microrna-29a and microrna-142-3p are regulators of - blood

14
PHAGOCYTES, GRANULOCYTES, AND MYELOPOIESIS MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia *Xiao-Shuang Wang, 1 *Jia-Nan Gong, 1 Jia Yu, 1 Fang Wang, 1 Xin-Hua Zhang, 2 Xiao-Lin Yin, 2 Zhen-Qing Tan, 3 Zi-Mian Luo, 3 Gui-Hua Yang, 1 Chao Shen, 1 and Jun-Wu Zhang 1 1 National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China; 2 Haematology Department, 303 Hospital, Nanning, China; and 3 Haematology Department, Central Hospital of Xiangtan, Xiangtan, China Although microRNAs (miRNAs) are in- creasingly linked to various physiologic processes, including hematopoiesis, their function in the myeloid development is poorly understood. We detected up- regulation of miR-29a and miR-142-3p dur- ing myeloid differentiation in leukemia cell lines and CD34 hematopoietic stem/ progenitor cells. By gain-of-function and loss-of-function experiments, we demon- strated that both miRNAs promote the phorbol 12-myristate 13-acetate–induced monocytic and all-trans-retinoic acid- induced granulocytic differentiation of HL- 60, THP-1, or NB4 cells. Both the miRNAs directly inhibited cyclin T2 gene, prevent- ing the release of hypophosphorylated retinoblastoma and resulting in induction of monocytic differentiation. In addition, a target of miR-29a, cyclin-dependent ki- nase 6 gene, and a target of miR-142-3p, TGF-–activated kinase 1/MAP3K7 bind- ing protein 2 gene, are involved in the regulation of both monocytic and granulo- cytic differentiation. A significant de- crease of miR-29a and 142-3p levels and an obvious increase in their target protein levels were also observed in blasts from acute myeloid leukemia. By lentivirus- mediated gene transfer, we demonstrated that enforced expression of either miR- 29a or miR-142-3p in hematopoietic stem/ progenitor cells from healthy controls and acute myeloid leukemia patients down-regulated expression of their tar- gets and promoted myeloid differentia- tion. These findings confirm that miR-29a and miR-142-3p are key regulators of nor- mal myeloid differentiation and their re- duced expression is involved in acute myeloid leukemia development. (Blood. 2012;119(21):4992-5004) Introduction Acute myeloid leukemia (AML) is a heterogeneous group of blood cancers characterized by increased, uncontrolled proliferation of hematopoietic progenitors, and a blockage in myeloid differentia- tion is the major characteristic of AML. 1 According to the French-American-British classification system, AMLs involving the monocytic and granulocytic lineage account for 85% (M1 to M5 subtypes) of adult patients. 2 Under normal conditions, mono- cytes and granulocytes develop from long-term hematopoietic stem cells (LT-HSCs) in BM under the influence of a complex network of cytokins such as G-CSF, GM-CSF, and M-CSF, and transcrip- tion factors such as PU.1, C/EBP, IFN consensus sequence binding protein/IFN regulatory factor 8, Kru ¨ppel-like factor 4, c-Maf, and C/EBP. 3-6 MicroRNAs (miRNAs) that negatively regulate gene expres- sion at posttranscriptional level 7 have also been identified as crucial regulators in normal and malignant myeloid differentiation. Expres- sion and function analyses have unraveled their important regula- tory roles during hematopoiesis. 8 In a previous study, we demonstrated a significantly decreased expression of miR-29a and 142-3p in the peripheral blood mononu- clear cells (PBMNCs) from AML patients (French-American- British M1 to M5 subtypes). 9 These 2 miRNAs were also reported to be down-regulated in a variety of tumors and to act as tumor suppressors. 10-12 The miR-29a cluster is one of the most studied miRNA clusters. Down-regulation of miR-29a was observed in AML samples with deletions of 7q (del7q). 10 Several genes have been reported to be silenced by miR-29, most of which are potential oncogenes, such as SKI, Tcl1, the p53 upstream inhibitors p85a and CDC42, and the Bcl2 family members Bcl2 and Mcl1. 11 Mean- while, miR-142-3p, first identified as being uniquely expressed in hematopoietic system, is aberrantly expressed in T-cell and B-cell leukemia. 12 In addition, increased miR-142-3p expression has been observed at different stages of normal granulocytopoiesis. 13 Validated targets of miR-142-3p include ADCY9, 14 CD133, 15 IL-6, 16 and the RAC1. 17 In this study, we sought to investigate role of these 2 miRNAs in monocytic and granulocytic differentiation (also called myeloid differentiation), and to test whether their down-regulation is related to the differentiation block in AML blasts. Using the leukemia cell lines, NB4, HL-60, and THP-1 18-23 we observed up-regulation of miR-29a and miR-142-3p expression during all-trans-retinoic acid (ATRA)–induced granulocytic differentiation and phorbol 12- myristate 13-acetate (PMA)–induced monocytic differentiation, and examined effects of overexpression or knockdown of each miRNA on myeloid differentiations. Moreover, we identified targets of both miRNAs and examined direct effect of the target genes on myeloid differentiation. Similar results were also obtained in myeloid induction cultures of CD34 hematopoietic stem/ progenitor cells (HSPCs) derived from normal human umbilical cord blood (UCB) and BM from healthy donors and AML patients. Submitted October 16, 2011; accepted March 22, 2012. Prepublished online as Blood First Edition paper,April 4, 2012; DOI 10.1182/blood-2011-10-385716. *X.-S.W. and J.-N.G. contributed equally to this study. The online version of this article contains a data supplement. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ‘‘advertisement’’ in accordance with 18 USC section 1734. © 2012 by The American Society of Hematology 4992 BLOOD, 24 MAY 2012 VOLUME 119, NUMBER 21 For personal use only. on December 24, 2018. by guest www.bloodjournal.org From

Upload: others

Post on 11-Feb-2022

3 views

Category:

Documents


0 download

TRANSCRIPT

PHAGOCYTES, GRANULOCYTES, AND MYELOPOIESIS

MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiationand acute myeloid leukemia*Xiao-Shuang Wang,1 *Jia-Nan Gong,1 Jia Yu,1 Fang Wang,1 Xin-Hua Zhang,2 Xiao-Lin Yin,2 Zhen-Qing Tan,3 Zi-Mian Luo,3

Gui-Hua Yang,1 Chao Shen,1 and Jun-Wu Zhang1

1National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union MedicalCollege, Beijing, China; 2Haematology Department, 303 Hospital, Nanning, China; and 3Haematology Department, Central Hospital of Xiangtan, Xiangtan,China

Although microRNAs (miRNAs) are in-creasingly linked to various physiologicprocesses, including hematopoiesis, theirfunction in the myeloid development ispoorly understood. We detected up-regulation of miR-29a and miR-142-3p dur-ing myeloid differentiation in leukemiacell lines and CD34� hematopoietic stem/progenitor cells. By gain-of-function andloss-of-function experiments, we demon-strated that both miRNAs promote thephorbol 12-myristate 13-acetate–inducedmonocytic and all-trans-retinoic acid-induced granulocytic differentiation of HL-

60, THP-1, or NB4 cells. Both the miRNAsdirectly inhibited cyclin T2 gene, prevent-ing the release of hypophosphorylatedretinoblastoma and resulting in inductionof monocytic differentiation. In addition, atarget of miR-29a, cyclin-dependent ki-nase 6 gene, and a target of miR-142-3p,TGF-�–activated kinase 1/MAP3K7 bind-ing protein 2 gene, are involved in theregulation of both monocytic and granulo-cytic differentiation. A significant de-crease of miR-29a and 142-3p levels andan obvious increase in their target proteinlevels were also observed in blasts from

acute myeloid leukemia. By lentivirus-mediated gene transfer, we demonstratedthat enforced expression of either miR-29a or miR-142-3p in hematopoietic stem/progenitor cells from healthy controlsand acute myeloid leukemia patientsdown-regulated expression of their tar-gets and promoted myeloid differentia-tion. These findings confirm that miR-29aand miR-142-3p are key regulators of nor-mal myeloid differentiation and their re-duced expression is involved in acutemyeloid leukemia development. (Blood.2012;119(21):4992-5004)

Introduction

Acute myeloid leukemia (AML) is a heterogeneous group of bloodcancers characterized by increased, uncontrolled proliferation ofhematopoietic progenitors, and a blockage in myeloid differentia-tion is the major characteristic of AML.1 According to theFrench-American-British classification system, AMLs involvingthe monocytic and granulocytic lineage account for 85% (M1 toM5 subtypes) of adult patients.2 Under normal conditions, mono-cytes and granulocytes develop from long-term hematopoietic stemcells (LT-HSCs) in BM under the influence of a complex networkof cytokins such as G-CSF, GM-CSF, and M-CSF, and transcrip-tion factors such as PU.1, C/EBP, IFN consensus sequence bindingprotein/IFN regulatory factor 8, Kruppel-like factor 4, c-Maf, andC/EBP�.3-6

MicroRNAs (miRNAs) that negatively regulate gene expres-sion at posttranscriptional level7 have also been identified as crucialregulators in normal and malignant myeloid differentiation. Expres-sion and function analyses have unraveled their important regula-tory roles during hematopoiesis.8

In a previous study, we demonstrated a significantly decreasedexpression of miR-29a and 142-3p in the peripheral blood mononu-clear cells (PBMNCs) from AML patients (French-American-British M1 to M5 subtypes).9 These 2 miRNAs were also reportedto be down-regulated in a variety of tumors and to act as tumorsuppressors.10-12 The miR-29a cluster is one of the most studiedmiRNA clusters. Down-regulation of miR-29a was observed in

AML samples with deletions of 7q (del7q).10 Several genes havebeen reported to be silenced by miR-29, most of which are potentialoncogenes, such as SKI, Tcl1, the p53 upstream inhibitors p85a andCDC42, and the Bcl2 family members Bcl2 and Mcl1.11 Mean-while, miR-142-3p, first identified as being uniquely expressed inhematopoietic system, is aberrantly expressed in T-cell and B-cellleukemia.12 In addition, increased miR-142-3p expression has beenobserved at different stages of normal granulocytopoiesis.13Validatedtargets of miR-142-3p include ADCY9,14 CD133,15 IL-6,16 andthe RAC1.17

In this study, we sought to investigate role of these 2 miRNAs inmonocytic and granulocytic differentiation (also called myeloiddifferentiation), and to test whether their down-regulation is relatedto the differentiation block in AML blasts. Using the leukemia celllines, NB4, HL-60, and THP-118-23 we observed up-regulation ofmiR-29a and miR-142-3p expression during all-trans-retinoic acid(ATRA)–induced granulocytic differentiation and phorbol 12-myristate 13-acetate (PMA)–induced monocytic differentiation,and examined effects of overexpression or knockdown of eachmiRNA on myeloid differentiations. Moreover, we identifiedtargets of both miRNAs and examined direct effect of the targetgenes on myeloid differentiation. Similar results were also obtainedin myeloid induction cultures of CD34� hematopoietic stem/progenitor cells (HSPCs) derived from normal human umbilicalcord blood (UCB) and BM from healthy donors and AML patients.

Submitted October 16, 2011; accepted March 22, 2012. Prepublished online asBlood First Edition paper, April 4, 2012; DOI 10.1182/blood-2011-10-385716.

*X.-S.W. and J.-N.G. contributed equally to this study.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 USC section 1734.

© 2012 by The American Society of Hematology

4992 BLOOD, 24 MAY 2012 � VOLUME 119, NUMBER 21

For personal use only.on December 24, 2018. by guest www.bloodjournal.orgFrom

Based on these examinations, we demonstrated that miR-29a andmiR-142-3p are important regulators of normal myeloid differentia-tion and AML development and that they function at least partiallythough direct suppression of their common target gene, cyclin T2(CCNT2), and their individual target genes cyclin-dependentkinase 6 (CDK6) and TGF-� activated kinase 1/MAP3K7 bindingprotein 2 (TAB2), respectively.

Methods

Cell culture and differentiation induction

The human leukemia cell lines NB4, HL-60, and THP-1 and humanembryonic kidney cell lines 293T and 293TN were maintained in propermedia (supplemental Methods, available on the Blood Web site; see theSupplemental Materials link at the top of the online article).

The granulocytic differentiation of NB4 and HL-60 was induced withATRA (Sigma-Aldrich) and the monocytic differentiation of THP-1 andHL-60 with PMA (Sigma-Aldrich; supplemental Methods). The mRNAexpression levels of cell surface markers CD11b, CD14, and CSF1R formonocytic and CD11b, CSF3R, and MPO for granulocytic differentiationwere determined by real-time PCR. CD11b and CD14 levels were alsodetermined by flow cytometry on a FACSAria (BD Biosciences) or AccuriflowC6 (BD Biosciences). Morphology was evaluated by conventionallight-field microscopy of May-Grunwald-Giemsa–stained cytospins usingthe Olympus BX51 (Japan) optical microscopy. Cells were also stainedusing the nitroblue tetrazolium assay (supplemental Methods).

RNA extraction, cDNA synthesis of mRNAs and miRNAs, andreal-time PCR assays

See supplemental Methods.

PAGE-northern and Western blot

Northern blot analysis of miRNAs and Western blot analysis of proteinswere performed as described previously.24 Monoclonal mouse anti-CCNT2antibody (Abcam), monoclonal mouse anti-CDK6 antibody, monoclonalrabbit anti-TAB2 antibody, polyclonal rabbit anti-pRb antibody (Ser807/811), and monoclonal mouse anti-Rb antibody (Cell Signaling Technol-ogy) were used for Western analysis. The immunoblots were quantifiedusing Gelpro Version 4.0 software.

Cell transfection with miRNA mimics and siRNAs

For cell transfection with miRNA mimics and miRNA inhibitors, or withsmall interfering RNAs (siRNAs) against the miRNA target mRNAs, seesupplemental Methods. For each cell transfection, 2 or 3 replicationexperiments were performed.

Luciferase miRNA target reporter assay

The full length of the 3�-untranslated regions (UTRs) of human CCNT2,CDK6, and TAB2 mRNAs was each PCR amplified and cloned into thepMIR-REPORT Luciferase Reporter Vector (Ambion) to generate thereporters. Mutations of the predicted seed regions in these 3 mRNAsequences were created using primers, including the mutated sites. Theluciferase miRNA target reporter assay is described in supplementalMethods.

Cell viability and cell cycle assay

Cellular proliferation and cell cycle were analyzed using the Cell CountingKit-8 (Dojindo Laboratories), and intracellular DNA was measured via flowcytometry as described in supplemental Methods.

CD34� cell separation and granulocytic and monocyticinduction culture

Human UCB was obtained from normal full-term deliveries from BeijingHospital. The peripheral blood samples and BM samples from normalvolunteers and AML patients were obtained from the 303 Hospital and theCentral Hospital of Xiangtan. The informed consent was obtained from allof the examined subjects in accordance with the Declaration of Helsinki,and the related studies were approved by the ethics committees of theparticipating hospitals and institute. MNC fractions were isolated from thesamples by Percoll density gradient (d � 1.077; GE Healthcare), and theMNC fractions were enriched for CD34� HSPCs using magnetic-activatedcell sorting technology according to the manufacturer’s recommendations(Miltenyi Biotec). The CD34� cells were cultured in granulopoietic andmonocytopoietic induction cultures as described in supplemental Methods.The developmental maturation of the differentiated cells was confirmed byobserving cytospins as well as the expression of cell surface markers.

Lentiviral production and transduction

The self-inactivating transfer vector pMIRNA1, control plasmid, and thepackaging kit System Biosciences were used according to the manufactur-er’s instructions. DNA fragments 500 bp in size containing the pre-miR-29aor pre-miR-142 were inserted under the CMV promoter in pMIRNA1. Aftervirus packaging, the recombinant lentivirus particles were harvested andtitrated as described in supplemental Methods. The CD34� cells weretransduced after 24 hours of expansion with IL-3 and SCF by addinglentivector supernatants (Lenti-miR-29a, Lenti-miR-142, and Lenti-control) to the cells at an MOI of 50 in the presence of polybrene(5 �g/mL). The cells were washed the next day with PBS and plated forcolony-forming experiments (supplemental Methods) and liquid cultures.

Results

miR-29a and miR-142-3p expression increases during myeloiddifferentiation of THP-1, NB4, and HL-60

To investigate whether miR-29a and miR-142-3p participate inmyeloid differentiation, we first examined their expression duringmyeloid differentiation of THP-1, NB4, and HL-60. Cell differen-tiation was monitored by evaluating the expression of myeloid-specific surface markers CD11b (a marker of both granulocytic andmonocytic cell differentiation), and CD14 (a marker of monocyticcell differentiation), by flow cytometry (supplemental Figure 1A).Real-time PCR analysis revealed a gradual increase in the expres-sion of both the 2 miRNAs during PMA-induced monocyticdifferentiation of THP-1 and ATRA-induced granulocytic differen-tiation of NB4 cells (supplemental Figure 1B). The same resultswere also observed in HL-60 cells subjected to monocytic andgranulocytic differentiation (supplemental Figure 1B). These expres-sion patterns were also confirmed by Northern blot analysis usingthe antisense DNA sequences of mature miR-29a and miR-142-3pas probes (supplemental Figure 1C).

miR-29a and miR-142-3p promote ATRA- and PMA-inducedmyeloid differentiation

To examine the functional relevance of miR-29a and miR-142-3pwith myeloid differentiation, we transiently transfected THP-1,HL-60, and NB4 cells with either miR-29a or miR-142-3p mimicsor a scramble control. Transfection efficiency was confirmed bysemiquantitative PCR and real-time PCR (Figure 1A; supplementalFigure 2A). After miR-29a or miR-142-3p mimic transfection, flow

miR-29a AND miR-142-3p AND MYELOID DIFFERENTIATION 4993BLOOD, 24 MAY 2012 � VOLUME 119, NUMBER 21

For personal use only.on December 24, 2018. by guest www.bloodjournal.orgFrom

cytometric analysis revealed a significantly higher percentage ofCD11b-positive cells among the PMA-treated THP-1 cells, as wellas among the ATRA-treated NB4 cells compared with the cellstransfected with the scramble control (Figure 1B; supplementalFigure 2B).

miR-29a or miR-142-3p mimic transfection also induced mor-phologic changes in the cells after monocytic and granulocyticdifferentiation. May-Grunwald-Giemsa staining showed slightly

morphologic changes in untreated cell populations of THP-1 andNB4 cell populations transfected with miR-29a or miR-142-3pmimic. However, after induction for 72 hours with PMA, THP-1cells transfected with miR-29a or miR-142-3p mimic showed asignificantly greater fraction of more mature monocytes (Figure 1Cleft). Similarly, after induction for 72 hours with ATRA, NB4 cellstransfected with miR-29a or miR-142-3p showed a significantlygreater fraction of more mature granulocytic cells (Figure 1C

Figure 1. Transfection of THP-1 and NB4 cells with miRNA mimics or antisense inhibitors for miR-29a and miR-142-3p affects myeloid differentiation. (A) Theexpression levels of miR-29a and miR-142-3p were detected by RT-PCR (left) and real-time PCR (right) in THP-1 and NB4 cells that were transiently transfected with miRNAmimics or mimic scramble control. U6 snRNA was used as an internal control. The values of each group were expressed as mean � SD for 3 real-time PCR assays.(B) Expression of CD11b in THP-1 and NB4 cells was analyzed by flow cytometry. Numbers in the graphs represent the percentages of positively stained cells at 72 hours ofPMA or 96 hours of ATRA treatment (black histograms) compared with untreated cells (red histograms). A representative experiment of 3 is presented. The differences arestatistically significant (see supplemental Figure 2B). (C) Representative May-Grunwald-Giemsa staining of THP-1 and NB4 cells transfected with microRNA mimics orscramble, and treated with PMA or ATRA for 72 hours. Images were captured at room temperature using 20� objective with numeric aperture 0.5 and acquired through CCDDP72 camera and Cellsens Standard Version 1.2.1 software (Olympus). The mild differentiated cells were annotated by gray arrows. Mature macrophages and segmentedneutrophils after 72 hours of differentiation were annotated by black arrows, and their percentages were marked. More mature monocytes show bluish-gray cytoplasm and asaddle-shaped nucleus. More mature granulocytic cells show polylobular nuclei, a decreased ratio of nuclear area to cytoplasmic area, and decreased cytoplasm staining,corresponding to band cells and metamyelocytes. (D) Expression levels of CD14 or CD11b mRNA were analyzed by real-time PCR. Comparative real-time PCR wasperformed in triplicate and normalized to GAPDH mRNA. Error bars represent SD. The expression of the mRNA in untreated scramble-transfected cell was normalized as 1.(E) Nitroblue tetrazolium assay in NB4 and HL-60 cells transfected with miR-29a mimic, miR-142-3p mimic, or mimic control. (F) Real-time PCR analysis of the expressionlevels of miR-29a and miR-142-3p in THP-1 and NB4 cells transfected with miR-29a inhibitor (anti-29a), miR-142-3p inhibitor (anti–142-3p), or inhibitor control (anticontrol). U6snRNA was used as an internal control. (G) Expression levels of CD14 in THP-1 cells and CD11b in HL-60 cells were analyzed by FACS. A representative experiment of 3 ispresented. The differences are statistically significant (see supplemental Figure 2F). *P .05 (Student t test). **P .01 (Student t test).

4994 WANG et al BLOOD, 24 MAY 2012 � VOLUME 119, NUMBER 21

For personal use only.on December 24, 2018. by guest www.bloodjournal.orgFrom

right). These morphologic changes were corroborated by examin-ing stained HL-60 cells transfected with miR-29a mimic, miR-142-3p mimic, or scramble control and treated with PMA or ATRAfor 72 hours (supplemental Figure 2C).

We next analyzed the effect of miR-29a and miR-142-3p on themRNA levels of differentiation markers by real-time PCR (Figure1D). The results were consistent with flow cytometry and morpho-logic observation. In summary, transfection with miR-29a ormiR-142-3p mimic resulted in an elevated expression of CD11b,CD14, CSF1R (supplemental Figure 2D), and CSF3R (supplemen-tal Figure 2E) mRNAs. In contrast, a decreased level of myeloper-oxidase mRNA, which is expressed only during the late myeloblastand promyelocyte stages and disappears during later stages ofneutrophilic development, was observed in the cells transfectedwith miR-29a or miR-142-3p mimic (supplemental Figure 2E). Theacceleration of maturation resulting from miR-29a or miR-142-3ptransfection was also confirmed by nitroblue tetrazolium staining(Figure 1E), which showed enhanced oxidative ability in ATRA-treated NB4 and HL-60 cells. These results suggested that theenforced expression of miR-29a and miR-142-3p promotes thePMA-induced monocytic and ATRA-induced granulocyticdifferentiation.

Antisense inhibitors directed against either miR-29a or miR-142-3p (anti-29a or anti–142-3p) were then transfected into THP-1and NB4 cells, and knockdown of the targeted miRNA wasconfirmed (Figure 1F). The THP-1 cells were then allowed toundergo PMA treatment for 72 hours, and reduced expression ofCD14 antigen was observed in anti–miR-29a– and anti–miR-142-3p–treated cells (Figure 1G top; supplemental Figure 2F). Thepercentage of CD11b-positive cells was also modestly decreased inATRA-induced NB4 cells transfected with miR-29a or miR-142-3pinhibitors (Figure 1G bottom; supplemental Figure 2F). Takentogether, the reduced expression of miR-29a and miR-142-3psuppresses the myeloid differentiation of THP-1 and NB4 cells.

We next verified whether transfection of the 2 miRNAssimultaneously could enhance their ability to promote differentia-tion. As determined by flow cytometry, cotransfection of the2 miRNAs exhibited a more obvious effect on the differentiation ofboth lineages than single transfection before induced differentia-tion, but after 72 hours treatment with PMA or ATRA the enhancedeffect of promoting differentiation by the cotransfection is notsignificant (supplemental Figure 3).

CCNT2 was identified as a common target of miR-29a andmiR-142-3p

We identified several potential targets of miR-29a or miR-142-3pusing the prediction programs TargetScan and Pictar, including theCCNT2 mRNA, which contains sequence motifs that match withthe “seed” sequence of miR-142-3p and miR-29a in its 3�-UTR(Figure 2A). To test whether miR-29a and miR-142-3p are able toregulate CCNT2 directly, we made a reporter construct carrying thewild-type CCNT2 3�-UTR (pCCNT2_WT), constructs carrying theCCNT2 3�-UTR sequences with mutation in the putative miR-29aand miR-142-3p recognition elements (pCCNT2_142-3pM andpCCNT2_29aM, respectively), and a construct with both themutations (pCCNT2_DM). When miR-29a or miR-142-3p mimicwas transfected into 293T cells the luciferase activity ofpCCNT2_WT was reduced by 30%. Meanwhile, single mutationand double mutations abolished the repression by miR-29a ormiR-142-3p, demonstrating that miR-29a and miR-142-3p couldspecifically target their binding sites in the 3�-UTRs of CCNT2(Figure 2B). Interestingly, transfection with both microRNAs

simultaneously reduced the reporter luciferase activity more signifi-cantly, and this reduction could be abolished by mutations of theboth sites, indicating that miR-29a and miR-142-3p functionindependently in targeting the CCNT2 3�-UTR (Figure 2C).

To validate that CCNT2 is a real target of both miR-29a andmiR-142-3p, we quantified the expression of the 2 CCNT2 proteinisoforms, CCNT2b and CCNT2a, in the cells by Western blotting.A significant reduction of endogenous CCNT2 protein levels wasdetected in the cells transfected with miR-29a or miR-142-3pmimic (Figure 2D). Inversely, CCNT2 proteins increased as a resultof miR-29a or miR-142-3p inhibitor transfection in THP-1 cells(Figure 2E). Noticeably, the CCNT2b protein expression appears tobe more sensitive to the expression changes of miR-29a andmiR-142-3p than CCNT2a protein.

CCNT2 counteracts monocytic differentiation of PMA-inducedHL-60 and THP-1 cells

We then examined whether the function of CCNT2 is linked tomyeloid differentiation. By Western blot analysis, we found that theCCNT2 proteins, which are highly expressed in untreated THP-1cells, were rapidly down-regulated on PMA treatment; meanwhile,this down-regulation was gradual in PMA-induced HL-60 cells(Figure 2F). On the contrary, CCNT2 protein levels are almostunchanged during ATRA-induced NB4 cell differentiation (datanot shown).

We also examined the effect of CCNT2 expression levels onmonocytic differentiation. Transfection of HL-60 and THP-1 cellswith siRNAs targeting CCNT2 mRNA (si_CCNT2) resulted in asignificant decrease of endogenous protein levels (Figure 2G). Inthe presence of PMA, this resulted in a strong increase in the levelsof both CD11b and CD14 surface antigens compared with the cellstransfected with a nontargeting si_control, as judged by flowcytometry analysis (Figure 2H; supplemental Figure 4A). In bothHL-60 and THP-1 cells, the down-regulation of CCNT2 alsoresulted in increased mRNA levels of CD11b, CD14, and themonocyte terminal differentiation marker CSF1R (M-CSFr; supple-mental Figure 4B-C). Morphologic analysis using May-Grunwald-Giemsa staining and a cell adherence assay showed that si_CCNT2transfection increased the adherence ability of PMA-inducedTHP-1 cells greatly, indicating that more macrophage cells weregenerated (supplemental Figure 4D-E).

CCNT2 inhibits the monocytic differentiation of HL-60 andTHP-1 cells by increasing proliferation

CCNT2 is a component of positive transcription elongation factor b(P-TEFb), a complex that is composed of cyclin-dependent kinase9 and a cyclin T (CDK9/CCNT). CDK9 activation requires bindingof a T family cyclin (CCNT1, CCNT2a, or CCNT2b) or CCNK.25-27

CCNT2 was not previously reported to be involved in myeloiddifferentiation. We performed a proliferation assay on THP-1 cells.PMA induced the cells to differentiation, but an obvious reductionof cell amplification was observed in the cells transfected withCCNT2 siRNA compared with the control cells (Figure 2I); a similarreduction in proliferation was not observed in uninduced cells.

Previous studies demonstrated that CDK9 is able to phosphory-late p56/Rb in vitro,26 and CDK9/CCNT2 can bind to Rb andphosphorylate the Rb region spanning amino acids 793 to 834in vivo.27 To confirm this interaction between CCNT2 and Rb inTHP-1 and HL-60 cells, we detected phosphorylated Rb (pRb) byWestern blot analysis. A decreased pRb level was detected in the

miR-29a AND miR-142-3p AND MYELOID DIFFERENTIATION 4995BLOOD, 24 MAY 2012 � VOLUME 119, NUMBER 21

For personal use only.on December 24, 2018. by guest www.bloodjournal.orgFrom

Figure 2. CCNT2 is a target gene of both miR-29a and miR-142-3p, and CCNT2 plays a role in inhibiting PMA-induced monocytic differentiation in THP-1 and HL-60 cells.(A) The nucleotide sequences of miR-29a, miR-142-3p, and their complementary sequences in CCNT2 mRNA. The predicted miR-29a and miR-142-3p binding sites in the CCNT23�-UTR and the mutated nucleotides (gray boxes) of the 3�-UTR site are shown. CCNT2_WT is a reporter construct containing the entire wild-type 3�-UTR sequence of CCNT2.CCNT2_29aM, CCNT2_142-3pM, and CCNT2_DM are reporter constructs containing mutations in the miR-29a binding site, the miR-142-3p binding site mutation, and both sites.(B) Luciferase activity in 293T cells cotransfected with one of the 4 reporter constructs and either one of the miRNA(miR-29a or miR-142-3p) mimics or scramble control, as indicated. Thereporter constructs with perfect complementary sites for miR-29a and miR-142-3p (p29a_site and p142-3p site, respectively) were used as positive controls. The activities were calculatedas a ratio of firefly to renilla luciferase activity, and are expressed as mean � SD of 3 separate experiments. *P .05 (Student t test). **P .01 (Student t test). (C) Luciferase activity in293T cells cotransfected with either the wild-type or double mutant, and both miR-29a and miR-142-3p mimics simultaneously or scramble control. (D) Western blot analysis of CCNT2expression in 293Tand myeloid cell linesTHP-1, HL-60, and NB4 transfected with miR-29a mimic, miR-142-3p mimic, or scramble control. Densitometric values normalized on the basis ofGAPDH expression are indicated below the corresponding lanes and shown as fold changes. N.D indicates a densitometric value cannot be given because the band is almostundetectable. (E) Western blot analysis of CCNT2 expression in monocytic cell line THP-1 transfected with an inhibitor for miR-29a (anti-29a), an inhibitor for miR-142-3p (anti–142-3p), oran inhibitor control. (F) Western blot analysis showing expression of the 2 CCNT2 isoforms during PMA-induced monocytic differentiation in THP-1 and HL-60 cells; GAPDH was detectedfor checking equal protein loading. (G) Western blot analysis showing the expression of CCNT2 in HL-60 and THP-1 cells that were transfected with CCNT2 siRNA(si_CCNT2) or siRNAcontrol (si_control) and subsequently treated with PMAfor 72 hours. (H) Flow cytometric analysis of CD11b in THP-1 cells transfected with si_CCNT2 or si_control. Numbers in the graphsrepresent the percentages of positively stained after 72 hours of differentiation (the right peak) compared with untreated cells (the left peak).A representative experiment of 3 is presented.(I) Cell proliferation assay ofTHP-1 cells transfected with si_CCNT2 or si_control using cell counting kit 8.The cells were cultured in medium with or without PMA. (J) Western blots showingpRb and Rb protein levels in THP-1 cells after transfection with si_CCNT2 or si_control, and in HL-60 cells after transfection with miR-29a mimic, miR-142-3p mimic, or scramble control.

4996 WANG et al BLOOD, 24 MAY 2012 � VOLUME 119, NUMBER 21

For personal use only.on December 24, 2018. by guest www.bloodjournal.orgFrom

cells transfected with CCNT2 siRNA compared with the cellstransfected with the control, whereas the level of total Rb remainedconsistent (Figure 2J top), which is consistent with the observationthat the ectopic expression of both miRNAs decreased the pRblevel (Figure 2J bottom). Given that Rb is well known for itsinvolvement in mediating the cell cycle, we examined intracellularDNA content using flow cytometry and failed to see any obviousG1 arrest induced by si_CCNT2 in the uninduced cells. However,after PMA treatment for 24 hours, a significantly increasedpercentage of cells in G1 was detected among these cells trans-fected with si_CCNT2 compared with the cells transfected withsi_control (supplemental Figure 5).

These results demonstrated that the CCNT2 down-regulation andthe resulting decrease in pRb levels and cell proliferation are one of thepathways by which miR-29a and miR-142-3p promote monocytopoiesis.

Validation of CDK6 as a target of miR-29a in THP-1 and NB4cells

CDK6 was previously reported as a target of miR-29a in mantle celllymphoma, in which CDK6, expressed at elevated levels, cooper-ates with cyclin D1 to further promote cell cycle progression.28 Wethus hypothesized that CDK6 may also function as a target ofmiR-29a in promyelocytic cell lines. We constructed 4 pMIR-

REPORT constructs that contained either the wild-type sequence ofCDK6 3�-UTR (pCDK6_WT) or the sequences of the 3�-UTR withmutation in the first (pCDK6_MUT1), or second binding site(pCDK6_MUT2) or in both (pCDK6_DM; Figure 3A). As ex-pected, miR-29a mimic transfection decreased the activity of thepCDK6_WT reporter markedly, with little effect on the activity ofpCDK6_MUT1 or pCDK6_MUT2 reporters and no effect on theactivity of the CDK6_DM reporter (Figure 3B). miR-29a mimictransfection also obviously decreased the CDK6 protein levels in293T, THP-1, and NB4 cells (Figure 3C top), whereas knockdownof miR-29a by inhibitor transfection resulted in increased CDK6levels in NB4 and THP-1 cells (Figure 3C bottom). These resultsdemonstrated CDK6 is a direct target of miR-29a in these cell lines.

TAB2 was identified as a target of miR-142-3p in THP-1 andNB4 cells

We also constructed 2 pMIR-REPORT constructs containing either thewild-type sequence of the TAB2 3�-UTR (pTAB2_WT) or a sequencewith a mutation in the miR-142-3p binding site (pTAB2_MUT; Figure3D). Significantly reduced luciferase activity was detected in the cellscotransfected with the pTAB2_WT and miR-142-3p mimics but not inthe cells cotransfected with the pTAB2_MUT and miR-142-3p mimics

Figure 3. Validation of CDK6 and TAB2 as targetgenes for miR-29a and miR-142-3p, respectively.(A) The predicted miR-29a binding sites in the 3�-UTR ofCDK6 mRNA are shown. CDK6_WT is the pMIR-REPORT construct containing the entire 3�-UTR se-quence of CDK. CDK6_MUT1, MUT2, and DM are thepMIR-REPORT constructs containing mutated nucleo-tides in the first, second, and both miR-29a binding sites,respectively. (B) Luciferase reporter assays. Relativeluciferase levels in 293T cells cotransfected with theCDK6_WT, CDK6_MUT1/2, or CDK6_DM report con-struct and either miR-29a mimic or scrambled control.*P .05 (Student t test). **P .01 (Student t test).(C) Western blot analysis of CDK6 levels in 293T, THP-1,and NB4 cells. miR-29a mimic, inhibitor (anti-29a), or acontrol was transfected into the indicated cells. A repre-sentative Western blot from 3 independent experimentsis shown. (D) The predicted miR-142-3p binding site inthe 3�-UTR of TAB2 mRNA is shown. TAB2_WT is thereporter construct containing the entire 3�-UTR sequenceof TAB2, and TAB2_MUT is the reporter construct contain-ing mutated nucleotides at the miR-142-3p binding site.The mutated nucleotides of the 3�-UTR site are indicatedby gray boxes. (E) Relative luciferase expression levelsin 293T cells cotransfected with TAB2_WT or TAB2_MUTwith either miR-142-3p mimic or a scrambled control.(F) Western blot analysis of TAB2 levels in 293T, THP-1,and NB4 cells. miR-142-3p mimic, a miR-142-3p inhibitor(anti–142-3p), or a control was transfected into theindicated cells. A representative Western blot from 3 inde-pendent experiments is shown.

miR-29a AND miR-142-3p AND MYELOID DIFFERENTIATION 4997BLOOD, 24 MAY 2012 � VOLUME 119, NUMBER 21

For personal use only.on December 24, 2018. by guest www.bloodjournal.orgFrom

(Figure 3E). Western blot analysis indicated that transfection of miR-142-3p mimic reduced the TAB2 level in the transfected cells (Figure 3Ftop), whereas knockdown of miR-142-3p by inhibitor transfectionincreased TAB2 level (Figure 3F bottom). Taken collectively, these dataindicated that TAB2 is a direct target of miR-142-3p.

Effects of CDK6 and TAB2 on granulocytic and monocyticdifferentiation

By Western blot analysis, we observed gradually decreasedlevels of both TAB2 and CDK6 proteins during the ATRA-induced granulocytic differentiation of NB4 and the PMA-induced monocytic differentiation of THP-1. The decrease inTAB2 levels appeared to be more rapid than that of CDK6(Figure 4A). To further elucidate the roles of TAB2 and CDK6in myeloid differentiation, we transfected NB4 and THP-1 cellswith TAB2-siRNA (si_TAB2), CDK6-siRNA (si_CDK6), ornontargeting siRNA (si_control). The decrease of CDK6 andTAB2 protein levels via RNA interference was confirmed byWestern blot (Figure 4B). The suppressed expression of TAB2or CDK6 promoted both the PMA-induced monocytic differen-tiation and ATRA-induced granulocytic differentiation, as evalu-ated by the up-regulation of CD11b and CD14 mRNA expres-

sion (Figure 4C), morphologic changes (supplemental Figure 6),and the CD11b antigens levels (Figure 4D-E).

Expression inhibition of the target genes partially rescues theanti–miR-29a– and anti–miR-142-3p–mediated myeloiddifferentiation block

To test whether the regulation of myeloid differentiation bymiR-29a and miR-142-3p is mediated via the targets identifiedin the preceding 5 paragraphs, NB4 and THP-1 cells weretransfected with either CCNT2 or CDK6 siRNA after anti–miR-29a transfection, or with CCNT2 or TAB2 siRNA after anti–miR-142-3p transfection. As shown in supplemental Figure 7A,transfection of the target siRNAs repressed the up-regulation oftarget expression that should have been induced by anti–miR-29a or anti–miR-142-3p transfection. Flow cytometric analysisshowed that the expression up-regulation of myeloid differentia-tion markers was partially rescued by knocking down the targetgenes (supplemental Figure 7B). These data further demonstratethat miR-29a and miR-142-3p promote myeloid differentiationvia directly targeting their common target CCNT2 and indi-vidual targets, CDK6 and TAB2, respectively.

Figure 4. Expression inhibition of CDK6 and TAB2 increases ATRA-induced differentiation in NB4 cells and PMA-induced differentiation in THP-1 cells. (A) THP-1cells treated with 50 ng/mL PMA (left) and NB4 cells treated with 2�M ATRA (right) were collected at the indicated time points for Western blot analysis of TAB2 and CDK6protein levels. GAPDH was used as a loading control. (B) THP-1 and NB4 cells were transiently transfected with control siRNA (si_control), TAB2 siRNA (si_TAB2), or CDK6siRNA (si_CDK6). A representative Western blot detecting TAB2 and CDK6 is shown. (C) Real-time PCR analysis of the CD11b mRNA level in the transfected NB4 cells afterATRA induction, and real-time PCR analysis of the CD14 mRNA level in the transfected THP-1 cells after PMA induction. Error bars represent mean � SD (n � 3). (D) Flowcytometric analysis of CD11b levels. The cells were transfected with the indicated inhibitors and then treated for 72 hours with ATRA (for NB4 cells) or PMA (for THP-1 cells).A representative experiment of 3 is presented. (E) Statistical analysis of the flow cytometric assays. Data are mean � SD from 3 independent experiments. *P .05 (Studentt test). **P .01 (Student t test).

4998 WANG et al BLOOD, 24 MAY 2012 � VOLUME 119, NUMBER 21

For personal use only.on December 24, 2018. by guest www.bloodjournal.orgFrom

Role of miR-29a, miR-142-3p, and their targets in the normalhematopoiesis

To study the role of miR-29a and miR-142-3p in the normalhematopoietic program, we analyzed their levels in monocyte/macrophage and granulocytic induction cultures of UCB CD34�

HSPCs. Figure 5A shows a gradual increase of miR-29a andmiR-142-3p during the induction cultures.

The role of miR-29a and miR-142-3p in the myeloiddifferentiation was validated by ectopic expression of them ininduction cultures. Overexpression of the miRNAs was accom-plished by infecting CD34� cells with Lenti-29a or Lenti-142.After 4 days of viral infection, the miR-142-3p level reached1.9-fold in Lenti-142–infected cells and the miR-29a levelreached 2-fold in Lenti-29a–infected cells compared withLenti-control–infected cells, and this overexpression persisteduntil the differentiation cultures were terminated. The differen-tiation status of cells was evaluated by morphologic analysis,colony-forming assay, and real-time PCR analysis, particularlyfor the appearance and rising expression of monocytic/macrophage (CD11b and CD14) and granulocytic (CD11b andCSF3R) differentiation markers. Compared with the cells in-fected with the Lenti-control, overexpression of either miR-29aor miR-142-3p resulted in an increased expression of differentia-tion markers that began 7 days after induction (Figure 5B;supplemental Figure 8A). Colony-forming assay showed thatboth miR-29a and miR-142 promote the forming of CFU-GM,CFU-G, and CFU-M, increasing both clone number and size(Figure 5C). Giemsa staining also confirmed an increasedpercentage of more mature cells in the monocytic and granulo-cytic induction cultures of CD34� cells infected with eitherLenti-29a or Lenti-142 compared with the control cultures(Figure 5D-E; supplemental Figure 8B).

Western blot analysis showed the levels of the 3 miRNAtarget proteins decrease beginning on day 7 after differentiationinduction (Figure 6A-B). To prove the interaction of themiRNAs and their targets in the differentiating cells, the targetprotein levels in the induction cultures were analyzed. De-creased CCNT2 levels were detected in the monocytic inductioncultures of CD34� cells infected with either Lenti-29a orLenti-142 compared with the cells infected with Lenti-control(Figure 6C). Decreased CDK6 and TAB2 levels were alsodetected in the monocytic and granulocytic induction cultures ofCD34� cells infected with Lenti-29a and Lenti-142, respec-tively, compared with control cells (Figure 6D-E).

It should be noted that the precursor of miR-142 can beprocessed into 2 mature miRNAs: miR-142-3p and miR-142-5p.29

By real-time PCR analysis, we found that the expression level ofmiR-142-5p was always much lower than that of miR-142-3pduring the monocytic and granulocytic differentiation of CD34�

cells (supplemental Figure 9). An increased expression of miR-142-3p, but not of miR-142-5p, was detected in the induction culturesinfected with Lenti-142 compared with the controls. These sug-gested that the promotion of myeloid differentiation effected by thelentivirus-mediated gene transfer primarily reflected the action ofmiR-142-3p.

Validation that miR-29a and miR-142-3p negatively regulate thetarget genes in AML PBMNCs

We previously demonstrated significantly decreased expression ofmiR-29a and miR-142-3p in PBMNCs by comparing 100 normalsubjects and 52 primary diagnosed AML patients.9 To validate thatthese miRNAs regulate their identified targets in AML, we recently

collected PBMNCs from 30 healthy donors and 20 primarydiagnosed AML patients (supplemental Table 1). Again, weobserved that expression of the 2 miRNAs significantly decreasedin AML patients (supplemental Figure 10A). Western blot analysisrevealed that levels of the target proteins CCNT2, CDK6, andTAB2 were, to a great extent, inversely correlated with miR-29a andmiR-142-3p expression in the samples (supplemental Figure 10B).

Enforced expression of miR-29a or miR-142-3p in AML BMblasts improved myeloid differentiation

We expanded our analysis to BM CD34� cells derived from partialprojects and observed significantly decreased miR-29a and miR-142-3p expression in the AML patients compared with healthydonors (Figure 7A). We also stochastically selected BM CD34�

cells derived from an AML M5 patient (patient 12) and an M3bpatient (patient 4; supplemental Table 1) to examine their expres-sion levels after myeloid differentiation induction. Up-regulationof the 2 miRNAs during granulocytic and monocytic inductioncultures was detected (supplemental Figure 11), but the ratios ofexpression increase were much less than that in the normal CD34�

cells (Figure 5A).To test whether ectopic expression of the miRNAs could

promote myeloid maturation of AML BM blasts, we infected BMCD34� cells derived from 3 AML patients with the recombinationviruses. Using flow cytometry, we observed that the ectopicexpression of miR-29a or miR-142-3p partially overcame differen-tiation arrest in AML blasts (Figure 7B-E). Western blot analysisfurther confirmed that the ectopic expression of miR-29a ormiR-142-3p decreased their target protein levels (Figure 7F).

Discussion

In this study, we demonstrated a positive regulation of miR-29a andmiR-142-3p on the monocytic and granulocytic differentiationsand validated their 3 targets. In addition, we showed that abnormalexpression of these 2 miRNAs and the 3 identified targets isinvolved in AML development.

Expression signature of miR-142-3p had been previouslyimplicated in hematopoietic differentiation of AML cells, but somecontradict results were reported, which may be resulted from thedifferences in cell induction and RNA isolation methods.13,29-31 ThemiR-142-3p expression was reported to increase by 2.5-fold after2 weeks of granulocytic differentiation in UCB-derived CD34�

cells.13 A study compared miRNA expression among HSPCs andperipheral blood leukocytes also identified miR-142-3p as the mostup-regulated miRNA in peripheral blood leukocytes.30 Meanwhile,previous reports regarding miR-29a did not demonstrate its involve-ment in myeloid differentiation but focused on its abnormalexpression in many kinds of leukemia.32 Moreover, we previouslydetected a significantly decreased expression of miR-29a andmiR-142-3p in the PBMNCs of AML patients,9 and confirmed theresult in AML BM blast cells in the current study.

We confirmed CCNT2 as a common target of miR-29a andmiR-142-3p. CCNT2 is a component of the positive transcriptionelongation factor b (P-TEFb).33 In mammals, P-TEFb is essentialfor the elongation of transcription and cotranscriptional processingby RNA polymerase II, and different P-TEFb complexes canregulate subsets of distinct genes that are important for embryonicdevelopment.34

We observed inhibition of proliferation in THP-1 cells and adecreased level of phosphorylated Rb by siRNA-mediated knock-down of CCNT2, which is consistent with previous findings.27 We

miR-29a AND miR-142-3p AND MYELOID DIFFERENTIATION 4999BLOOD, 24 MAY 2012 � VOLUME 119, NUMBER 21

For personal use only.on December 24, 2018. by guest www.bloodjournal.orgFrom

Figure 5. Role of miR-29a and miR-142-3p in CD34� HSPCs derived from UCB. CD34� cells purified from human UCB samples were infected with Lenti-control, Lenti-29a, orLenti-142. Cells were cultured for 17 days. Granulocytic differentiation was induced by human G-CSF (20 ng/mL) and rhIL-6 (10 ng/mL), and monocytic differentiation by rhM-SCF(50 ng/mL), rhIL-6 (1 ng/mL), and rhFlt-3L (100 ng/mL). Cells were collected for morphologic, colony formation, and myeloid marker expression analysis. (A) Real-time PCR analysis ofmiR-29a and miR-142-3p expression during differentiation. Samples were collected at the indicated days after induction. The relative expression of miR-142-3p and miR-29a wasnormalized to the expression level at day 0. (B) CD14 mRNA levels for monocytic differentiation and CD11b mRNA levels for granulocytic differentiation were analyzed by real-time PCR.(C) Colony formation assay after an 11-day colony formation period.A total of 1 � 104 of infected CD34� cells were plated in complete methylcellulose medium without erythropoietin. Thearrows indicate typical CFU-GM (colony-forming unit-granulocyte/macrophage), CFU-G (colony-forming unit-granulocyte), and CFU-M (colony-forming unit-macrophage; left).Quantification of formation of blood colonies was shown (right). Images were captured with microscopy Nikon eclipse TS100 using 10� phase-contrast objective with numeric aperture0.3 and acquired through CCD DS-Qi1 camera and NIS-Elements F Version 3.0 software (Nikon) at room temperature. (D) Changes in the morphology of May-Grunwald-Giemsa–stainedCD34� cells infected with the viruses at day 13 of the differentiation culture. Images were captured at room temperature with Olympus BX51 microscope using 20� with numeric aperture0.5. (E) The number of cells in various differentiation stages. In total, 100 cells were counted per sample. The averages from 2 independent experiments are shown. MB indicatesmyeloblasts; PM, promyelocytes; MM, metamyelocytes; Band and Seg, band neutrophils and segmented neutrophils for granulocytic differentiation; Mob, monoblasts; PMo,promonocytes; Mo, monocytes; and Ma, macrophage for monocytic differentiation.

5000 WANG et al BLOOD, 24 MAY 2012 � VOLUME 119, NUMBER 21

For personal use only.on December 24, 2018. by guest www.bloodjournal.orgFrom

also showed that the ectopic expression of miR-29a or miR-142-3pcould decrease pRb level, which might be a consequence of thereduction of CCNT2 expression.

Previous studies showed that the monocytic differentiation ofBM cells is correlated to high levels of pRb, whereas duringneutrophilic differentiation, pRb levels are low.35 It has beendemonstrated that the importance of Rb phosphorylation in differ-ent pathways other than the cell cycle.36 In contrary, mechanisticstudies of the response of THP-1 cells to PMA treatment showedthat high levels of pRb are indispensible, and growth arrest at theG1 phase of the cell cycle is involved.37 Thus, reduced levels of pRbare indispensible for the monocytic differentiation of both promy-eloid cell lines and BM HSPCs, whether G1 phase arrest is inducedor not. This also accounts for our observation that the CCNT2protein levels decreased only during monocytic induction cultures butremained almost unchanged during granulocytic induction cultures.

We validated CDK6 as another target of miR-29a duringmyeloid differentiation. In vitro, CDK6 levels rapidly decreaseafter the onset of terminal granulocytic differentiation in 32Dcl3cells.38 In vivo, loss of CDK6 in the mouse affected the productionof terminally differentiated myeloid and erythroid cells.39 It is clearthat CDK6 blocks myeloid differentiation by interfering withRunx1� ability to bind DNA and with Runx1-C/EBP interactionin immature proliferating cells.40 Down-regulation of CDK6 there-fore provides a molecular switch that allows the differentiation-promoting activity of Runx proteins to be selectively activated interminally differentiating cells.

We also identified TAB2 as another target of miR-142-3p. TAB2has been shown to play a role in vitro as an adapter protein in thereceptor proximal signaling events of the proinflammatory IL-1and of several TNF-family members, including RANKL, toactivate the NF-�B pathway via binding to ubiquitinated TRAF2and TRAF6 in the cytoplasm.41-43 In this way, RANKL stimulatesosteoclast differentiation from hematopoietic precursor cells. Wepropose that the up-regulation of miR-142-3p blocks TAB2 expres-sion, which results in inhibition of osteoclast development, and as aconsequence, drives monocytic precursors to differentiate intomature macrophages. This miRNA-based mechanism underlyingthe commitment of monocyte progenitors to a single-cell fate was alsoobserved by Mann et al.44 They demonstrated that miR-155 drives thecommitment of monocyte progenitors toward activated macrophages byinterfering with the expression of MITF, which propels the sameprogenitors toward osteoclast differentiation.44 In the current study, wefound that knockdown of TAB2 also induced granulocytic differen-tiation in NB4 cells on ATRA treatment. Future studies are neededto explain the mechanism underlying this regulation of TAB2.

Because miR-29a and miR-142-3p have many proven andpotential targets, we could have expected to find that their functionin promoting myeloid differentiation is implemented by varioustargets. However, given the pro-myelopoiesis effects of CCNT2,CDK6, and TAB2 siRNA transfection in the current study, and thatthe expression of these proteins in the CCNT2, CDK6, and TAB2knockdown assays can be rescued by anti–miR-29a or anti–miR-142-3p transfection, it is therefore conceivable that miR-29a and

Figure 6. CDK6, TAB2, and CCNT2 expression levelsin monocytic and granulocytic induction cultures ofCD34� cells. (A-B) Western blot analysis of CDK6,TAB2, and CCNT2 in extracts from monocytic (A) andgranulocytic (B) induction cultures at the indicated times.Whole cell extracts were incubated with anti-CDK6,TAB2, or CCNT2 antibodies. (C) CCNT2 protein expres-sion was detected by Western blot analysis at day 4, 9,and 11 of monocytic induction cultures of CD34� cellsinfected with Lenti-29a, Lenti-142, or Lenti-control.(D) CDK6 protein expression was detected by Westernblot analysis at day 4, 9, and 11 of monocytic andgranulocytic induction cultures of CD34� cells infectedwith Lenti-29a or Lenti-control. (E) TAB2 protein expres-sion was detected by Western blot analysis at day 4, 9,and 11 of monocytic and granulocytic induction culturesof CD34� cells infected with Lenti-142 or Lenti-control.For the Western blots shown in this figure, GAPDHantibody was used to assess equal protein loading. Thesignal in each lane was quantified using Gelpro software,and the ratio of CCNT2, CDK6, and TAB2 to GAPDH wasdetermined. Because the CD34� cells are a mixturecontaining various HPCs and the sample amount waslimited, the 0-day samples was not included in theWestern blot analysis.

miR-29a AND miR-142-3p AND MYELOID DIFFERENTIATION 5001BLOOD, 24 MAY 2012 � VOLUME 119, NUMBER 21

For personal use only.on December 24, 2018. by guest www.bloodjournal.orgFrom

Figure 7. Expression of miR-29a and miR-142-3p in AML BM blasts and their function in promoting myelopoiesis. (A) Expression levels of miR-29a and miR-142-3p weredetermined by real-time PCR analysis of BM CD34� cells fromAMLpatients and healthy donors using TaqMan probes. The differences were shown to be significant by one-tailed unpairedMann-Whitney U nonparametric t test analysis. (B-E) BM CD34� cells from 3AMLpatients (patients 11, 5, and 6 were diagnosed as French-American-British M5, M2, and M4, respectively;all demonstrated low expression levels of miR-29a and miR-142-3p; supplemental Table 1) were infected with Lenti-miR-29a, Lenti-miR-142-3p, or Lenti-control. After infection for24 hours, the cells were cultured in monocytic or granulocytic induction medium for the indicated number of days, after which the cells were collected and the GFP-positive cells wereanalyzed for myeloid marker expression. The expression levels of CD14 (B) and CD11b (C) in lentivirus-infected blast cells (#11 and #5 of supplemental Table 1) were analyzed by FACSanalysis compared with their expression levels in CD34� BM cells from normal donors (N13 and N12). Red histogram represents unstained CD34� cells; and black histogram, cellsinduced for the indicated number of days. The monocytic (D) and granulocytic (E) differentiation of CD34� cells from AML patient 6 were determined by FACS analysis. (F) Western blotanalysis of the target proteins, CCNT2, CDK6, and TAB2. The protein extracts were obtained from the cells at day 6 of granulocytic and monocytic induction cultures of BM CD34� cellsderived fromAML 6, and infected with Lenti-29a, Lenti-142-3p, or Lenti-control. GAPDH was detected as the loading control.

5002 WANG et al BLOOD, 24 MAY 2012 � VOLUME 119, NUMBER 21

For personal use only.on December 24, 2018. by guest www.bloodjournal.orgFrom

miR-142-3p promote myeloid differentiation primarily by affectingthese 3 targets. Moreover, significantly increased levels of thetarget proteins were detected in the AML blasts with abnormallyreduced expression of miR-29a and miR-142-3p, which furthersuggested that the 2 miRNAs regulate myeloid differentiation andAML development via the 3 target genes.

Collectively, our data demonstrated that miR-29a and miR-142-3p play a key role in promoting granulopoiesis and monopo-iesis, and the abnormally decreased expression of these 2 miRNAsis responsible for the differentiation block of blast cells in someAML patients, whereas enforced expression of either miR-29a ormiR-142-3p could improve the differentiation status. Because wehave observed the universal down-regulation of these 2 miRNAs inAML M1 to M5 subtypes, increasing endogenous expression orectopic implantation of miR-29a and miR-142-3p may be apotential strategy for AML treatment.

Acknowledgments

The authors thank Dr Shao-Wei Wang from Beijing Hospital forassistance in UCB collection.

This work was supported by the National Natural ScienceFoundation of China (30970616 and 31171311; J.-W.Z.) and theSpecific Fund of National Laboratory of China (3060204; J.-W.Z.).

Authorship

Contribution: X.-S.W. designed and performed experiments, ana-lyzed data, and wrote the paper; J.-N.G. performed experimentsand analyzed data; J.Y., F.W., G.-H.Y., and C.S. performedexperiments; X.-H. Z., X.-L.Y., Z.-Q.T., and Z.-M.L. provided theexperimental materials of AML patients and healthy donors; andJ.-W.Z. designed the research, analyzed data and wrote the paper.

Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.

Correspondence: Jun-Wu Zhang, National Laboratory of Medi-cal Molecular Biology, Institute of Basic Medical Sciences,Chinese Academy of Medical Sciences and Peking Union MedicalCollege, 5 Dong Dan San Tiao, Beijing 100005, China; e-mail:[email protected].

References

1. Frohling S, Scholl C, Gilliland DG, Levine RL. Ge-netics of myeloid malignancies: pathogenetic andclinical implications. J Clin Oncol. 2005;23:6285-6295.

2. Vardiman JW, Harris NL, Brunning RD. TheWorld Health Organization (WHO) classificationof the myeloid neoplasms. Blood. 2002;100(7):2292-2302.

3. Metcalf D. Hematopoietic cytokines. Blood. 2008;111(2):485-491.

4. Friedman AD. Transcriptional control of granulo-cyte and monocyte development. Oncogene.2007;26(47):6816-6828.

5. Auffray C, Sieweke MH, Geissmann F. Bloodmonocytes: development, heterogeneity, and re-lationship with dendritic cells. Annu Rev Immunol.2009;27:669-692.

6. Bhana N. Granulocyte colony-stimulating factorsin the management of chemotherapy-inducedneutropenia: evidence based review. Curr OpinOncol. 2007;19(4):328-335.

7. Bartel DP. MicroRNAs: genomics, biogenesis,mechanism, and function. Cell. 2004;116(2):281-297.

8. Monticelli S, ed. MicroRNAs and the immunesystem: methods and protocols. Walker JM,ed. Methods in Molecular Biology, Vol. 667.Lausanne, Switzerland: Humana Press; 2010.

9. Wang F, Wang XS, Yang GH, et al. Down-regula-tion of miR-29a and miR-142-3p and their diag-nostic implication in human acute myeloid leuke-mia. Mol Biol Rep. 2012;39(3):2713-2722.

10. Garzon R, Volinia S, Liu CG, et al. MicroRNA sig-natures associated with cytogenetics and progno-sis in acute myeloid leukemia. Blood. 2008;111(6):3183-3189.

11. Marcucci G, Mrozek K, Radmacher MD, GarzonR, Bloomfield CD. The prognostic and functionalrole of microRNAs in acute myeloid leukemia.Blood. 2011;117(4):1121-1129.

12. Bellon M, Lepelletier Y, Hermine O, Nicot C.Deregulation of microRNA involved in hemato-poiesis and the immune response in HTLV-Iadult T-cell leukemia. Blood. 2009;113(20):4914-4917.

13. Careccia S, Mainardi S, Pelosi A, et al. A re-stricted signature of miRNAs distinguishes APLblasts from normal promyelocytes. Oncogene.2009;28(45):4034-4040.

14. Huang B, Zhao J, Lei Z, et al. miR-142-3p re-

stricts cAMP production in CD4�CD25� T cellsand CD4�CD25� TREG cells by targeting AC9mRNA. EMBO Rep. 2009;10(2):180-185.

15. Bissels U, Wild S, Tomiuk S, et al. Combinedcharacterization of microRNA and mRNA profilesdelineates early differentiation pathways ofCD133� and CD34� hematopoietic stem andprogenitor cells. Stem Cells. 2011;29(5):847-857.

16. Sun Y, Varambally S, Maher CA, et al. Targetingof microRNA-142-3p in dendritic cells regulatesendotoxin induced mortality. Blood. 2011;117(23):6172-6183.

17. Wu L, Cai C, Wang X, Liu M, Li X, Tang H. Mi-croRNA-142-3p, a new regulator of RAC1, sup-presses the migration and invasion of hepatocel-lular carcinoma cells. FEBS Lett. 2011;585(9):1322-1330.

18. Lanotte M, Martin-Thouvenin V, Najman S,Balerini P, Valensi F, Berger R. NB4, a maturationinducible cell line with t(15;17) marker isolatedfrom a human acute promyelocytic leukemia(M3). Blood. 1991;77(5):1080-1086.

19. Melnick A, Licht JD. Deconstructing a disease:RAR alpha, its fusion partners, and their roles inthe pathogenesis of acute promyelocytic leuke-mia. Blood. 1999;93(10):3167-3215.

20. Tsuchiya S, Yamabe M, Yamaguchi Y,Kobayashi Y, Konno T, Tada K. Establishmentand characterization of a human acute mono-cytic leukemia cell line (THP-1). Int J Cancer.1980;26(2):171-176.

21. Pierce A, Heyworth CM, Nicholls SE, et al. Anactivated protein kinase C alpha gives a differen-tiation signal for hematopoietic progenitor cellsand mimicks macrophage colony-stimulating fac-tors-stimulated signaling events. J Cell Biol.1998;140(6):1511-1518.

22. Breitman TR, Sclonick SE, Collins SJ. Inductionof differentiation of the human promyelocyticleukemia cell line (HL-60) by retinoic acid. ProcNatl Acad Sci U S A. 1980;77(5):2936-2940.

23. Huberman E, Callaham MF. Induction of terminaldifferentiation in human promyelocytic leukemiacells by tumor-promoting agents. Proc Natl AcadSci U S A. 1979;76(3):1293-1297.

24. Yang GH, Wang F, Yu J, Wang XS, Yuan JY,Zhang JW. MicroRNAs are involved in erythroiddifferentiation control. J Cell Biochem. 2009;107:548-556.

25. de Falco G, Giordano A. CDK9 (PITALRE): a mul-

tifunctional cdc2-related kinase. J Cell Physiol.1998;177(4):501-506.

26. De Luca A, Esposito V, Baldi A, et al. CDC2-re-lated kinase PITALRE phosphorylates pRb exclu-sively on serine and is widely expressed in hu-man tissues. J Cell Physiol. 1997;172(2):265-273.

27. Simone C, Bagella L, Bellan C, Giordano A.Physical interaction between pRb and cdk9/cy-clinT2 complex. Oncogene. 2002;21(26):4158-4165.

28. Zhao JJ, Lin J, Lwin T, et al. microRNA expres-sion profile and identification of miR-29 as a prog-nostic marker and pathogenetic factor by target-ing CDK6 in mantle cell lymphoma. Blood. 2010;115(13):2630-2639.

29. Kasashima K, Nakamura Y, Kozu T. Altered ex-pression profiles of microRNAs during TPA-in-duced differentiation of HL-60 cells. Biochem Bio-phys Res Commun. 2004;322(2):403-410.

30. Jin P, Wang E, Ren J, et al. Differentiation of twotypes of mobilized peripheral blood stem cells bymicroRNA and cDNA expression analysis.J Transl Med. 2008;6:39.

31. Chen A, Luo MY, Yuan GH, et al. Complementaryanalysis of microRNA and mRNA expression dur-ing phorbol 12-myristate 13-acetate (TPA)-in-duced differentiation of HL-60 cells. BiotechnolLett. 2008;30:2045-2052.

32. Han Y-C, Park CY, Bhagat G, et al. microRNA-29a induces aberrant self-renewal capacity in he-matopoietic progenitors, biased myeloid develop-ment, and acute myeloid leukemia. J Exp Med.2010;207(3):475-489.

33. Wei P, Gender ME, Fang SM, Fisher WH,Jones KA. A novel CDK9-associated C-type cy-clin interacts directly with HIV-1 Tat and mediatesits high-affinity, loop-specific binding to TAR RNA.Cell. 1998;92(4):451-462.

34. Kohoutek J, Li Q, Blazek D, Luo Z, Jiang H,Peterlin BM. Cyclin T2 is essential for mouse em-bryogenesis. Mol Cell Biol. 2009;29(12):3280-3285.

35. Bergh G, Ehinger M, Olsson I, Jacobsen SE,Gullberg U. Involvement of the retinoblastomaprotein in monocytic and neutrophilic lineagecommitment of human bone marrow progenitorcells. Blood. 1999;94(6):1971-1978.

36. Sebastian T, Malik R, Thomas S, Sage J,Johnson PF. C/EBPbeta cooperates with RB:

miR-29a AND miR-142-3p AND MYELOID DIFFERENTIATION 5003BLOOD, 24 MAY 2012 � VOLUME 119, NUMBER 21

For personal use only.on December 24, 2018. by guest www.bloodjournal.orgFrom

E2F to implement Ras(V12)-induced cellularsenescence. EMBO J. 2005;24(18):3301-3312.

37. Traore K, Trush MA, George M Jr,Spannhake EW, Anderson W, Asseffa A. Signaltransduction of phorbol 12-myristate 13-acetate(PMA)-induced growth inhibition of human mono-cytic leukemia THP-1 cells is reactive oxygen de-pendent. Leuk Res. 2005;29(8):863-879.

38. Rane SG, Mangan JK, Amanullah A, et al. Activa-tion of the Jak3 pathway is associated with granu-locytic differentiation of myeloid precursor cells.Blood. 2002;100(8):2753-2762.

39. Malumbres M, Sotillo R, Santamaria D, et al.Mammalian cells cycle without the D-type cyclin-

dependent kinases Cdk4 and Cdk6. Cell. 2004;118(4):493-504.

40. Fujimoto T, Anderson K, Jacobsen SEW,Nishikawa S-I, Nerlov C. Cdk6 blocks myeloiddifferentiation by interfering with Runx1 DNAbinding and Runx1-C/EBPa interaction. EMBO J.2007;26(9):2361-2370.

41. Morlon A, Munnich A, Munnich AS. TAB2, TRAF6and TAK1 are involved in NF-kappaB activationinduced by the TNF-receptor, Edar and its adap-tor Edaradd. Hum Mol Genet. 2005;14(23):3751-3757.

42. Takaesu G, Kishida S, Hiyama A, et al. TAB2, anovel adaptor protein, mediates activation of

TAK1 MAPKKK by linking TAK1 to TRAF6 in theIL-1 signal transduction pathway. Mol Cell. 2000;5(4):649-658.

43. Mizukami J, Takaesu G, Akatsuka H, et al. Re-ceptor activator of NF-kappaB ligand (RANKL)activates TAK1 mitogen-activated protein kinasekinase kinase through a signaling complex con-taining RANK, TAB2, and TRAF6. Mol Cell Biol.2002;22(4):992-1000.

44. Mann M, Barad O, Agami R, Geiger B,Hornstein E. miRNA-based mechanism for thecommitment of multipotent progenitors to a singlecellular fate. Proc Natl Acad Sci U S A. 2010;107(36):15804-15809.

5004 WANG et al BLOOD, 24 MAY 2012 � VOLUME 119, NUMBER 21

For personal use only.on December 24, 2018. by guest www.bloodjournal.orgFrom

online April 4, 2012 originally publisheddoi:10.1182/blood-2011-10-385716

2012 119: 4992-5004  

Tan, Zi-Mian Luo, Gui-Hua Yang, Chao Shen and Jun-Wu ZhangXiao-Shuang Wang, Jia-Nan Gong, Jia Yu, Fang Wang, Xin-Hua Zhang, Xiao-Lin Yin, Zhen-Qing differentiation and acute myeloid leukemiaMicroRNA-29a and microRNA-142-3p are regulators of myeloid 

http://www.bloodjournal.org/content/119/21/4992.full.htmlUpdated information and services can be found at:

(672 articles)Phagocytes, Granulocytes, and Myelopoiesis    (1897 articles)Myeloid Neoplasia   

Articles on similar topics can be found in the following Blood collections

http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requestsInformation about reproducing this article in parts or in its entirety may be found online at:

http://www.bloodjournal.org/site/misc/rights.xhtml#reprintsInformation about ordering reprints may be found online at:

http://www.bloodjournal.org/site/subscriptions/index.xhtmlInformation about subscriptions and ASH membership may be found online at:

  Copyright 2011 by The American Society of Hematology; all rights reserved.of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society

For personal use only.on December 24, 2018. by guest www.bloodjournal.orgFrom