulation of hundreds of target genes via seed-pairing in their 3’ untranslated region. In fact, miRNAs tightly regulate fundamental stem cell processes, like self-renewal, proliferation and differentiation; therefore, miRNA deregulation may contribute to the development of solid tumours and haematological malignancies.


ABSTRACT
MicroRNAs are key regulators of gene expression that control stem cell fate by posttranscriptional down-regulation of hundreds of target genes via seed-pairing in their 3' untranslated region.In fact, miRNAs tightly regulate fundamental stem cell processes, like self-renewal, proliferation and differentiation; therefore, miRNA deregulation may contribute to the development of solid tumours and haematological malignancies.
MiR-382-5p has been found to be upregulated in patients with myeloid neoplasms but its role in normal hematopoiesis is still unknown.Here we demonstrated that miR-382-5p overexpression in CD34+ hematopoietic stem/progenitor cells leads to a significant decrease of megakaryocyte precursors coupled to increase of granulocyte ones.Furthermore, by means of a computational analysis using different prediction algorithms, we identified several putative mRNA targets of miR-382-5p that are downregulated upon miRNA overexpression (i.e.FLI1, GATA2, MAF, MXD1, RUNX1 and SGK1).Among these, we validated MXD1 as real target of miR-382-5p by luciferase reporter assay.Finally, we showed that MXD1 knockdown mimics the effects of miR-382-5p overexpression on granulocyte and megakaryocyte differentiation of CD34+ cells.
Overall, our results demonstrated that miR-382-5p expression favours the expansion of granulocyte lineage and impairs megakaryocyte commitment through MXD1 downregulation.Therefore, our data showed for the first time that the miR-382-5p/MXD1 axis plays a critical role in myelopoiesis by affecting the lineage choice of CD34+ hematopoietic stem/progenitor cells.

Stem Cells and Development
MiR-382-5p controls hematopoietic stem cell differentiation through the downregulation of MXD1 (doi: 10.1089/scd.2016.0150)This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction.The final published version may differ from this proof.

INTRODUCTION
MicroRNAs (miRNAs) are a large class of small noncoding RNAs that act as posttranscriptional regulators of gene expression (1).miRNAs simultaneously modulate the expression of several targets by binding to the 3'-untraslated region (3'-UTR) of the mRNAs, resulting in mRNA destabilization and/or translational inhibition (2).
In the last decade, growing evidence has unravelled the role of miRNAs as key regulators of stem cell fate by modulating essential processes like self-renewal, proliferation and differentiation.In particular, the combinatorial expression of miRNAs can precisely characterize specific cell types and regulate different phases of differentiation (3,4).Moreover, recent advancements in computational and molecular biology have highlighted the existence of regulatory networks where miRNAs modulate the expression of transcription factors and transcription factors regulate miRNA levels to promote or maintain some important cell functions (1).On the other side, miRNA expression deregulation may contribute to the development of different human diseases, including cancer (5).
Despite an abnormal expression level of miR-382-5p has been described in these hematological malignancies, its role in normal hematopoiesis has not been described so far.Therefore, in order to characterize the function of miR-382-5p in myeloid development, we evaluated the effects of its overexpression on the commitment of human CD34+ hematopoietic stem/progenitor cells (HSPCs).Interestingly, we identified MAX dimerization protein 1 (MXD1) as a target of miR-382-5p, and  demonstrated that miR-382-5p favours granulocyte lineage at the expense of megakaryocyte one through MXD1 downregulation.

Electroporation of CD34+ cells
Human CD34+ cells were transfected by using the 4D-Nucleofector System (Lonza Group Ltd, Basel, Switzerland) as previously reported (12).Briefly, after 24 hours of incubation in the cell-culture condition described above, CD34+ cells were nucleofected twice, once every 24 hours, with mirVana miR-382-5p mimic or mirVana the program DS112.After each transfection, CD34+ cells were transferred into prewarmed fresh medium in 24-well plates (Euroclone) and maintained in the same culture conditions as described above.
For small interfering RNAs (siRNAs) transfection, each sample was electroporated 3 times, once every 24 hours, with 3 µg of a mix of 3 Silencer Select siRNA targeting human MXD1 (supplemental Table 1) (Life Technologies).As previously described, CD34+ cells were nucleofected 24 hours after immunomagnetic purification, by using the P3 Primary Cell Solution (Lonza) and the electroporation protocol DS112.To exclude nonspecific effects caused by interfering RNA (RNAi) nucleofection, a sample transfected with a non-targeting siRNA negative control (ss-NegCTR; Silencer Select Negative Control #2 siRNA; Life Technologies) was included.
Cells were analyzed 24 and 48 hours after the last nucleofection for both cell viability and miR-382-5p and/or MXD1 expression.
Viability measurement was assessed by trypan blue exclusion assay 24 hours after the last nucleofection (14).In a Neubauer chamber, at least 100 cells were microscopically analyzed in duplicate for viability.The mean percentage of living cells of the two analyses was calculated.

Differentiation of CD34+ cells was monitored by morphological analysis of May-
Grunwald-Giemsa-stained cytospins and by flow cytometry analysis of CD34, CD14, CD66b, CD15, Glycophorin A (GPA), CD41 and CD42b surface antigen expression at day 5, 7, 10, and 12 after the last nucleofection.Images were captured by using an Ax10scopeA1 microscope equipped with AxioCam ERc 5S Digital Camera and Axion software 4.8 (all Carl Zeiss MicroImaging Inc.; Thornwood, NY, USA).The images were then processed with Adobe Photoshop 11.0.2software.
CD34+ cells differentiation was monitored by morphological analysis of MGG-stained cytospins and by flow-cytometric analysis of differentiation marker expression.The medium was replaced every 2 days.MiR-382-5p expression level was detected by qRT-PCR at different time points (i.e.days 1, 2, 3, 5, 7, 9 and 12) after seeding cells in erythrocyte, megakaryocyte, granulocyte or mono-macrophage unilineage cultures.The day 0 sample was designated as the calibrator.

Methylcellulose and collagen clonogenic assays
Two different clonogenic assays were performed at 24 hours after the last nucleofection.The methylcellulose assay was carried out by plating CD34+ cells in MethoCult TM H4434 Classic (StemCell Technologies Inc.; Vancouver), as previously described (15).In addition, MK colony forming units (CFU-MK) were assayed in collagen-based medium, using a commercial MK assay detection kit (MegaCult-C; StemCell Technologies Inc.) as previously reported (16).

RNA purification and quantitative reverse transcription polymerase chain reaction (qRT-PCR)
Total cellular RNA, including small RNAs, was isolated from CD34+ cells using the miRNeasy mini RNA isolation kit (Qiagen, Hilden, Germany) following the manufacturer's recommendations.The purity and integrity of RNA samples were  determined by using disposable RNA chips (Agilent RNA 6000 Nano LabChip kit) and the Agilent 2100 Bioanalyzer (Agilent Technologies; Waldbrunn, Germany).
NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies; Wilmington, DE) was used to evaluate the RNA sample concentration, while 260/280 nm and 260/230 nm ratios were used to assess the purity of RNA.cDNA was reverse-transcribed from total RNA (100 ng per sample) using the High-Capacity cDNA Reverse Transcription Kit (Life technologies; Carlsbad, CA, USA), and real-time PCR was carried out in triplicate with TaqMan gene expression assays and Fast advanced master mix (all reagents from Life Technologies) by using an AB 7900HT Fast Real-Time PCR System (Applied Biosystems) (17).Gene expression relative quantification (RQ) was achieved using the comparative cycle threshold (CT) method using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the housekeeping gene.The ss-NegCTR sample was considered as the calibrator, the RQ value was expressed as 2−ΔΔCT.
For miRNA cDNA synthesis, 5 ng of total RNA was reverse-transcribed using the Taqman microRNA Reverse Transcription Kit and specific primers included in TaqMan MicroRNA assays.qRT-PCR was performed using TaqMan MicroRNA assays (Life Technologies) as described above and U6 snRNA as a housekeeping control.

Luciferase reporter assays
Predicted target sites of miR-382-5p were cloned into the Pme1 and XbaI sites of pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega Italia, Milano, Italy), following manufacturer's instructions.Every cloned plasmid contains the firefly luciferase gene upstream of a given miRNA binding site, and the Renilla luciferase gene acting as a normalizer gene.Annealing oligonucleotides containing wild type miRNA binding sites were designed based on TargetScan 7.0 sequences (Supplemental Table 2).Mutated miRNA binding sites were obtained from the wildtype miRNA binding sites by changing nucleotides 3-5 in the miRNA seed regions.K562 cells were electroporated by means of the Amaxa 4D-NucleofectorTM System, according to the manufacturer's instructions.Briefly, K562 cells were cultured at a density of 3×10 5 cells/mL 2 days before nucleofection.Cells were then transiently conucleofected with either a miR-382-5p or m-NegCTR at a concentration of 3.6 μM and with either empty vector or miRNA binding site-containing construct at a concentration of 200 ng/sample.For each electroporation, 1x10 6 cells were resuspended in 100 μL of SF Cell line Solution (Lonza) and pulsed with the program FF120.Firefly and Renilla luciferase activities were measured 24 hours after electroporation using the Dual-Luciferase Reporter Assay System (Promega), and luminescence was recorded on a GloMax®-Multi+ Detection System with Intinct TM Software (Promega), according to the manufacturer's protocol.
In order to analyze the data, three levels of normalization were performed as previously described: firstly, firefly luciferase activity was normalized to that of Renilla luciferase as the internal control for nucleofection efficiency; secondly, the data were normalized to the effect of the miRNA mimic on the empty vector control reporter; lastly, the luminescence signals were normalized to the effect of the m-NegCTR on the corresponding miRNA binding site-containing construct (12).

Statistical analysis
The statistics used for data analysis in overexpression/silencing experiments and luciferase reporter assays were based on 2-tailed Student t tests for averages comparison in paired samples.Data were analyzed by using Microsoft Excel (Microsoft Office, 2008 release) and are reported as mean ± standard error of the mean (SEM).P < .05 was considered significant.
Flow cytometry analysis of MK differentiation markers performed on TPO unilineage culture at days 5, 7, 10, and 12 showed that miR-382-5p overexpression induces a significant decrease in the CD41+ cell fraction compared with the m-NegCTR control (Figure 1B).In addition, the mature megakaryocytic CD41+/CD42b+ population was similarly reduced at days 7 and 10 (Figure 1C).On the contrary, miR-382-5p overexpression in GCSF unilineage culture led to a significant increase in the percentage of CD66b+ and CD15+ cells at days 10 and 12 after the last nucleofection (Figure 1D-E  1H, Panels i-iv).Similarly, miR-382-5p upregulation in multilineage culture conditions further confirmed these results (Figure 2).Flow cytometry analysis of monomacrophage (CD14) and erythrocyte (GPA) markers did not highlight any significant modulation of expression between miR-382-5p CD34+ cells and the m-NegCTR sample (Figure S1, Panels B-D).
Furthermore, the collagen-based assay that supports the growth of MK progenitors in vitro showed that miR-382-5p overexpression causes a significant decrease in the CFU-MK percentage coupled with a significant increase of non-MK colonies (Figure 3B).
Since miR-382-5p interferes with lineage choice of CD34+ cells, we assessed its expression level in CD34+ and differentiated cells obtained from granulocyte, monocyte-macrophage, erythrocyte, and megakaryocyte unilineage culture conditions.As shown in figure S2, miR-382-5p expression level was decreased during the initial phase of megakaryocyte and erythroid unilineage cultures, while its expression increased in the last phases of differentiation.On the contrary, miR-382-5p results upregulated during the granulocyte and monocyte-macrophage differentiation (Figure S2).
Overall, these results indicated that miR-382-5p overexpression in CD34+ HSPCs enhances granulocyte differentiation while negatively interferes with the megakaryocyte one.

MXD1 silencing in CD34+ cells
To better characterize the role of miR-382-5p/MXD1 axis in myelopoiesis, we investigated whether MXD1 silencing could reproduce the effects mediated by miR-382-5p overexpression on GN and MK differentiation.Therefore, CD34+ cells were transfected with a mixture of three Silencer Select siRNAs targeting MXD1 mRNA (supplemental Table 1) and with a non-targeting siRNA as a negative control (ss-NegCTR).As shown in Figure S3 Panel A, cell viability was > 90% in all samples, without any significant differences between ss-NegCTR and MXD1-siRNA.The expression level of MXD1 in control samples and MXD1-siRNA cells was assessed by qRT-PCR and Western Blot analysis.As shown in Figure 5A, we obtained a significant MXD1 downregulation of MXD1 mRNA at 24 and 48 hours after the last nucleofection (RQ ± SEM, 0.34 ± 0.03, P < .001and 0.48 ± 0.10, P < .01,respectively).Accordingly, the MXD1 protein level is remarkably decreased at 24 hours post-nucleofection (Figure 5B).
As observed for the miR-382-5p overexpression, MXD1 silencing led to a substantial decrease in the percentage of CD41+ and CD41+/CD42b+ cells at day 7 and 10 after the last nucleofection in MK unilineage culture (Figure 5C-D could be appreciated also in MGG-stained cytospins obtained from multilineage culture (Figure 5J, Panels i-iv).
Conversely, we did not observe any modulation of GPA and CD14 expression upon MXD1 knockdown (Figure S3, Panels B-D).
Finally, according to results obtained by means of clonogenic assay on miR-382-5poverexpressing cells, we found a significant increase in the percentage of CFU-G, while CFU-M, BFU-E/CFU-E and CFU-GM colonies were not significantly affected by MXD1 down-regulation (Figure 6A).In addition, the collagen-based assay confirmed that MXD1 silencing induces a decrease of MK progenitors as showed by a lower CFU-MK percentage in MXD1-siRNA sample compared with control (Figure 6B).
Since the expression levels of the megakaryocyte transcription factors GATA2, FLI1, RUNX1, and MAF and of the kinase SGK1 were decreased upon miR-382-5p overexpression, although they have not been previously identified as functional targets, we still wondered whether MXD1 silencing could modulate their expression.By means of qRT-PCR, we showed that the mRNA levels of GATA2, RUNX1 and FLI1 were significantly downregulated in MXD1-silenced cells (Figure 7).Conversely, MAF and SGK1 expression levels seem not to be affected by MXD1 knockdown.

DISCUSSION
Growing evidence shows that miRNAs regulate stem cell-fate decision by modulating fundamental processes like self-renewal, proliferation and differentiation.For this reason, dysregulation of miRNA expression represents a common mechanism of tumorigenesis and cancer progression.MiR-382-5p overexpression has been described in some hematological malignancies, such as PMF (12) and Acute Myeloid Leukemia (AML) (10,11), even if its role in normal hematopoiesis was still unknown.
Here we showed for the first time that miR-382-5p plays a critical role in hematopoiesis by influencing the lineage choice of CD34+ cells.In particular, we demonstrated that miR-382-5p supports the expansion of granulocyte lineage whereas negatively interferes with megakaryocyte commitment, as demonstrated by a significant increase of granulocyte precursors coupled to decrease of megakaryocyte ones in miR-382-5p-overexpressing cells (Figure 1-3).
MXD1, also known as MAD1, is a transcriptional repressor involved in the MYC/MAX/MAD pathway, one of the best known network that controls cell proliferation and differentiation (23).In fact, MXD1 interacts with MAX antagonizing MYC/MAX dimer activity (23).Queva et al. showed that MXD1 is rapidly induced upon cell differentiation, when MYC expression is downregulated and cells begin to exit the cell cycle acquiring a differentiated phenotype (24).
Here we demonstrated that MXD1 knockdown reproduces the effects of miR-382-5p overexpression on CD34+ HSPC differentiation leading to a significant decrease of megakaryocyte precursors coupled to increase of granulocyte ones (Figure 5 and 6).
According to our results, the increased expression of MXD1 has been previously reported also in megakaryocytes obtained in vitro from human CD34+ cells (21).
Therefore, the MXD1 expression in CD34+ cells seems to be essential for megakaryocyte differentiation; in fact, the MK transcription factors GATA2, FLI1 and RUNX1 were significantly decreased after MXD1 silencing (Figure 7) (18), suggesting that the expression of these genes could be regulated by MXD1 in a direct or indirect way.For instance, the downregulation of GATA2, FLI1 and RUNX1 might be mediated by the MXD1-driven inhibition of a transcriptional repressor (i.e.BCL6) (25).Moreover, MXD1 can also heterodimerize with other proteins in addition to MAX (i.e.MLX), even if the function of these complexes as well as their targets has not been defined so far (26,27).
Two independent studies reported that MXD1 induces erythroid differentiation in human (28)   of granulocyte differentiation, resulting in increased frequency of granulocytic precursors in the bone marrow (20)        m-NegCTR transfection.(C) Western blot analysis of MXD1 protein levels in lysates from miR-382-5p and m-NegCTR transfected CD34+ cells at 24 hours after the last nucleofection.β-actin protein level is reported as loading control.
Values are reported as mean ± SEM; *, P < .05versus mutated miRNA binding site.
Results come from three independent experiments performed in duplicate.This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction.The final published version may differ from this proof.

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Cells and DevelopmentMiR-382-5p controls hematopoietic stem cell differentiation through the downregulation of MXD1 (doi: 10.1089/scd.2016.0150)This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction.The final published version may differ from this proof.
miRNA mimic Negative Control #1 (m-NegCTR) (Life Technologies, Carlsbad, CA, USA).For each electroporation, 4 × 10 5 CD34+ cells were resuspended in 100 µL of P3 Primary Cell Solution (Lonza) containing 3 µg of mimic miRNA, and pulsed with Page 6 of 42 Stem Cells and Development MiR-382-5p controls hematopoietic stem cell differentiation through the downregulation of MXD1 (doi: 10.1089/scd.2016.0150)Thisarticle has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction.The final published version may differ from this proof.

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Cells and DevelopmentMiR-382-5p controls hematopoietic stem cell differentiation through the downregulation of MXD1 (doi: 10.1089/scd.2016.0150)This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction.The final published version may differ from this proof.

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Cells and DevelopmentMiR-382-5p controls hematopoietic stem cell differentiation through the downregulation of MXD1 (doi: 10.1089/scd.2016.0150)This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction.The final published version may differ from this proof.

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Cells and DevelopmentMiR-382-5p controls hematopoietic stem cell differentiation through the downregulation of MXD1 (doi: 10.1089/scd.2016.0150)This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction.The final published version may differ from this proof.

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Cells and DevelopmentMiR-382-5p controls hematopoietic stem cell differentiation through the downregulation of MXD1 (doi: 10.1089/scd.2016.0150)This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction.The final published version may differ from this proof.

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Cells and DevelopmentMiR-382-5p controls hematopoietic stem cell differentiation through the downregulation of MXD1 (doi: 10.1089/scd.2016.0150)This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction.The final published version may differ from this proof.
).Similarly, miR-382-5p upregulation in multilineage culture conditions further confirmed these results (data not shown).Flow cytometry analysis of mono-macrophage and erythrocyte differentiation markers did not highlight any significant modulation of expression between miR-382-5p CD34+ cells and the m-NegCTR sample (data not shown).According to flow cytometry results, the morphologic analysis of MGG-stained cytospins obtained from TPO unilineage culture showed a remarkable reduction in MK precursors at different stages of maturation in miR-382-5p-overexpressing cells compared with control (Figure 1G, Panels i-iv).Moreover, morphological evaluation of GCSF-treated cells clearly displayed a considerable enrichment in granulocytes in miR-382-5p sample (Figure Page 13 of 42 Stem Cells and Development MiR-382-5p controls hematopoietic stem cell differentiation through the downregulation of MXD1 (doi: 10.1089/scd.2016.0150)This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction.The final published version may differ from this proof.

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Cells and DevelopmentMiR-382-5p controls hematopoietic stem cell differentiation through the downregulation of MXD1 (doi: 10.1089/scd.2016.0150)This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction.The final published version may differ from this proof.
Figure 4A, all mRNAs were downregulated in miR-382-5p-overexpressing cells at 24 ).At the same time, MXD1 silencing induces a significant increase in the percentage of CD66b+ and CD15+ cells compared with the ss-NegCTR sample (Figure 5E-F).Furthermore, the morphological analysis of MGG-stained cytospins showed a significant reduction in MK precursors (Figure 5H, Panels i-iv) and a significant expansion of granulocytes in MXD1-siRNA cells (Figure 5I, Panels i-iv).The same morphological differences Page 16 of 42 Stem Cells and Development MiR-382-5p controls hematopoietic stem cell differentiation through the downregulation of MXD1 (doi: 10.1089/scd.2016.0150)Thisarticle has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction.The final published version may differ from this proof.

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Cells and DevelopmentMiR-382-5p controls hematopoietic stem cell differentiation through the downregulation of MXD1 (doi: 10.1089/scd.2016.0150)This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction.The final published version may differ from this proof.

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Cells and DevelopmentMiR-382-5p controls hematopoietic stem cell differentiation through the downregulation of MXD1 (doi: 10.1089/scd.2016.0150)This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction.The final published version may differ from this proof.
and mouse (29) erithroleukemia cells.Here we showed that MXD1 silencing did not impair erythroid differentiation in normal CD34+ cells, as demonstrated by the expression level of GPA (Figure S3, Panel C-D) and the percentage of red colonies (Figure 6A), which did not change after MXD1 knockdown.A possible explanation for this different cellular behaviour is that normal CD34+ cells might express some proteins that could replace MXD1 during erythroid differentiation.Furthermore, our data demonstrated that MXD1 plays an important role in granulocyte differentiation; in fact, the MXD1 knockdown enhances the fraction of granulocyte precursors both in liquid and semisolid cultures (Figure 5 and 6).In agreement with our data, Foley et al. demonstrated that MXD1 knockout in mice affects the granulocyte lineage by reducing cell cycle withdrawal during the late stage Page 19 of 42 Stem Cells and Development MiR-382-5p controls hematopoietic stem cell differentiation through the downregulation of MXD1 (doi: 10.1089/scd.2016.0150)This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction.The final published version may differ from this proof.

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FIGURE LEGENDS

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Cells and DevelopmentMiR-382-5p controls hematopoietic stem cell differentiation through the downregulation of MXD1 (doi: 10.1089/scd.2016.0150)This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction.The final published version may differ from this proof.

Figure S3 .
Figure S3.Effect of MXD1 silencing in CD34+ HSPC on cell viability and -5p controls hematopoietic stem cell differentiation through the downregulation of MXD1 (doi: 10.1089/scd.2016.0150)This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction.The final published version may differ from this proof.Development MiR-382-5p controls hematopoietic stem cell differentiation through the downregulation of MXD1 (doi: 10.1089/scd.2016.0150)been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction.The final published version may differ fr -5p controls hematopoietic stem cell differentiation through the downregulation of MXD1 (doi: 10.1089/scd.2016.0150) . Moreover, another mechanism by which MXD1 inhibition could favour the expansion of granulocyte fraction is mediated by the ACKNOWLEDGEMENTThis work was supported by Associazione Italiana per la Ricerca sul Cancro (AIRC), project number #10005 "Special Program Molecular Clinical Oncology 5x1000" to