Embed Size (px)
Citation preview
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/yexcr
E X P E R I M E N T A L C E L L R E S E A R C H ] ( ] ] ] ] ) ] ] ] – ] ] ]
http://dx.doi.org/10.10014-4827/& 2014 E
nCorresponding auE-mail addresses
[email protected]@suda.edu.cn
1 These authors c
Please cite this ar(2014), http://dx.d
Research Article
miR-34a expands myeloid-derived suppressor cellsvia apoptosis inhibition
Anfei Huanga,1, Haitao Zhangb,1, Si Chena, Fei Xiaa, Yi Yanga, Fulu Donga, Di Suna,Sidong Xionga, Jinping Zhanga,n
aInstitutes of Biology and Medical Sciences, Soochow University, Suzhou 215123, Jiangsu Province, People's Republic of ChinabDepartment of General Surgery, The First Affiliated Hospital of Soochow University, Suzhou 215021, Jiangsu Province, People's Republic of China
a r t i c l e i n f o r m a t i o n
Article Chronology:
Received 19 January 2014Received in revised form21 March 2014Accepted 16 April 2014
Keywords:
Myeloid-derived suppressor cellsmiR-34aExpansionApoptosis
016/j.yexcr.2014.04.010lsevier Inc. All rights reser
thor.: [email protected] (A(F. Xia), [email protected](S. Xiong), j_pzhang@sudontributed equally.
ticle as: A. Huang, et al.,oi.org/10.1016/j.yexcr.201
a b s t r a c t
Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population and show significant
expansion under pathological conditions. microRNA plays important roles in many biological processes,whether microRNAs have a function in the expansion of MDSCs is still not very clear. In this study,miR-34a overexpression can induce the expansion of MDSCs in bone marrow chimera and transgenicmice model. The experimental results suggest that miR-34a inhibited the apoptosis of MDSCs but didnot affect the proliferation of MDSCs. The distinct mRNAmicroarray profiles of MDSCs of wild type andmiR-34a over-expressing MDSCs combined with the target prediction of miR-34a suggest that miR-34amay target genes such as p2rx7, Tia1, and plekhf1 to inhibit the apoptosis of MDSCs. Taken together,miR-34a contributes to the expansion of MDSCs by inhibiting the apoptosis of MDSCs.
& 2014 Elsevier Inc. All rights reserved.
Introduction
Myeloid-derived suppressor cells (MDSC) are a heterogeneouspopulation of immature myeloid cells that include precursors ofmonocytes, granulocytes, and dendritic cells. They are generatedunder nearly all acute and chronic inflammation conditions suchas tumor, auto-immune disease, sepsis, parasite infection, and soon. Under these pathophysiological conditions, immature myeloidcells are arrested at different stages of maturation. Moreover,these differentiation-terminated myeloid cells with suppressiveactivity accumulate in the BM or spleen.
In recent decades, much effort has been given to explain howMDSCs differentiate and expand. Granulocyte-macrophage-colony-
ved.
. Huang), [email protected] (Y. Yang), [email protected] (J. Zhang).
miR-34a expands myeloi4.04.010
stimulating factor (GM-CSF) functions at earlier stages of thislineage commitment in steady state by regulating the expansionand maturation of early hematopoietic progenitors [1]. Highconcentrations of GM-CSF produced by activated T cells [2],NK-cells [3], and DC [4] during immune responses may lead tothe expansion, redistribution, and gain-of-function of Gr-1þCD11bþ
cells. Studies have demonstrated that GM-CSF functions differentlyon the two subsets of MDSCs, namely, the CD11bþGr-1int andCD11bþGr-1low population. In tumor-bearing mice, GM-CSFexpands both subsets in spleen, whereas it only expands theCD11bþGr-1low subset in the bone marrow [5]. Prolonged CpGoligodeoxynucleotide (CpG ODN) stimulation can inhibit the devel-opment of DCs and induce differentiation from BM precursor cells
om (H. Zhang), [email protected] (S. Chen),6.com (F. Dong), [email protected] (D. Sun),
d-derived suppressor cells via apoptosis inhibition, Exp Cell Res
Table 1 – Primers used to detect the expression of particulargenes via real-time PCR.
E X P E R I M E N T A L C E L L R E S E A R C H ] ( ] ] ] ] ) ] ] ] – ] ] ]2
to MDSCs [6]. Stimulation by monophosphoryl lipid A (MPL)derived from Lipopolysaccharides (LPS) at the beginning of celldifferentiation disturbs the development of DCs and leads to theaccumulation of MDSCs [7].The accumulation of MDSCs in cancer is due to changes not
only in differentiation and expansion but also in apoptosis. ABT-737, which is an inhibitor of Bcl-2, Bcl-xL, and Bcl-w, therapeu-tically increases the spontaneous apoptosis of MDSCs and thusdecreases the accumulation of MDSCs in tumor-bearing mice.Cimetidine, a histamine type-2 receptor antagonist, can induceFas and FasL expression in MDSCs and sequentially regulate thecaspase-dependent apoptosis pathway, thereby inhibiting tumorgrowth by modulating MDSC apoptosis instead of MDSC differ-entiation [8].miRNAs are small, single-stranded, non-coding RNAs of
approximately 22 nucleotides. Ample evidence have suggestedthat they have pivotal functions in many biological processesincluding myeloid cell biology [9–11] and myeloproliferativedisorders by affecting the differentiation and accumulation ofprogenitors and immature cells [10,12–14]. miR-29a, miR-21, andmiR-196b are involved in myeloid progenitor expansion [15,16].miR-223 promotes granulopoiesis [17–19]. miR-146a deficiency inmice induces chronic myeloproliferative disorders [11], whereasmiR-17-5p, miR-20a, and miR-106a enhance blast proliferationand inhibit monocytic differentiation and maturation of humanCD34þ hematopoietic progenitor cells [20]. Given that miRNAscan regulate myeloid differentiation and maturation processes;aberrant expressions of miRNAs may possibly contribute to theexpansion of MDSCs under pathophysiological conditions. How-ever, the mechanism by which microRNAs affect the accumulationor expansion of MDSCs has not been studied thus far.In this study, miR-34a overexpression can increase the number
of Gr1þCD11bþ MDSCs both in the BM and spleen by establishingthe miR-34a over-expressing chimera and transgenic mice mod-els. In addition, our results demonstrated that miR-34a affects theapoptosis but not proliferation of MDSCs. Based on miRwalksoftware and gene expression profile of wild type and miR-34a-over-expressing MDSCs, we proposed purinergic receptor p2x,ligand-gated ion channel, 7 (p2rx7), T-cell-restricted intracellularantigen-1 (Tia1), and pleckstrin homology domain-containingprotein, family F, member 1 (plekhf1) as the potential targets ofmiR-34a that may be involved in the apoptosis of MDSCs.
Gene Sequence
Cadm3-F 50-TGGTGGCACAGTGGTTCTCAA-30
Cadm3-R 50-TCATCGGCCAGCGCCACATT-30
Creb1-F 50-ACCACTGTAACAGTGCCAACC-30
Creb1-R 50-TGCATACTGTAGAATGGTAGTAC-30
Dkk2-F 50-GGGAGACTCCTGCTCAGTCA-30
Dkk2-R 50-TGCAGGCTGATGATCCTTGGT-30
Ednrb-F 50-GTAAGCTGGTGCCCTTCATAC-30
Ednrb-R 50-AAAACCTATGGCTTCGGGGAC-30
Hoxb4-F 50-TGGATGCGCAAAGTTCACGTG-30
Hoxb4-R 50-TTGATCTGGCGCTCGGACAG-30
Iqcc-F 50-TCAGCACTCTTCAGTGGACTGG-30
Iqcc-R 50-GGCTCTTTTCTGTCTTCTTCTG-30
Jrk-F 50-TGGAGTGTATGGGTTAAGCAAC-30
Jrk-R 50-AGGAGAGCTACCCTGGACTG-30
Klhdc5-F 50-GCGAGTGCAAGGGGAAGATTT-30
Klhdc5-R 50-GACACCCACCCACGATGTAG-30
Kctd12b-F 50-AACTACTTTGATTTCTTGGATCTC-30
Kctd12b-R 50-AACCAGGAATGCTGATCAAAGTA-30
Materials and methods
Mice
Six- to eight-week-old C57/BL6 mice were purchased from ShanghaiSLAC Laboratory Animal Co. The miR-34a transgenic mice used in thisstudy were bred by Cyagen Bioscience, Inc., as described below. Allmice were maintained in a barrier facility at Soochow University. Allanimal experiments were approved by the Institutional Animal Careand Use Committee of Soochow University.
Antibodies
The following antibodies were purchased from Biolegend Inc.:anti-CD11b-pacific blue (M1/70), anti-Gr-1-PE-cy7 (RB6-8C5),Annexin V-FITC, and anti-Brdu-APC (BU20A).
Please cite this article as: A. Huang, et al., miR-34a expands myeloi(2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.010
Cell staining and flow cytometry
Mouse BM and spleen cells were stained with anti-CD11b andanti-Gr-1 antibodies to detect the percentage of MDSCs. Forapoptosis assay, the spleen cells were first stained with anti-CD11b, anti-Gr-1 antibodies and then stained with annexin V inan annexin V binding buffer. For the BrdU incorporation assay,each mouse was injected with 1 mg of BrdU through i.p. at 12 and4 h before they were sacrificed. The spleen cells were harvestedand processed according to the manufacturer's protocol by usinganti-BrdU-APC antibody and antibodies against surface markersincluding anti-CD11b and anti-Gr-1. After staining, the cells wereanalyzed using BD FACS AriaIII or BD FACS CantoII. FACS data wereanalyzed using the FlowJo software (Tree Star, Inc.).
Real time PCR assay
Total RNAs were extracted from sorted MDSCs or 293T cells byusing RNAiso Plus reagent (TAKARA Biotechnology Co. LTD,DALIAN). To detect miR-34a expression, the total RNAs werereversed using an MMLV reverse transcriptase with miR-34aspecific RT primer 50-CTCA ACTG GTGT CGTG GAGT CGGC AATTCA GTT GAG ACA ACC AG-30. The resulting cDNAs were then usedas templates to perform real-time PCR with specific PCR primers:F: 50-ACA CTC CAG CTG GG TGG CAG TGT CTT AGC T-30, R: 50-CTCAAC TGG TGT CGT GGA-30 by using a Roche real-time PCR kit. Todetect the expression of other genes, the total RNAs were reversedusing an MMLV reverse transcriptase with Oligo(dT). The tran-scripts were quantified via real-time quantitative polymerasechain reaction (RT-PCR) and normalized to the amount ofexpressed GAPDH mRNA. The primers for the confirmation ofmicroarray results are presented in Table 1.
Microarray analysis
Spleen cells from WT or miR-34a TG mouse were stained withanti-Gr1 and anti-CD11b antibodies, and the Gr1þCD11bþ cells
d-derived suppressor cells via apoptosis inhibition, Exp Cell Res
E X P E R I M E N T A L C E L L R E S E A R C H ] ( ] ] ] ] ) ] ] ] – ] ] ] 3
were then sorted using FACS AriaIII. The total RNAs of these sortedMDSCs were extracted using a TRIzol reagent (Invitrogen) accord-ing to the manufacturer's description and quantified usingNanoDrop ND-1000. An AgilentSurePrint G3 Mouse GE 8�60KMicroarray Kit was used to detect the level of mRNA expression.The slides were scanned using an Affymetrix GeneChip Scanner3000 (Affymetrix, USA), and mRNA data were analyzed using themRNA QC Tool (Affymetrix).
Statistical analysis
Statistical analysis was performed for all experiments by usingStudent's t-test. P values less than 0.05 were considered statisti-cally significant.
Fig. 1 – Overexpression of miR-34a increases the percentage of Gr1þ
in 293T cells transfected with (miR-34a) or without (control samplThe expression levels were normalized by GAPDH expression. (B) Exwithout (control sample) pMDH-PGK-GFP-miR-34a plasmid detectemiR-34a chimera mice; GFPþ represents miR-34a-over-expressed BMof Gr1highCD11bþ and Gr1lowCD11bþ MDSCs in the BM (D, E) and spover-expressed cells, and GFP- represents normal cells. * means pomore than three repeats.
Please cite this article as: A. Huang, et al., miR-34a expands myeloi(2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.010
Results
miR-34a overexpression increases the percentageof MDSCs in chimeric mice
To explore the effects of miR-34a on the development of immunecells, we first constructed a miR-34a over-expressing vectornamed pMDH-PGK-GFP-miR-34a. After we confirmed the properprocessing and expression of miR-34a by northern blot andquantitative RT-PCR (Fig. 1A and B), we then established a bonemarrow chimeric mice model by transferring bone marrow cellsexpressing miR-34a into irradiated recipient mice. Based on theFACS analysis results, miR-34a overexpression in mouse hemato-poietic stem cells can reduce the percentage of B cells in BM,
CD11bþ MDSCs in BM chimera mice. (A) Expression of miR-34ae) pMDH-PGK-GFP-miR-34a plasmid detected via real time PCR.pression of miR-34a in 293T cells transfected with (miR-34a) ord via northern blot. (C) Percentage of B220þ cells in the BM ofcells, and GFP- represents normal BM cells. (D–G) Percentages
leen (F, G) of miR-34a chimera mice; GFPþ represents miR-34a-0.05, ** means po0.01, and *** means po0.001. Data represent
d-derived suppressor cells via apoptosis inhibition, Exp Cell Res
E X P E R I M E N T A L C E L L R E S E A R C H ] ( ] ] ] ] ) ] ] ] – ] ] ]4
which is consistent with the results of Baltimore's study [21](Fig. 1C). Moreover, the percentages of MDSCs in the BM (Fig. 1Dand E) and in the spleen (Fig. 1F and G) increased in thechimera model.
The percentage and absolute number of MDSCs areincreased in miR-34a transgenic mice
To further determine the effect of miR-34a on the development ofMDSCs, we established an miR-34a transgenic mice model. Thegenotyping PCR results indicate that the miR-34a-coding genewas successfully inserted into the mice genome (data not shown).The results from real-time PCR with miR-34a specific primersshow that mature miR-34a can be highly expressed in miR-34a TGmice (Fig. 2A). The results from FACS analysis also demonstratethat the amount of B cells in miR-34a TG mice BM decreasedcompared with that in WT mice (Fig. 2B), which suggests that themiR-34a TG mice model was successfully established. To confirmthe effect of miR-34a on the development of MDSCs in miR-34aTG mice, a single spleen cell from miR-34a TG mice was preparedand stained with anti-Gr1 and anti-CD11b antibodies. Flow
Fig. 2 – Phenotype screen of miR-34a transgenic mice. (A) Expressi(TG). (B) Percentage of B220þ cells in miR-34a TG mice. (C–E) Perctransgenic mice. (F) Percentages of CD4þ and CD8þ T cells in the sp** means po0.01, and *** means po0.001. Data represent more th
Please cite this article as: A. Huang, et al., miR-34a expands myeloi(2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.010
cytometry analysis results indicate that the percentage of MDSCsfrom the spleen of miR-34a TG mice was remarkably up-regulatedcompared with that of wild-type mice (Fig. 2C and D). Theabsolute number of MDSCs also increased in the spleen of miR-34aTGmice (Fig. 2E). This phenotype was similar to that in miR-34a over-expressing chimera mice. However, the percentages of CD4þ andCD8þ T cells were not affected in the spleen and thymus of miR-34aTG mice (Fig. 2F). Collectively, the phenotypes of miR-34a TG micewere consistent with those of miR-34a chimera mice.
miR-34a inhibits the apoptosis of MDSCs
The increase in the absolute and relative numbers of MDSCs maybe due to changes in apoptotic or proliferative activity or evenself-renewal. As such, we tried to distinguish these possibilities.First, we stained MDSCs from wild-type and transgenic mice withAnnexin V (Fig. 3A and B). The flow cytometry results indicatethat miR-34a significantly reduced the apoptotic activity ofMDSCs. Furthermore, BrdU cooperation assay was performed toevaluate the proliferation or self-renewal status of MDSCs. Flowcytometry analysis results demonstrated that the percentage of
on of miR-34a in wild-type (WT) and miR-34a transgenic miceentage of Gr1highCD11bþ and Gr1lowCD11bþ MDSCs in miR-34aleen and thymus of miR-34a transgenic mice. * means po0.05,an five repeats.
d-derived suppressor cells via apoptosis inhibition, Exp Cell Res
Fig. 3 – Apoptosis and proliferation of MDSCs in miR-34a transgenic mice. (A, B) Percentages of Annexin V in Gr1highCD11bþ andGr1lowCD11bþ MDSCs in wild-type (WT) and miR-34a transgenic mice (TG). (C, D) Proliferation of Gr1highCD11bþ and Gr1lowCD11bþ
MDSCs in wild-type (WT) and miR-34a transgenic mice (TG) detected via BrdU cooperation assay. * means po0.05, ** meanspo0.01. Data represent more than three repeats.
E X P E R I M E N T A L C E L L R E S E A R C H ] ( ] ] ] ] ) ] ] ] – ] ] ] 5
BrdU-positive MDSCs was similar in wild-type and miR-34a over-expressing mice (Fig. 3C and D), which indicate that miR-34a didnot affect the proliferation or self-renewal of MDSCs. Takentogether, these results suggest that miR-34a increased the num-ber of MDSCs by impairing the balance of apoptosis but not byaltering the proliferation of MDSCs.
The target prediction of miR-34a in MDSCs
To further explore how miR-34a inhibits the apoptosis of MDSCs,we first purified the MDSCs from the spleen of miR-34a TG andwild-type mice. We then performed the mRNA assay to screengenes with different expressions (Fig. 4A), some of which wereconfirmed via real-time PCR (Fig. 4B). We selected the down-regulated genes in miR-34a MDSCs and compared them withthose in wild-type MDSCs because microRNA can bind to mRNAand affect the stability of mRNA. Meanwhile, we predicted thetarget genes of miR-34a by using the miRwalk software. After
Please cite this article as: A. Huang, et al., miR-34a expands myeloi(2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.010
overlapping the above two cluster genes, we selected 9 genes(Table 2). Furthermore, according to the descriptions of thefunction of genes in OMIM and based on the results of severalstudies, we learned that plekhf1, p2Rx7, and Tia1 have pivotalfunctions in apoptosis processes. Thus, we deduced that miR-34amay inhibit the apoptosis of MDSCs through these three genes.However, more experiments should be conducted to confirm thefunctional targets of miR-34a on MDSCs.
Discussion
Hematopoietic stem cells (HSCs) are widely believed to differ-entiate into common myeloid progenitor (CMP) cells and theninto immature myeloid cells (IMCs). Physiologically, these IMCsmigrate to peripheral organs and proceed to differentiate intodendritic cells, macrophages, or granulocytes. However, thecytokines produced in a tumor or infection microenvironment
d-derived suppressor cells via apoptosis inhibition, Exp Cell Res
Fig. 4 – mRNA expression detected using an Agilent mRNA microarray and confirmed via real-time PCR. (A) Selected mRNAexpression profile in MDSCs frommiR-34a transgenic mice and fromwild-type mice. Data represent two repeats. (B) Expressions ofparticular genes as confirmed via real-time PCR. Data represent more than three repeats.
Table 2 – Predicted target genes of miR-34a in MDSCs.
Genes Expression ratio (WT/miR-34a)
Tns4 2.76Plekhf1 2.83P2rx7 2.96Rarb 3.16Igf1 3.21Tia1 3.27Ank2 3.51Tgfb2 4.40Gjb6 42.65
E X P E R I M E N T A L C E L L R E S E A R C H ] ( ] ] ] ] ) ] ] ] – ] ] ]6
cause the IMCs to accumulate in these sites, but their maturationinto DCs, macrophages, or granulocytes becomes impaired. Theseaccumulated cells then induce the accumulation or expansion ofregulatory T cells to repress immune response.Recently, studies have shown several factors that contribute
to the expansion of MDSCs, such as cyclooxygenase-2 (COX2)[22–24], prostaglandins [25–27], stem-cell factor (SCF) [26],macrophage colony-stimulating factor (M-CSF), IL-6 [25], granu-locyte/macrophage colony-stimulating factor (GM-CSF) [27], andvascular endothelial growth factor (VEGF) [28]. Most of thesefactors affect the expansion of MDSCs via signal transducer andactivator of transcriptions (STATs) signaling [29–31], which repre-sents key signals that are involved in cell proliferation, survival,apoptosis [32], and differentiation. Collectively, abnormal STATsprevent the differentiation of IMC into mature myeloid cells andthereby induce the expansion of MDSCs.Apoptosic inhibition is one of the important reasons for the
accumulation of MDSCs. Recently, researchers have demonstratedthat FasLþ-activated T cells directly affect the apoptosis of MDSCsvia Fas-STAT6 signaling [33]. Moreover, IL-4-mediated apoptosisin MDSCs involves the IL-4R-STAT6 signaling pathway [34].
Please cite this article as: A. Huang, et al., miR-34a expands myeloi(2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.010
A study has also demonstrated that miR-34a has vital functionsin cell apoptosis. The in vitro overexpression of miR-34a inducesapoptosis in B-CLL (chronic lymphocytic leukemia) cells and inthe MEC1 cell line in a p53-dependent manner [35]. Activatingthe p53/miR-34a/SIRT1 tumor suppressor network induces theapoptosis of CLL cells [36]. Moreover, miR-34a directly targetsProtein phosphatase 1 nuclear targeting subunit (PNUTS) toreduce cardiomyocytes apoptosis [37]. The transcription factorMYCN, which is also a target of miR-34a, regulates cell apoptosis [38].Furthermore, the suppression of SIRT1 by miR-34a ultimately leads toapoptosis in human colon cancer cells but not in human colon cancercells that lack p53 [39]. These results indicate that miR-34a isimportant in regulating cell apoptosis although it has paradoxicalfunctions in different cells.
This research demonstrates that miR-34a overexpression caninhibit the apoptosis of MDSCs, which results in the accumulationof MDSCs in the spleen and BM. This result may be due to thedown-regulation of the translation and stability of the mRNA ofplekhf1, p2Rx7, and Tia1. P2rx7 is similar to the platelet P2Treceptor, and this similarity may be caused by the combination ofP2Y and P2X-dependent responses [40,41]. The P2X7 receptorcontributes to the apoptosis of lymphocytes in individuals withCLL, which results in an increased number of B-cells [42]. Cao'sand colleagues have demonstrated that plekhf1 (LAPF) recruitsphosphorylated p53 to lysosomes and triggers lysosomal desta-bilization in apoptosis in L929 cells [43]. Tian et al. (1991) havedemonstrated that TIA1 is a nucleic acid-binding protein thatpreferentially recognizes poly(A) homopolymers and induces DNAfragmentation in permeabilized thymocytes [44]. Their studiessuggested that TIA1 may be involved in the induction of apoptosisin CTL targets. However, whether these genes are also involved inthe apoptosis in MDSC cells is not clear. More experimentalevidence is needed to verify this issue further.
d-derived suppressor cells via apoptosis inhibition, Exp Cell Res
E X P E R I M E N T A L C E L L R E S E A R C H ] ( ] ] ] ] ) ] ] ] – ] ] ] 7
Acknowledgments
This work is supported by grants from the Priority AcademicDevelopment Program of Jiangsu Higher Education Institutions(PAPD), Program for Changjiang Scholars and Innovative ResearchTeam in University (PCSIRT) (IRT1075), Jiangsu Key Laboratory ofInfection and Immunity, Institutes of Biology and Medical Sciences ofSoochow University, and intramural research funding to JinpingZhang (Q413401810, Q313406711) from Soochow University, NationalNature Science Foundation of China to Jinping Zhang (31270939),Fei Xia (31200660), and Fulu Dong (81300553), Natural ScienceFoundation of Jiangsu Province to Jinping Zhang (BK2012617), andKey University Science Research Project of Jiangsu Province to JinpingZhang (13KJA310004).
r e f e r e n c e s
[1] D.R. Barreda, P.C. Hanington, M. Belosevic, Regulation of myeloiddevelopment and function by colony stimulating factors, Dev.Comp. Immunol. 28 (2004) 509–554.
[2] A.O. Abdalla, S. Kiaii, L. Hansson, E.D. Rossmann, M. Jeddi-Tehrani,F. Shokri, A. Osterborg, H. Mellstedt, H. Rabbani, Kinetics ofcytokine gene expression in human CD4þ and CD8þ T-lymphocyte subsets using quantitative real-time PCR, Scand. J.Immunol. 58 (2003) 601–606.
[3] M.A. Morris, K. Ley, Trafficking of natural killer cells, Curr. Mol.Med. 4 (2004) 431–438.
[4] B. de Saint-Vis, I. Fugier-Vivier, C. Massacrier, C. Gaillard, B.Vanbervliet, S. Ait-Yahia, J. Banchereau, Y.J. Liu, S. Lebecque, C.Caux, The cytokine profile expressed by human dendritic cells isdependent on cell subtype and mode of activation, J. Immunol.160 (1998) 1666–1676.
[5] L. Dolcetti, E. Peranzoni, S. Ugel, I. Marigo, A. Fernandez Gomez,C. Mesa, M. Geilich, G. Winkels, E. Traggiai, A. Casati, F. Grassi, V.Bronte, Hierarchy of immunosuppressive strength amongmyeloid-derived suppressor cell subsets is determined by GM-CSF, Eur. J. Immunol. 40 (2010) 22–35.
[6] J. Chen, C. Deng, Q. Shi, J. Jiang, Y. Zhang, W. Shan, W. Sun, CpGoligodeoxynucleotide induces bone marrow precursor cells intomyeloid-derived suppressor cells, Mol. Med. Rep. 8 (2013) 1149–1154.
[7] J. Chen, B. Sun, X. Zhao, D. Liang, J. Liu, Y. Huang, W. Lei, M. Chen,W. Sun, Monophosphoryl lipid A induces bone marrow precursorcells to differentiate into myeloid-derived suppressor cells, Mol.Med. Rep. 8 (2013) 1074–1078.
[8] Y. Zheng, M. Xu, X. Li, J. Jia, K. Fan, G. Lai, Cimetidine suppresseslung tumor growth in mice through proapoptosis of myeloid-derived suppressor cells, Mol. Immunol. 54 (2013) 74–83.
[9] M.P. Boldin, K.D. Taganov, D.S. Rao, L. Yang, J.L. Zhao, M. Kalwani,Y. Garcia-Flores, M. Luong, A. Devrekanli, J. Xu, G. Sun, J. Tay, P.S.Linsley, D. Baltimore, miR-146a is a significant brake on auto-immunity, myeloproliferation, and cancer in mice, J. Exp. Med.208 (2011) 1189–1201.
[10] R.M. O’Connell, J.L. Zhao, D.S. Rao, MicroRNA function in myeloidbiology, Blood 118 (2011) 2960–2969.
[11] J.L. Zhao, D.S. Rao, M.P. Boldin, K.D. Taganov, R.M. O’Connell, D.Baltimore, NF-kappaB dysregulation in microRNA-146a-deficientmice drives the development of myeloid malignancies, Proc. Natl.Acad. Sci. U.S.A. 108 (2011) 9184–9189.
[12] D. Baltimore, M.P. Boldin, R.M. O’Connell, D.S. Rao, K.D. Taganov,MicroRNAs: new regulators of immune cell development andfunction, Nat. Immunol. 9 (2008) 839–845.
Please cite this article as: A. Huang, et al., miR-34a expands myeloi(2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.010
[13] G. Marcucci, K. Mrozek, M.D. Radmacher, R. Garzon, C.D.Bloomfield, The prognostic and functional role of microRNAs inacute myeloid leukemia, Blood 117 (2011) 1121–1129.
[14] E. Pelosi, C. Labbaye, U. Testa, MicroRNAs in normal andmalignant myelopoiesis, Leuk. Res. 33 (2009) 1584–1593.
[15] Y.C. Han, C.Y. Park, G. Bhagat, J. Zhang, Y. Wang, J.B. Fan, M. Liu, Y.Zou, I.L. Weissman, H. Gu, microRNA-29a induces aberrant self-renewal capacity in hematopoietic progenitors, biased myeloiddevelopment, and acute myeloid leukemia, J. Exp. Med. 207(2010) 475–489.
[16] C.S. Velu, A.M. Baktula, H.L. Grimes, Gfi1 regulates miR-21 andmiR-196b to control myelopoiesis, Blood 113 (2009) 4720–4728.
[17] F. Fazi, S. Racanicchi, G. Zardo, L.M. Starnes, M. Mancini, L.Travaglini, D. Diverio, E. Ammatuna, G. Cimino, F. Lo-Coco, F.Grignani, C. Nervi, Epigenetic silencing of the myelopoiesisregulator microRNA-223 by the AML1/ETO oncoprotein, CancerCell 12 (2007) 457–466.
[18] F. Fazi, A. Rosa, A. Fatica, V. Gelmetti, M.L. De Marchis, C. Nervi, I.Bozzoni, A minicircuitry comprised of microRNA-223 and tran-scription factors NFI-A and C/EBPalpha regulates human granu-lopoiesis, Cell 123 (2005) 819–831.
[19] J.B. Johnnidis, M.H. Harris, R.T. Wheeler, S. Stehling-Sun, M.H.Lam, O. Kirak, T.R. Brummelkamp, M.D. Fleming, F.D. Camargo,Regulation of progenitor cell proliferation and granulocytefunction by microRNA-223, Nature 451 (2008) 1125–1129.
[20] L. Fontana, E. Pelosi, P. Greco, S. Racanicchi, U. Testa, F. Liuzzi, C.M. Croce, E. Brunetti, F. Grignani, C. Peschle, MicroRNAs 17-5p-20a-106a control monocytopoiesis through AML1 targeting andM-CSF receptor upregulation, Nat. Cell. Biol. 9 (2007) 775–787.
[21] D.S. Rao, R.M. O’Connell, A.A. Chaudhuri, Y. Garcia-Flores, T.L.Geiger, D. Baltimore, MicroRNA-34a perturbs B lymphocytedevelopment by repressing the forkhead box transcription factorFoxp1, Immunity 33 (2010) 48–59.
[22] M. Fujita, G. Kohanbash, W. Fellows-Mayle, R.L. Hamilton, Y.Komohara, S.A. Decker, J.R. Ohlfest, H. Okada, COX-2 blockadesuppresses gliomagenesis by inhibiting myeloid-derived sup-pressor cells, Cancer Res. 71 (2011) 2664–2674.
[23] N. Obermajer, R. Muthuswamy, J. Lesnock, R.P. Edwards, P.Kalinski, Positive feedback between PGE2 and COX2 redirects thedifferentiation of human dendritic cells toward stable myeloid-derived suppressor cells, Blood 118 (2011) 5498–5505.
[24] J.D. Veltman, M.E. Lambers, M. van Nimwegen, R.W. Hendriks, H.C. Hoogsteden, J.G. Aerts, J.P. Hegmans, COX-2 inhibitionimproves immunotherapy and is associated with decreasednumbers of myeloid-derived suppressor cells in mesothelioma.Celecoxib influences MDSC function, BMC Cancer 10 (2010) 464.
[25] S.K. Bunt, L. Yang, P. Sinha, V.K. Clements, J. Leips, S. Ostrand-Rosenberg, Reduced inflammation in the tumor microenviron-ment delays the accumulation of myeloid-derived suppressorcells and limits tumor progression, Cancer Res. 67 (2007) 10019–10026.
[26] P.Y. Pan, G.X. Wang, B. Yin, J. Ozao, T. Ku, C.M. Divino, S.H. Chen,Reversion of immune tolerance in advanced malignancy: mod-ulation of myeloid-derived suppressor cell development byblockade of stem-cell factor function, Blood 111 (2008) 219–228.
[27] P. Serafini, R. Carbley, K.A. Noonan, G. Tan, V. Bronte, I. Borrello,High-dose granulocyte-macrophage colony-stimulating factor-producing vaccines impair the immune response through therecruitment of myeloid suppressor cells, Cancer Res. 64 (2004)6337–6343.
[28] D. Gabrilovich, T. Ishida, T. Oyama, S. Ran, V. Kravtsov, S. Nadaf, D.P. Carbone, Vascular endothelial growth factor inhibits thedevelopment of dendritic cells and dramatically affects thedifferentiation of multiple hematopoietic lineages in vivo, Blood92 (1998) 4150–4166.
[29] M. Kortylewski, M. Kujawski, T. Wang, S. Wei, S. Zhang, S. Pilon-Thomas, G. Niu, H. Kay, J. Mule, W.G. Kerr, R. Jove, D. Pardoll, H.Yu, Inhibiting Stat3 signaling in the hematopoietic system elicits
d-derived suppressor cells via apoptosis inhibition, Exp Cell Res
E X P E R I M E N T A L C E L L R E S E A R C H ] ( ] ] ] ] ) ] ] ] – ] ] ]8
multicomponent antitumor immunity, Nat. Med. 11 (2005)1314–1321.
[30] Y. Nefedova, M. Huang, S. Kusmartsev, R. Bhattacharya, P. Cheng,R. Salup, R. Jove, D. Gabrilovich, Hyperactivation of STAT3 isinvolved in abnormal differentiation of dendritic cells in cancer,J. Immunol. 172 (2004) 464–474.
[31] Y. Nefedova, S. Nagaraj, A. Rosenbauer, C. Muro-Cacho, S.M. Sebti,D.I. Gabrilovich, Regulation of dendritic cell differentiation andantitumor immune response in cancer by pharmacologic-selective inhibition of the janus-activated kinase 2/signal trans-ducers and activators of transcription 3 pathway, Cancer Res. 65(2005) 9525–9535.
[32] J. Bromberg, Stat proteins and oncogenesis, J. Clin. Invest. 109(2002) 1139–1142.
[33] P. Sinha, O. Chornoguz, V.K. Clements, K.A. Artemenko, R.A.Zubarev, S. Ostrand-Rosenberg, Myeloid-derived suppressor cellsexpress the death receptor Fas and apoptose in response to Tcell-expressed FasL, Blood 117 (2011) 5381–5390.
[34] F. Roth, A.C. De La Fuente, J.L. Vella, A. Zoso, L. Inverardi, P.Serafini, Aptamer-mediated blockade of IL4Ralpha triggersapoptosis of MDSCs and limits tumor progression, Cancer Res. 72(2012) 1373–1383.
[35] D. Asslaber, J.D. Pinon, I. Seyfried, P. Desch, M. Stocher, I.Tinhofer, A. Egle, O. Merkel, R. Greil, microRNA-34a expressioncorrelates with MDM2 SNP309 polymorphism and treatment-free survival in chronic lymphocytic leukemia, Blood 115 (2010)4191–4197.
[36] V. Audrito, T. Vaisitti, D. Rossi, D. Gottardi, G. D’Arena, L. Laurenti,G. Gaidano, F. Malavasi, S. Deaglio, Nicotinamide blocks prolif-eration and induces apoptosis of chronic lymphocytic leukemiacells through activation of the p53/miR-34a/SIRT1 tumor sup-pressor network, Cancer Res. 71 (2011) 4473–4483.
Please cite this article as: A. Huang, et al., miR-34a expands myeloi(2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.010
[37] R.A. Boon, K. Iekushi, S. Lechner, T. Seeger, A. Fischer, S. Heydt, D.Kaluza, K. Treguer, G. Carmona, A. Bonauer, A.J. Horrevoets, N.Didier, Z. Girmatsion, P. Biliczki, J.R. Ehrlich, H.A. Katus, O.J.Muller, M. Potente, A.M. Zeiher, H. Hermeking, S. Dimmeler,MicroRNA-34a regulates cardiac ageing and function, Nature 495(2013) 107–110.
[38] E. Sotillo, T. Laver, H. Mellert, J.M. Schelter, M.A. Cleary, S.McMahon, A. Thomas-Tikhonenko, Myc overexpression bringsout unexpected antiapoptotic effects of miR-34a, Oncogene 30(2011) 2587–2594.
[39] M. Yamakuchi, M. Ferlito, C.J. Lowenstein, miR-34a repression ofSIRT1 regulates apoptosis, Proc. Natl. Acad. Sci. U.S.A. 105 (2008)13421–13426.
[40] J. Jin, V.R. Dasari, F.D. Sistare, S.P. Kunapuli, Distribution of P2Yreceptor subtypes on haematopoietic cells, Br. J. Pharmacol. 123(1998) 789–794.
[41] V. Ralevic, G. Burnstock, Receptors for purines and pyrimidines,Pharmacol. Rev. 50 (1998) 413–492.
[42] J.S. Wiley, L.P. Dao-Ung, B.J. Gu, R. Sluyter, A.N. Shemon, C. Li, J.Taper, J. Gallo, A. Manoharan, A loss-of-function polymorphicmutation in the cytolytic P2X7 receptor gene and chroniclymphocytic leukaemia: a molecular study, Lancet 359 (2002)1114–1119.
[43] N. Li, Y. Zheng, W. Chen, C. Wang, X. Liu, W. He, H. Xu, X. Cao,Adaptor protein LAPF recruits phosphorylated p53 to lysosomesand triggers lysosomal destabilization in apoptosis, Cancer Res.67 (2007) 11176–11185.
[44] Q. Tian, M. Streuli, H. Saito, S.F. Schlossman, P. Anderson, Apolyadenylate binding protein localized to the granules ofcytolytic lymphocytes induces DNA fragmentation in target cells,Cell 67 (1991) 629–639.
d-derived suppressor cells via apoptosis inhibition, Exp Cell Res
