miRNA-130a Targets ATG2B and DICER1 to Inhibit Autophagy ... · A total of 1 107 primary B cells were transfected with the human B-cell Nucleofector Kit and program U-015 with 1 mmol/L

Molecular and Cellular Pathobiology

miRNA-130a Targets ATG2B and DICER1 to InhibitAutophagy and Trigger Killing of Chronic LymphocyticLeukemia Cells

Valentina Kovaleva1, Rodrigo Mora2, Yoon Jung Park3,6, Christoph Plass3, Abhilash I. Chiramel4,Ralf Bartenschlager4, Hartmut D€ohner5, Stephan Stilgenbauer5, Armin Pscherer1,Peter Lichter1, and Martina Seiffert1

AbstractToxicity and relapses from the immunochemotherapy used to treat chronic lymphocytic leukemia (CLL)

prompt continued interest in gentle but effective targeted treatment options for the mainly elderly populationsuffering from this disease. Here, we report the definition of critical CLL cell survival pathways that can betargeted by ectopic reexpression of the miRNA genes miR-130a and miR-143 which are widely downregulated inCLL. Notably, miR-130a inhibited autophagy by reducing autophagosome formation, an effect mediated bydownregulation of the genesATG2B andDICER1, the latter of which is amajor component of themiRNA silencingmachinery. In support of the concept of a fundamental connection between miRNA disregulation and alteredautophagic flux in this cancer, we showed that RNA interference–mediated knockdown ofDICER1 expressionwassufficient to reduce autophagy in primary or established cultures of CLL cells. Together, our findings show thatmiR-130a modulates cell survival programs by regulating autophagic flux, and they define roles for miR-130a andDicer1 in a regulatory feedback loop that mediates CLL cell survival. Cancer Res; 72(7); 1763–72. �2012 AACR.

IntroductionB-cell chronic lymphocytic leukemia (CLL) is characterized

by the expansion of CD5þ/CD19þ/CD23þ B lymphocytes andshows diverse pathogenic mechanisms and a heterogeneousclinical course (1, 2). Inherent defects in cell death of CLL Blymphocytes that are nourished by a small proliferative pool inlymph nodes, spleen, and bone marrow are responsible for themassive accumulation of malignant cells. Prolonged CLL cellsurvival is induced by autocrine signaling pathways and sur-vival stimuli from the microenvironment (3, 4).

miRNAs are endogenously expressed small RNA moleculesthat mediate posttranscriptional gene silencing and have thecapacity to simultaneously regulate tens to hundreds of targetgenes (5). Thereby, these noncoding RNAs are involved in theregulation of multiple cellular processes including prolifera-tion, apoptosis, development, and differentiation (6), and havealso been associated with cancer. SpecificmiRNAswere shownto be differentially expressed between normal and tumor cells,including CLL (7, 8) and are therefore potential targets foranticancer therapy.

Autophagy is one of the key protective cellular pathways thatmediate stress-induced adaptation and damage control.Macroautophagy (hereafter referred to as autophagy) is amajor type of autophagy, which sequesters organelles andlong-lived proteins in membrane-coated vesicles, so calledautophagosomes. This survival mechanism, induced by vari-ous types of stress, can block intrinsic and extrinsic apoptoticpathways (9), and cancer cells exploit autophagy to overcomestarvation and hypoxia. Interestingly, anticancer therapy suchas DNA-damaging drugs or hormone antagonists induceautophagy as prosurvival response (10, 11) and the combina-tion of standard therapies with chloroquine, an inhibitor ofautophagy, has been shown to enhance treatment efficacy (12).Therefore, the ability to modulate autophagy is extensivelystudied in cancer research. However, only few studies haveinvestigated autophagy regulation on the miRNA level (13, 14).The involvement of autophagy in CLL has been reported in theliterature, in which induction of autophagy in CLL cells upondasatinib or dexamethasone treatment was described (15, 16).In addition, it was shown that chloroquine induces apoptosis

Authors' Affiliations: 1MolecularGenetics; 2IntegrativeBioinformatics andSystems Biology; 3Epigenomics and Cancer Risk Factors, German CancerResearch Center; 4Department for Infectious Diseases - Molecular Virol-ogy, University of Heidelberg, Heidelberg; 5Internal Medicine III, Universityof Ulm, Ulm, Germany; and 6Department of Nutritional Science and FoodManagement, College of Health Science, EwhaWomans University, Seoul,Republic of Korea

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Current address for R. Mora: Department of Medical Virology, Faculty ofMicrobiology, University of Costa Rica, San Pedro, Costa Rica.

Current address for A. Pscherer: BioRN Cluster Management GmbH,Heidelberg, Germany.

Corresponding Author: Peter Lichter, German Cancer Research Center,Department for Molecular Genetics, Im Neuenheimer Feld 280, 69120Heidelberg, Germany. Phone: 49-6221-42-4619; Fax: 49-6221-42-4639;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-11-3671

�2012 American Association for Cancer Research.

CancerResearch

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on April 5, 2021. © 2012 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst February 20, 2012; DOI: 10.1158/0008-5472.CAN-11-3671

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in CLL cells in vitro (17) and might therefore enhance theefficacy of drugs currently used for CLL treatment.

In this study, we investigate the effects of 5 miRNAs that aredownregulated in CLL on the regulation of cell death/cellsurvival programs. We show that enforced expression ofmiR-130a impairs cell viability and inhibits autophagic fluxin CLL cells, suggesting a new miRNA-autophagy regulatoryaxis for modulating survival mechanisms in CLL cells.

Materials and MethodsCell lines

HEK293T and HS-5 cells were purchased from AmericanType Culture Collection and retested and authenticated in03.2011 and 05.2008, respectively. MEC-1 cells were obtainedfrom DSMZ (Braunschweig, Germany) in 2009 and not pas-saged longer than 6 months continuously. Cells were culturedin Dulbecco's Modified Eagle's Medium (Invitrogen) supple-mented with 10% fetal calf serum (Biochrom) and 1% peni-cillin/streptomycin (Gibco BRL, Invitrogen; referred to ascomplete medium) at 37�C and 10% CO2.

Primary cellsPeripheral blood (PB) samples were collected from CLL

patients (Supplementary Table S1) and healthy donors afterinformed consent. All cases matched standard diagnosticcriteria (18). Mononuclear cells were isolated and cultured asreported before (19).

RNA and DNA isolationTotal RNAwith orwithoutmiRNA fractionwas isolatedwith

miRNeasy or RNeasy Mini Kit, respectively (QIAGEN). Geno-mic DNA was isolated with Blood & Cell Culture DNAMidi Kit(QIAGEN).

miRNA expression analysisTotal RNA (700 ng) was employed for miRNA expression

analysis using Bead-based miRNA expression assay (Illumina).The array data were analyzed by BeadStudio v3.2 software(Illumina) and are available at the National Center for Bio-technology Information Gene Expression Omnibus (GEO)database, accession no. GSE31599. For further validation ofmiRNA expression, quantitative real-time PCR (qRT–PCR)analysiswas carried out byTaqManMicroRNAAssays (AppliedBiosystems).

miRNA precursor molecules, miRNA inhibitors, andsiRNAs

All syntheticmiRs and anti-miRs including Negative Controlpre-miRNA (AM17110) and Negative Control anti-miR Inhib-itor (AM17010) were purchased from Applied Biosystems.Silencer Select Negative Control siRNA (4390843) and DICER1siRNA (s23755) were obtained from Applied Biosystems.

Nucleofection of cell lines and primary CLL cellsA total of 1 � 107 primary B cells were transfected with

the human B-cell Nucleofector Kit and program U-015 with1 mmol/L miRNA or siRNA according to the manufacturer's

instructions (Lonza). After nucleofection, primary cells werecultured in 4 mL of sterile filtered conditioned mediumobtained from HS-5 cell cultures. Transfection of MEC-1 cellswas carried out by nucleofection (solution V, program X-001)with 5 � 106 cells and 0.5 mmol/L miRNA or siRNA andsubsequent culture in complete medium.

Construction of DICER1 and ATG2B 30 untranslatedregion luciferase plasmids and reporter assay

The 30 untranslated region (UTR) fragments of DICER1 andATG2B containing the miRNA target sites were amplified fromMEC-1 genomic DNA by PCR using FastStart Taq DNA Poly-merase (Roche Diagnostics) and the primers listed in Supple-mentary Table S2. PCR products were cloned into pMIR-REPORT luciferase plasmid (Applied Biosystems) usingHindIIIand SpeI restriction sites. For reporter assays, 1� 105 HEK293Tcells were cotransfected with 5 ng of 30UTR reporter plasmid,50 ng pRL-TK Renilla luciferase reporter vector (Promega), and30 nmol/LmiR-130a orNegative ControlmiRNA using TransITtransfection reagent, according to the manufacturer's instruc-tions (Mirus BioLLC). Transfected cells were lysed 36 hoursafter transfection and luciferase activities were assayed by aDual-Luciferase Reporter System (Promega).

Cell viability analysisCell viability was assessed by flow cytometry after Annexin

V–phycoerythrin (PE) and 7-aminoactinomycin D (7-AAD)staining (BD Biosciences) as described before (20). Double-negative cells were counted as viable cells. Fluorescence-acti-vated cell sorting (FACS) analyses were carried out with aFACSCanto II flow cytometer equipped with FACSDiva V6.1.2software (BD Biosciences).

qRT-PCRFor mRNA quantification, total RNA was isolated from cells

48 hours after transfection and transcribed with SuperScript IIand anchored oligo-d(T)20 primer (Invitrogen). Amplificationand quantification of cDNA was carried out with SYBR GreenROX Mix (Abgene, Epsome) according to the manufacturer'sprotocol and as described before (20). qRT-PCRwas carried outin 7900 HT Fast Real-Time PCR System (Applied Biosystems).Relative quantification was done in relation to 2 housekeepinggenes (LAMINB1 and PGK1). The qRT-PCR primer sequencesare listed in Supplementary Table S2.

Western blottingCells were harvested 48 hours after transfection and lysed in

50 mmol/L Tris-HCl, pH 7.5; 150 mmol/L NaCl; 1% NP-40,containing Complete Mini protease inhibitor mixture (RocheDiagnostics). Protein extracts were electrophoresed on 4% to12% linear gradient Bis-Tris ready gels (Invitrogen) and trans-ferred to Immobilon-P polyvinylidene fluoride membranes(Millipore). Membranes were probed with antibodies specificfor Dicer1 (1:1,000, ab14601; Abcam), LC3-B (1:1,000, 2775 S;Cell Signaling, Inc.), a-tubulin (1:2,500; Sigma Aldrich), andglyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:2,500,CB1001; Calbiochem), and subsequently with horseradish per-oxidase–coupled anti-rabbit or anti-mouse antibodies (both

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1:2,500; Cell Signaling Inc.). Specific bands were visualized withECL Blotting Detection Reagents (AmershamBiosciences) andquantified with Image J software.

Generation of MEC-1/GFP-LC3 cellsTo generate a stable cell line expressing GFP-LC3 fusion

protein, MEC-1 cells were transduced with the pWPI lentiviralvector containing the GFP-LC3 sequence, using a titer of 2.6�106 for 5� 105 cells and selected in the presence of puromycin(Invitrogen) for 2 weeks.

Imaging of autophagic fluxAutophagic flux was quantified by an imaging flow cyt-

ometer (Image Stream, Amnis) employing 2� 106MEC-1/GFP-LC3 cells per analyzed sample. For starvation, cells weretransferred to Krebs-Henseleit buffer (Sigma-Aldrich) 24 hoursafter transfection and incubated for further 8 hours in thepresence or absence of 100 nmol/L lysosomal H1-ATPaseinhibitor bafilomycin A1 (Merck KGaA). Subsequently, cellswere harvested and resuspended in 100 mL of cold PBS/2%bovine serum albumin. To exclude dead cells from the analysis,TO-PRO3 (quinolinium, 4-[3-(3-methyl-2(3H)-benzothiazolyli-dene)-1-propenyl]-1-[3-(trimethylammonio)propyl], diiodide;Invitrogen) was added directly before acquisition at a finalconcentration of 1 mmol/L. For each sample, images of 5,000cells were acquired in the bright field channel and the fluo-rescence emission channels at 660 nm (TO-PRO-3) and 520 nm(GFP). The acquired images were analyzed with IDEAS soft-ware (Amnis) as described in Supplementary Material. Thestatistical significance of the differences between the autop-hagic flux distributions was assessed using a Kolmogorov-Smirnov test. In addition, the histogram channels of theautophagic flux distribution presenting a significant differencein frequencies were evidenced by the Student t test (seeSupplementary Material for details).

ResultsA set of miRNAs is downregulated in CLLPrevious studies have reported that the majority of miRNAs

are downregulated in CLL when compared with normal B cells(21). To identify deregulated miRNAs, we carried out miRNAexpression profiling on RNA samples of 18 CLL patients and onpooled RNAof PBB lymphocytes of 5 healthy donors bymiRNAarrays. MiR-126, miR-130a, miR-143, miR-181a, and miR-326were among the top 20 downregulatedmiRNAs in CLL patients(GEOno. GSE31599). To confirm this finding, we quantified therespective miRNAs by qRT-PCR in samples of 6 CLL patients.Both methods showed lower expression levels of these 5miRNAs in CLL cells compared with normal B cells andqRT-PCR values ranged from 1.5- to approximately 20-foldreduction (Fig. 1).To elucidate whether the lower expression levels of these

miRNAs in CLL is due to epigenetic transcriptional silencing,we assessed the methylation status of putative miRNA pro-moter regions by MassArray. Out of the tested miRNAs, onlymiR-126 showed a significant gain in methylation within itspredicted transcriptional start site in CLL cells compared with

healthy B-cell control (P¼ 0.007; Supplementary Fig. S1). Smallsubsets of patients showed hypermethylation of miR-326 andmiR-181a, but the average methylation values were not signif-icantly different compared with the controls. This result indi-cates that the majority of the tested sequences are not affectedby epigenetic regulation, and aberrant methylation is not themain reason for the deregulated expression of the studiedmiRNAs in our CLL patient cohort. However, we cannot ruleout that other, not yet identified miRNA regulating sequencesare under the influence of epigenetic regulation.

miR-130a and miR-143 induce cell death in CLL cellsTo investigate the function of the underrepresentedmiRNAs

in CLL, we first assessed their impact on cell viability becauseaccumulation of quiescent CLL cells is primarily due to defectsin apoptosis induction, rather than increased cell proliferationas reflected in gene expression (22, 23). Freshly isolated CLLcells were transiently transfected with one of the followingsynthetic miRNAs: miR-126, miR-130a, miR-143, miR-181a,miR-326, or with an unspecificmiRNA used as negative control(NC). Cell viability wasmeasured 48 hours after transfection byflow cytometry after Annexin V–PE/7-AAD staining. Introduc-tion of miR-130a, miR-143, and miR-181a led to an averagereduction in the number of viable cells of 21%, 15%, and 23%respectively, when compared with the NC set as 100% viability(PmiR-130a ¼ 0.01; PmiR-143 ¼ 0.02; PmiR-181a ¼ 0.03; Fig. 2A).Furthermore, we tested whether the above-mentioned 5miRNAs have an impact on the viability of the CLL cell lineMEC-1. Twenty-four hours after transfection of miR-130a ormiR-143, the number of viable MEC-1 cells was decreased by57% or 50%, respectively, when compared with unspecificmiRNA control (PmiR-130a ¼ 0.001; PmiR-143 ¼ 0.001; Fig. 2B).MiR-181a slightly, though not significantly, reduced cell via-bility of MEC-1 cells, whereas miR-126 and miR-326 did notaffect viability of either primary or cell line CLL cells. Theseresults indicate that miR-130a and miR-143 induce cell deathboth in primary CLL and MEC-1 cells.

Figure 1. miR-126, miR-130a, miR-143, miR-181a, and miR-326 aredownregulated in CLL cells. miRNA expression was assessed by arrayand qRT-PCR. Array data were normalized to the median of all miRNAexpression values within an individual sample. qRT-PCR data werenormalized to the average of RNU-6B and RNU-66. Mean log2 foldchanges andSDs (array: n¼18; qRT-PCR:n¼6) comparedwith apool of5 healthy donor (HD) B-lymphocyte samples are depicted.

miR-130a Regulates Autophagy in CLL

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Autophagy is induced in CLL cells under starvationWe next sought to investigate the mechanism of how miR-

130a and miR-143 impact survival-related pathways in CLL.Autophagy is one possible mechanism of how cancer cellsovercome cellular stress and maintain cell survival and resis-tance to apoptosis (11). To investigate the relevance of auto-phagy in CLL cell survival, we used chloroquine, a late stageinhibitor of autophagy, in primary CLL cultures and observeddecreased cell viability in a dose-dependent manner (Supple-mentary Fig. S2), which confirms results published earlier (17).In addition, chloroquine treatment resulted in enhanced flu-darabine-induced cell death in primary CLL cells (data notshown). To evaluate the role of autophagy in CLL cells in moredetail, we followed the specific autophagosome marker LC3, amicrotubule-associated protein 1 light chain 3, which isinvolved in the formation of autophagosomes and translocatesfroma soluble form, LC3-I, to the autophagosomalmembrane–bound form LC3-II (24). For this means, we generated a stableMEC-1 cell line expressing GFP-tagged LC3 (MEC-1/GFP-LC3).Accumulation of fluorescent dots, representing autophago-somes, was evaluated in these cells by imaging flow cytometryas described in the Materials and Methods section. Becauseautophagic flux can be measured as the difference in auto-phagosome formation between samples in the presence orabsence of inhibitors of autophagosome degradation (25),we quantified GFP-positive autophagosomes in cells treatedfor 8 hours with or wihout the lysosomal H1-ATPase inhibitorbafilomycin A1 (BaF).

In nonstress conditions, the basal level of autophagy can bevery low, but might be induced upon starvation. Therefore, wedetermined autophagic flux in MEC-1/GFP-LC3 cells culturedin complete medium or under starvation conditions. Repre-sentative images of analyzed cells are presented in Fig. 3A,

showing increased GFP-LC3 clustering in bafilomycin-treatedcells under starvation conditions. The level of GFP-LC3 clus-tering was extracted from the individual cellular images andrepresented by the bright detail intensity feature (Fig. 3A,bottom). A nonparametric description derived from the Kol-mogorov-Smirnov (K-S) statistics was applied for the quanti-fication and representation of these data, as described inSupplementary Material. Briefly, we calculated the autophagicflux of MEC-1/GFP-LC3 cells as the difference in bright detailintensities between bafilomycin-treated and bafilomycin-untreated states (Fig. 3B, left and middle). The obtained dataindicate that MEC-1 cells have a constitutively active autop-hagy in complete medium conditions, which is extensivelyupregulated upon starvation as indicated by the right shift ofthe curve (Fig. 3B, right).

Autophagy might therefore be a survival mechanism of CLLcells. All subsequent analyses of autophagic flux in MEC-1/GFP-LC3 cells were conducted under starvation conditions toobtain high assay sensitivity.

miR-130a inhibits autophagic flux in CLLTo investigate the impact of miR-130a and miR-143 on

autophagy in CLL cells, MEC-1/GFP-LC3 cells were transfectedwith these 2 synthetic miRNAs or a scrambled miRNA used asNC, and autophagic flux was analyzed. Introduction of miR-130a resulted in a robust change in the autophagic flux dis-tributions showing lower frequencies when compared with theNC. A summary of 4 independent experiments showed statis-tically significant reduction of autophagosome formation (Kol-mogorov-Smirnov test for each individual experiment, P < 0.05;mean histogram channels of 4 experiments with statisticaldifference are labeled with dots, Student t test, P < 0.05),indicating that miR-130a inhibits autophagic flux in MEC-1

Figure 2. miR-130a and miR-143induce cell death in primary CLLand MEC-1 cells. A, primary CLLcells were transfected withmiRNAs and cell viability wasquantified after 48 hours byAnnexin V–PE/7-AAD staining.One representative FACS data anda summary of 4 independentexperiments are depicted. Meanvalues and SDs of relative cellviability are shown (NC miRNA setto 100%). Paired t test was appliedto calculate statistical significance.�, P � 0.05; ��, P � 0.01. B,respective experiments werecarried out with MEC-1 cells, inwhich cell viability was assessed24 hours after transfection.

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cells (Fig. 3C). Transfection of cells withmiR-143 did not revealstatistically significant changes in the autophagic flux distri-bution (data not shown).To confirm our observations by an independent assay, we

conducted Western blot analysis for endogenous LC3 proteinbyMEC-1 cells treated under the same conditions as describedabove. Autophagic flux was assessed by calculating the differ-ence of LC3-II protein of samples in the presence or absence ofbafilomycin (DLC3-II) as reported (26). These experimentsrevealed decreased DLC3-II in miR-130a–transfected samples(Fig. 3D, left), which confirms reduced autophagic flux inMEC-

1 cells upon introduction of miR-130a. We further investigatedthe role of miR-130a in autophagy in primary CLL cells byWestern blot analysis. Similar as inMEC-1 cells, transfection ofprimary CLL cells with miR-130a resulted in a decrease inDLC3-II (Fig. 3D, right). These results show for the first timethat autophagy is an active process in primary CLL cells and isregulated by miR-130a.

DICER1 and ATG2B are direct targets of miR-130aTo identify genes that are regulated by miR-130a, we ana-

lyzed previously reported transcriptome data and searched for

Figure 3. miRNA-130a reduces autophagic flux in CLL cells. A, autophagy was assessed in MEC-1/GFP-LC3 cells with an imaging flow cytometer. Top,representative images of analyzed cells cultured in completemediumor under starvation conditions in the presence or absence of bafilomycin. Bottom, brightdetail intensity feature distribution representing GFP clustering. B, cumulative distribution function of bright detail intensity of MEC-1/GFP-LC3 cellsincubated with or without bafilomycin in complete medium (left) or under starvation (middle). Autophagic flux of MEC-1/GFP-LC3 cells cultured in completemedium (black), or under starvation conditions (red), calculated as the difference between the cumulative distribution functions between bafilomycin-treatedand untreated state. C, normalized autophagic flux of MEC-1/GFP-LC3 cells transfected with either NC miRNA (blue) or miR-130a (red), cultured understarvation conditions. Autophagic flux distributions are represented as average of 4 independent experiments (solid lines)� 1 SEM (dashed blue or red lines).The Student t test was conducted for each channel. Channels with significant difference between autophagic flux distributions are marked as dotted blacklines above the plots. D, Western blot analysis of LC3 protein with MEC-1 or primary CLL cells transfected with miR-130a or unspecificmiRNA (NC). Twenty-four hours after transfection, bafilomycin was added where indicated. One representative result is shown, in which numbers below the blot represent LC3-IIvalues normalized to a-tubulin. In the bar plots below, mean values� SDs of relative DLC3-II levels, which were calculated as the difference of LC3-II proteinbetween bafilomycin-treated and untreated states and were set to NC ¼ 1, are depicted (n ¼ 3). �, P � 0.1.






transcripts with higher expression levels in primary CLLsamples compared with healthy B cells (27). These differen-tially expressed geneswere analyzed formiR-130a binding sitesby Targetscan software. By this means, DICER1was selected asa novel potential miR-130a target gene because it was upre-gulated 2.3-fold in CLL cells relative to healthy donor controland has a highly conserved binding site for miR-130a. Inaddition, autophagy-related genes were screened for miR-130a binding sequences and thereby ATG2B, a mediator ofautophagy, was identified and selected for further studies.The miR-130a binding site within the 30UTR of DICER1 isconserved between human, mouse, rat, dog, and chicken; andof ATG2B between human, dog, and chicken (Fig. 4A).

To examine miR-130a–mediated regulation of the selectedgenes, we transfected primary CLL cells with synthetic miR-130a and assessed DICER1 and ATG2B mRNA levels by qRT-PCR 48 hours after transfection. This resulted in a 1.4-foldreduction of DICER1 (P ¼ 0.002) and a 1.8-fold reduction ofATG2B (P ¼ 0.2) mRNA levels when compared with transfec-tion with unspecific control miRNA (Fig. 4B).

Furthermore, to confirm that the downregulation of thetested mRNA is due to direct interaction between miR-130aand its predicted binding site, we cotransfected HEK293T cellswith miR-130a and pMiR luciferase reporter constructs con-taining 30UTR fragments of DICER1 and ATG2B. In the pres-ence of miR-130a, relative luciferase activity assessed 36 hoursafter transfection for the DICER1 and ATG2B constructs wasreduced by 25% (P¼ 0.09) and 37% (P¼ 0.03), respectively (Fig.4C). Importantly, inhibition of endogenous miR-130a by trans-

fection of anti–miR-130a (inhibitor of miR-130a) in HEK293Tcells increased the luciferase activities of reporter constructsfor DICER1 and ATG2B by 10% (P ¼ 0.05) and 33% (P ¼ 0.03),respectively (Fig. 4C).

These results show that miR-130a downregulates DICER1and ATG2BmRNAs by direct interaction with their 30UTRs andtherefore identifies these genes as novel targets formiR-130a inCLL cells.

miR-130a downregulates Dicer1 protein expression inprimary CLL and MEC-1 cells

We next assessed whether the observed regulation ofDICER1 and ATG2B by miR-130a is reflected by changes onprotein level. Due to the lack of ATG2B-specific antibodies,this could not be achieved for this protein so far. For Dicer1,Western blot analyses were conducted with lysates of pri-mary CLL cells transiently transfected with miR-130a orcontrol miRNA. Dicer1 protein levels were analyzed byquantifying the bands corresponding to full-length Dicer1protein of 218.7 kDa, which is the upper band in the blotsshown in Fig. 5A. In 3 analyzed samples of primary CLL cells,introduction of miR-130a resulted in reduced Dicer1 levelswhen compared with unspecific miRNA transfection (Fig.5A). A respective experiment using B lymphocytes of ahealthy donor showed reduced Dicer1 protein after miR-130a transfection in these cells as well (Fig. 5A). Further-more, introduction of miR-130a in MEC-1 cells resulted in adownregulation of Dicer1 by 2.3-fold relative to unspecificmiRNA control (Fig. 5B).

Figure 4. DICER1 andATG2B are direct targets ofmiR-130a. A, schematic representation of the 30UTRs ofDICER1 andATG2B indicating the fragmentswhichwere cloned into the luciferase reporter construct pMIR-REPORT. Evolutionary conserved regions containing the miR-130a binding sites are highlighted byframes in the sequence alignments of different species. B, expression level of DICER1 and ATG2BmRNA quantified by qRT-PCR 48 hours after miR-130atransfection in primary CLL cells. mRNA levels were normalized to 2 housekeeping genes (LAMINB1 and PGK1) and are presented as mean values � SEMrelative to NC results (DICER1, n ¼ 6; ATG2B, n ¼ 3). Paired t test was applied to assess statistical significance. PDICER1 ¼ 0.002; PATG2B ¼ 0.2. C, 30UTRluciferase reporter assay for DICER1 and ATG2B. HEK293T cells were cotransfected with pMIR-30UTR luciferase reporter construct and miR-130a oranti–miR-130a and respective controls (NC or anti-NC). Relative luciferase intensity was assessed as described in the Materials and Methods section and ispresented as mean � SEM of 4 independent experiments. �, P � 0.05.

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Knockdown of Dicer1 leads to reduced autophagy inprimary CLL and MEC-1 cellsTo investigate whether Dicer1 is involved in autophagy in

CLL cells, we transfected DICER1-specific siRNA inMEC-1 andprimary CLL cells, which resulted in a reduction of Dicer1protein 48 hours after transfection of around 75% and 60%,respectively. Autophagic flux was evaluated in the transfectedcells in the presence and absence of bafilomycin by Westernblotting of LC3 protein and revealed a considerable reductionof DLC3-II after knockdown of Dicer1 both in MEC-1 andprimary CLL cells (Fig. 6). This suggests that inhibition ofautophagy in CLL cells by miR-130a is mediated at least partlyby its target gene DICER1.Because Dicer1 is a key component of themiRNA biogenesis

machinery, it was likely that downregulation of Dicer1 by miR-130a results in reduced levels of miRNAs in general. Therefore,we measured expression levels of 7 different miRNAs (miR-21,miR-26a, miR-28, miR-146a, miR-148b, miR-193b, and let-7c)after transfection of miR-130a in MEC-1 cells. This resulted inreduced expression levels of all 7 tested miRNAs (between 0.4-and 0.8-fold) comparedwith transfections of unspecificmiRNA(data not shown). Therefore, the observed effect of miR-130aon cell survival and autophagy in CLL cells might be due tomajor changes in the miRNA profile of the cells.Taken together, our data show that miR-130a regulates

autophagic flux and cell viability in both MEC-1 and primaryCLL cells and suggest that its target geneDICER1 is involved inthis process.

DiscussionDeregulated expression of miRNAs is a common feature of

many tumor entities, including CLL. Here, specific miRNAsignatures were shown to differentiate patients with short andlong intervals to therapy (28, 29). The biological role of most ofthese miRNAs in tumor development and progression is

however still unclear. The aim of this study was therefore afunctional analysis of 5 deregulated miRNAs in CLL, miR-126,miR-130a, miR-143, miR-181a, andmiR-326, which were shownto be underrepresented in CLL cells compared with normal Bcells by us and others (21). For some of these deregulatedmiRNAs implications inCLLbiology have been suggested:miR-181a has been reported to regulate the CLL relevant oncogenes

Figure 5. Western blot analysisconfirms downregulation of Dicer1protein bymiR-130a. A,Western blotfor Dicer1 using primary CLLsamples as well as B cells from ahealthy donor (HD) transfected withmiR-130a or NC miRNA.Nontransfected B cells were used ascontrol (NT). Dicer1- and GAPDH-specific bands were quantified byImageJ software and Dicer1 proteinexpression levels were normalized toGAPDH values. B, respectiveexperiments were carried out withMEC-1 cells, of which 1representative result is shown. Meanvalues � SD of 3 independentexperiments are shown.

Figure 6. Knockdown of Dicer1 results in reduced autophagic flux inMEC-1 and primary CLL cells. MEC-1 (A) or primary CLL cells (B) weretransfected with DICER1-specific siRNA (siDicer) or Negative ControlsiRNA (siCo) and cultured for 40 hours in complete medium. MEC-1 cellswere then transferred to Krebs–Henseleit buffer, and bafilomycin wasadded where indicated (BaF) for further 8 hours. Results ofnontransfected MEC-1 cells either in complete medium (CM) or understarvation (KHS) are shown in addition. Western blot results for Dicer1and LC3-II were normalized to a-tubulin, and differences of LC3-II inBaF-treated and untreated samples were calculated (DLC3-II) and set tosiCo ¼ 1. Mean values � SDs of relative DLC3-II of 2 independentexperiments each are depicted on the right.






TCL1 and PLAG1 (21, 30), andmiR-143 which is downregulatedin several B-cell malignancies, was shown to target ERK5, amitogen—activated protein kinase protein (31).

One of the reasons for deregulated miRNA expression incancer is promoter silencing due to aberrant DNA methyl-ation. Therefore, we examined the promoter methylationstatus of the downregulated miRNAs in CLL patients andobserved a significant increase in promoter methylation ofmiR-126 in comparison with normal B cells. This is again inaccordance with data published by Pallasch and colleagues(21) who additionally showed a tendency for hypermethyla-tion of the promoter regions of miR-130a, miR-181a, andmiR-326.

Our primary interest was to elucidate a possible role of thedownregulated miRNAs in the persistent survival of CLL cells.By miRNA transfection studies, we showed that miR-130a andmiR-143 reduce viability of both primary CLL andMEC-1 cells.To investigate themolecular mechanism of how thesemiRNAsregulate survival of CLL cells, we proceeded with studying therole of autophagy in CLL cell survival and a possible involve-ment of miR-130a or miR-143 in its regulation.

Autophagy is a highly conserved prosurvival mechanism tomaintain cellular homeostasis upon different types of stressand plays a crucial role in cancer. In tumor cells with defects inapoptosis, autophagy was shown to prolong cell survival (32),but the interplay between autophagy and apoptosis and its rolein cancer are still poorly understood. Of crucial importance isto increase our knowledge on the regulation of autophagy incancer with the aim to modulate autophagy in tumor cells andthereby inhibit their survival. Inhibitors of autophagy thatsensitize apoptosis-resistant tumor cells to anticancer therapyare under current investigation in clinical trials (33). Thepotential regulation of autophagy by miRNAs may offer anefficient multitarget strategy to accomplish a robust auto-phagy inhibition unaffected by single target gene mutations,cell to cell heterogeneity or other hallmarks of cancer cellresistance. Some miRNAs have been reported to regulateautophagy-related genes, such as miR-30a, regulating BECN1and miR-196, regulating IRGM (13, 14).

In both primary CLL and MEC-1 cells, we monitored auto-phagic flux and showed for the first time that autophagy is anactive process in these cells. In addition, we showed thatMEC-1 cells were able to respond to starvation by an increasedautophagic flux. These data suggest that autophagy is a con-stitutivemechanism in CLL and is part of the stress response ofthese cells to the lack of nutrients. Because introduction ofmiR-130a and miR-143 reduced viability of both primary CLLand MEC-1 cells, we investigated their impact on autophagy.Indeed,miR-130a reduced autophagic flux in primary CLL cellsandMEC-1 cells, which for the first time identifiedmiR-130a asa regulator of autophagy in CLL. In accordance with ourresults, a recent systems biology–based computational anal-ysis study reported that miR-130a is one of 5 putative miRNAsthat regulate the autophagy-lysosomal pathway (34). Although,the role of miR-130a in CLL biology was unclear, in ovariancancer miR-130a was associated with anticancer drug resis-tance because it was found to be downregulated in cell linesresistant to paclitaxel and cisplatin (35).

To further elucidate themolecularmechanismofmiR-130a–mediated CLL cell survival and autophagy, we aimed at iden-tifying putative target genes of this miRNA. By luciferasereporter assay, we identified the autophagy-related geneATG2B as a direct target of miR-130a and showed downregula-tion of ATG2B expression in CLL cells transfected with miR-130a. ATG2B interacts with ATG2A and WDR45 and thus ispossibly involved in vesicle nucleation and the initial steps ofautophagosome formation (36). Interestingly, ATG2A wasfound to be 1.5-fold upregulated in CLL samples relative tohealthy donor controls (27). Association of ATG2B and cancerhas been reported: frameshift mutations were found in themononucleotide repeats in the coding sequence ofATG2B genein gastric and colorectal carcinomas (37). Our data imply thatATG2Bmight be involved inmiR-130a–mediated autophagy inCLL cells. Its functional role in this process has to be furtherinvestigated.

In addition, we identified DICER1 as a direct target gene ofmiR-130a and showed that knockdown of Dicer1 results inreduced autophagic flux in CLL cells. Dicer1 is a highlyconserved protein, with endonuclease RNase III activity,required for siRNA- andmiRNA-mediated silencing. Therefore,miR-130a is most likely involved in autophagy and cell surv-ival in CLL cells via regulating the maturation and activityof many miRNAs, multiplying the amount of indirect effectivetarget genes. Indeed, our results showed that reduction ofDicer1 levels by overexpression of miR-130a lead to reducedlevels of several miRNAs in CLL cells. The effects of miR-130ain CLL cells might therefore be mediated by a more globalchange in the miRNA profile of the cells and the presenceof feedback loops involving Dicer1 and several miRNAs arevery likely.

As a key enzyme in RNA-induced silencing, Dicer1 is essen-tial for maintaining many crucial cellular processes includingB-cell development. Koralov and colleagues showed that in theabsence of Dicer1, B-cell development is almost completelyblocked at the transition from pro-B to pre-B cells due tofailure in response to survival signals at this early stage ofdevelopment (38). Increased expression of DICER1 has beenshown in several tumor types including ovarian and prostatecancer (39, 40) and was often associated with tumor progres-sion and poor prognosis. In addition, deletion of Dicer1 in Bcells in a Myc-induced lymphoma model significantly inhib-ited lymphomagenesis (41).

Because the DICER1 gene harbors a long 30UTR (>4,000 bp)withmultiple sites for potentialmiRNAbinding, it is likely to betargeted by several miRNAs. In addition to our data showingregulation of DICER1 by miR-130a, the miRNA let-7 and themiR-103/107 cluster have been reported to regulate Dicer1protein (42, 43).

Taken together, our results show that miR-130a plays arole in CLL cell death/cell survival pathways including auto-phagy. We identified ATG2B, as an autophagy-related targetgene of miR-130a, which might be involved directly in CLLcell resistance to apoptosis. The regulatory network ofmiR-130a is presumably highly complex as it also targetsDicer1, an essential component of the miRNA biogenesismachinery, suggesting the presence of feedback loops and the

Kovaleva et al.





involvement of other miRNAs. Which miRNAs and respectivetarget genes are essentially involved in this regulation will be ofmajor interest on the way to identify novel drug targets.Our results also point toward a cancer-supporting role ofDicer1 in CLL because its downregulation by miR-130a leadsto a decrease in autophagy and induces cell death. Theunderstanding of these regulatory networks offers a greatpotential for the intelligentmultitargeted design of new cancertherapies.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

AcknowledgmentsThe authors thank Dr. Nathan Brady and Prof. Roland Eils, German Cancer

Research Center, for the possibility to work on the imaging flow cytometer, andSibylle Ohl for excellent technical assistance.

Grant SupportThis study was supported by grants from the Deutsche Jos�e Carreras

Leuk€amie-Stiftung (DJCLS R 0613v and DJCLS R 10/04) and a Roman Herzogresearch fellowship from the Hertie foundation.

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received November 6, 2011; revised January 19, 2012; accepted February 10,2012; published OnlineFirst February 20, 2012.

References1. Zenz T, Mertens D, K€uppers R, D€ohner H, Stilgenbauer S. From

pathogenesis to treatment of chronic lymphocytic leukaemia. Nat RevCancer 2010;10:37–50.

2. D€ohner H, Stilgenbauer S, Benner A, Leupolt E, Kr€ober A, Bullinger L,et al. Genomic aberrations and survival in chronic lymphocytic leuke-mia. N Engl J Med 2000;343:1910–6.

3. Seiffert M, Dietrich S, Jethwa A, GlimmH, Lichter P, Zenz T. Exploitingbiological diversity and genomic aberrations in chronic lymphocyticleukemia. Leuk Lymphoma 2011 Dec 6. [Epub ahead of print]

4. Caligaris-Cappio F, Ghia P. Novel insights in chronic lymphocyticleukemia: are we getting closer to understanding the pathogenesisof the disease? J Clin Oncol 2008;26:4497–503.

5. Bartel DP. MicroRNAs: target recognition and regulatory functions.Cell 2009;136:215–33.

6. Di Leva G, Calin GA, Croce CM. MicroRNAs: fundamental facts andinvolvement in human diseases. Birth Defects Res C Embryo Today2006;78:180–9.

7. Landgraf P, Rusu M, Sheridan R, Sewer A, Iovino N, Aravin A, et al. Amammalian microRNA expression atlas based on small RNA librarysequencing. Cell 2007;129:1401–14.

8. Volinia S, Galasso M, Costinean S, Tagliavini L, Gamberoni G, DruscoA, et al. Reprogramming of miRNA networks in cancer and leukemia.Genome Res 2010;20:589–99.

9. Kroemer G, Mari~no G, Levine B. Autophagy and the integrated stressresponse. Mol Cell 2010;40:280–93.

10. Dikic I, Johansen T, Kirkin V. Selective autophagy in cancer develop-ment and therapy. Cancer Res 2010;70:3431–4.

11. Mathew R, Karantza-Wadsworth V, White E. Role of autophagy incancer. Nat Rev Cancer 2007;72:961–7.

12. Calabretta B, Salomoni P. Inhibition of autophagy: a new strat-egy to enhance sensitivity of chronic myeloid leukemia stemcells to tyrosine kinase inhibitors. Leuk Lymphoma 2011;52:54–9.

13. Zhu H,Wu H, Liu X, Li B, Chen Y, Ren X, et al. Regulation of autophagyby a beclin 1-targeted microRNA, miR-30a, in cancer cells. Autophagy2009;5:816–23.

14. Brest P, Lapaquette P, Souidi M, Lebrigand K, Cesaro A, Vouret-Craviari V, et al. A synonymous variant in IRGM alters a binding site formiR-196 and causes deregulation of IRGM-dependent xenophagy inCrohn's disease. Nat Gen 2011;43:242–5.

15. Amrein L, Souli�eres D, Johnston JB, Aloyz R. p53 and autophagycontribute to dasatinib resistance in primary CLL lymphocytes. Leu-kemia Res 2011;35:99–102.

16. Laane E, Tamm KP, Buentke E, Ito K, Kharaziha P, Khahariza P,et al. Cell death induced by dexamethasone in lymphoid leukemia ismediated through initiation of autophagy. Cell Death Differ 2009;16:1018–29.

17. Lagneaux L, Delforge A, Carlier S, Massy M, Bernier M, Bron D.Early induction of apoptosis in B-chronic lymphocytic leukae-mia cells by hydroxychloroquine: activation of caspase-3 and

no protection by survival factors. Br J Haematol 2001;112:344–52.

18. Hallek M, Cheson BD, Catovsky D, Caligaris-Cappio F, Dighiero G,D€ohner H, et al. Guidelines for the diagnosis and treatment of chroniclymphocytic leukemia: a report from the International Workshop onChronic Lymphocytic Leukemia updating the National Cancer Insti-tute-Working Group 1996 guidelines. Blood 2008;111:5446–56.

19. Seiffert M, Stilgenbauer S, D€ohner H, Lichter P. Efficient nucleofectionof primary human B cells and B-CLL cells induces apoptosis, whichdepends on the microenvironment and on the structure of transfectednucleic acids. Leukemia 2007;21:1977–83.

20. Seiffert M, Schulz A, Ohl S, D€ohner H, Stilgenbauer S, Lichter P.Soluble CD14 is a novel monocyte-derived survival factor for chroniclymphocytic leukemia cells, which is induced by CLL cells in vitro andpresent at abnormally high levels in vivo. Blood 2010;116:4223–30.

21. Pallasch CP, Patz M, Park YJ, Hagist S, Eggle D, Claus R, et al. miRNAderegulation by epigenetic silencing disrupts suppression of theoncogene PLAG1 in chronic lymphocytic leukemia. Blood 2009;114:3255–64.

22. Caligaris-Cappio F, Hamblin TJ. B-cell chronic lymphocytic leukemia:a bird of a different feather. J Clin Oncol 1999;17:399–408.

23. Korz C, Pscherer A, Benner A, Mertens D, Schaffner C, Leupolt E, et al.Evidence for distinct pathomechanisms in B-cell chronic lymphocyticleukemia and mantle cell lymphoma by quantitative expression anal-ysis of cell cycle and apoptosis-associated genes. Blood 2002;99:4554–61.

24. Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T,et al. LC3, a mammalian homologue of yeast Apg8p, is localized inautophagosome membranes after processing. EMBO J 2000;19:5720–8.

25. Klionsky DJ, Abeliovich H, Agostinis P, Agrawal DK, Aliev G, AskewDS, et al. Guidelines for the use and interpretation of assays formonitoring autophagy in higher eukaryotes. Autophagy 2008;4:151–75.

26. Mizushima N, Yoshimori T. How to interpret LC3 immunoblotting.Autophagy 2007;3:542–5.

27. Farfsing A, Engel F, Seiffert M, Hartmann E, Ott G, Rosenwald A, et al.Gene knockdown studies revealed CCDC50 as a candidate gene inmantle cell lymphoma and chronic lymphocytic leukemia. Leukemia2009;23:2018–26.

28. VisoneR,Rassenti LZ, VeroneseA, Taccioli C, CostineanS,AgudaBD,et al. Karyotype-specific microRNA signature in chronic lymphocyticleukemia. Blood 2009;114:3872–9.

29. Rossi S, Shimizu M, Barbarotto E, Nicoloso MS, Dimitri F, Sampath D,et al. microRNA fingerprinting of CLL patients with chromosome 17pdeletion identify a miR-21 score that stratifies early survival. Blood2010;116:945–52.

30. Pekarsky Y, Santanam U, Cimmino A, Palamarchuk A, Efanov A,Maximov V, et al. Tcl1 expression in chronic lymphocytic leukemia isregulated by miR-29 and miR-181. Cancer Res 2006;66:11590–3.






31. AkaoY,NakagawaY,KitadeY, Kinoshita T, Naoe T.Downregulation ofmicroRNAs-143 and -145 in B-cell malignancies. Cancer Sci2007;98:1914–20.

32. Degenhardt K, Mathew R, Beaudoin B, Bray K, Anderson D, Chen G,et al. Autophagy promotes tumor cell survival and restricts necrosis,inflammation, and tumorigenesis. Cancer Cell 2006;10:51–64.

33. Amaravadi RK, Lippincott-Schwartz J, Yin X-M, Weiss WA, Takebe N,Timmer W, et al. Principles and current strategies for targeting autop-hagy for cancer treatment. Clin Cancer Res 2011;17:654–66.

34. Jegga AG, Schneider L, Ouyang X, Zhang J. Systems biology of theautophagy-lysosomal pathway. Autophagy 2011;7:477–89.

35. SorrentinoA, LiuC-G, Addario A, PeschleC, ScambiaG, Ferlini C. Roleof microRNAs in drug-resistant ovarian cancer cells. Gynecol Oncol2008;111:478–86.

36. Behrends C, Sowa ME, Gygi SP, Harper JW. Network organization ofthe human autophagy system. Nature 2010;466:68–76.

37. KangMR, KimMS, Oh JE, Kim YR, Song SY, Kim SS, et al. Frameshiftmutations of autophagy-related genes ATG2B, ATG5, ATG9B andATG12 in gastric and colorectal cancers with microsatellite instability.J Pathol 2009;217:702–6.

38. Koralov SB, Muljo SA, Galler GR, Krek A, Chakraborty T, Kanellopou-lou C, et al. Dicer ablation affects antibody diversity and cell survival inthe B lymphocyte lineage. Cell 2008;132:860–74.

39. Flavin RJ, Smyth PC, Finn SP, Laios A, O'Toole SA, Barrett C, et al.Altered eIF6 and Dicer expression is associated with clinicopatholog-ical features in ovarian serous carcinoma patients. Mod Pathol2008;21:676–84.

40. Chiosea S, Jelezcova E, Chandran U, Acquafondata M, McHale T,Sobol RW, et al. Up-regulation of Dicer, a component of themicroRNAmachinery, in prostate adenocarcinoma. Am J Pathol 2006;169:1812–20.

41. Arrate MP, Vincent T, Odvody J, Kar R, Jones SN, Eischen CM.MicroRNA biogenesis is required for Myc-induced B-cell lymphomadevelopment and survival. Cancer Res 2010;70:6083–92.

42. Tokumaru S, Suzuki M, Yamada H, Nagino M, Takahashi T. let-7regulates Dicer expression and constitutes a negative feedback loop.Carcinogenesis 2008;29:2073–7.

43. Martello G, Rosato A, Ferrari F, Manfrin A, Cordenonsi M, Dupont S,et al. A microRNA targeting dicer for metastasis control. Cell2010;141:1195–207.

Kovaleva et al.





2012;72:1763-1772. Published OnlineFirst February 20, 2012.Cancer Res Valentina Kovaleva, Rodrigo Mora, Yoon Jung Park, et al. Trigger Killing of Chronic Lymphocytic Leukemia Cells

to Inhibit Autophagy andDICER1 and ATG2BmiRNA-130a Targets

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miRNA-130a Targets ATG2B and DICER1 to Inhibit Autophagy ... · A total of 1 107 primary B cells were transfected with the human B-cell Nucleofector Kit and program U-015 with 1 mmol/L