Differential microRNA Profiles and Their Functional ...•ργασίες... · Chronic lymphocytic leukemia (CLL) was considered a homogeneous disease of naive, immune-incompetent,

INTRODUCTIONChronic lymphocytic leukemia (CLL)

was considered a homogeneous diseaseof naive, immune-incompetent, mini-

mally proliferating B cells that accumu-lated because of intrinsic apoptotic de-fects (1). More recently, however, thisview was essentially upturned: indeed,

currently, it is widely accepted that thedevelopment and evolution of CLL re-flects an interplay between genetics andmicroenvironment (2). Microenvironmen-tal stimuli specifically conveyed throughthe B-cell receptor (BcR) are critically im-plicated in the immunopathogenesis ofCLL as evidenced by (a) restrictions inthe immunoglobulin heavy variable(IGHV) gene repertoire (3); (b) differentprognosis of patients with different IGHVgene mutational status (4,5); and (c) theexistence of subsets of patients sharingBcRs with restricted, quasi-identical im-munoglobulin (IG) sequences (stereo-typed BcRs) in roughly 30% of cases(6–8). These observations may indicate

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Differential microRNA Profiles and Their FunctionalImplications in Different Immunogenetic Subsets of ChronicLymphocytic Leukemia

Nikos Papakonstantinou,1,2,3* Stavroula Ntoufa,2,3* Elisavet Chartomatsidou,2,3 Giorgio Papadopoulos,4

Artemis Hatzigeorgiou,4 Achiles Anagnostopoulos,2 Katerina Chlichlia,1 Paolo Ghia,5 Marta Muzio,6

Chrysoula Belessi,7 and Kostas Stamatopoulos2,3

1Laboratory of Molecular Immunobiology, Department of Molecular Biology and Genetics, Democritus University of Thrace,Alexandroupolis, Greece; 2Hematology Department and HCT Unit, G. Papanicolaou Hospital, Thessaloniki, Greece; 3Institute ofApplied Biosciences, Center for Research and Technology Hellas, Thessaloniki, Greece; 4Institute of Molecular Oncology, BiomedicalSciences Research Center “Alexander Fleming,” Vari, Greece; 5Laboratory of B cell Neoplasia and Unit of Lymphoid Malignancies,Department of Onco-Hematology and Division of Molecular Oncology, Istituto Scientifico San Raffaele and Università Vita-SaluteSan Raffaele, Milan, Italy; 6Cell Activation and Signaling Unit, Division of Molecular Oncology, Istituto Scientifico San Raffaele,Milan, Italy; and 7Hematology Department, Nikea General Hospital, Athens, Greece

Critical processes of B-cell physiology, including immune signaling through the B-cell receptor (BcR) and/or Toll-like receptors(TLRs), are targeted by microRNAs. With this in mind and also given the important role of BcR and TLR signaling and microRNAs inchronic lymphocytic leukemia (CLL), we investigated whether microRNAs could be implicated in shaping the behavior of CLLclones with distinct BcR and TLR molecular and functional profiles. To this end, we examined 79 CLL cases for the expression of 33microRNAs, selected on the following criteria: (a) deregulated in CLL versus normal B-cells; (b) differentially expressed in CLL sub-groups with distinct clinicobiological features; and, (c) if meeting (a) + (b), having predicted targets in the immune signaling path-ways. Significant upregulation of miR-150, miR-29c, miR-143 and miR-223 and downregulation of miR-15a was found in mutatedversus unmutated CLL, with miR-15a showing the highest fold difference. Comparison of two major subsets with distinct stereo-typed BcRs and signaling signatures, namely subset 1 [IGHV1/5/7-IGKV1(D)-39, unmutated, bad prognosis] versus subset 4 [IGHV4-34/IGKV2-30, mutated, good prognosis] revealed differences in the expression of miR-150, miR-29b, miR-29c and miR-101, all down-regulated in subset 1. We were also able to link these distinct microRNA profiles with cellular phenotypes, importantly showing that,in subset 1, miR-101 downregulation is associated with overexpression of the enhancer of zeste homolog 2 (EZH2) protein, whichhas been associated with clinical aggressiveness in other B-cell lymphomas. In conclusion, specific miRNAs differentially expressedamong CLL subgroups with distinct BcR and/or TLR signaling may modulate the biological and clinical behavior of the CLL clones.Online address: http://www.molmed.orgdoi: 10.2119/molmed.2013.00005

*NP and SN contributed equally to this work.

Address correspondence to Kostas Stamatopoulos, Hematology Department and HCT

Unit, G. Papanicolaou Hospital, 57010, Exohi, Thessaloniki, Greece. Phone and Fax: +30-

2313-307992; E-mail: [email protected]; or Paolo Ghia, Università Vita-

Salute San Raffaele, Via Olgettina 58, 20132 Milano, Italy. Phone: +39-02-2643-4797; Fax:

+39-02-2643-4723; E-mail: [email protected].

Submitted January 11, 2013; Accepted for publication April 16, 2013; Epub

(www.molmed.org) ahead of print April 16, 2013.

that a correspondingly restricted set ofantigens or structurally related epitopesmight be implicated in the selection ofclones carrying distinctive BcRs (9).

In addition to the BcR, B cells express awide repertoire of molecules involved inmicroenvironmental interactions, includ-ing Toll-like receptors (TLRs) (10). TLRshave important roles in both detectingpathogens and initiating inflammatoryprocesses that subsequently prime spe-cific adaptive immune responses duringinfection (11). Dual BcR and TLR engage-ment can fine-tune B-cell responses (12).However, in some instances, it may alsoresult in aberrant activation and loss oftolerance (13), alluding to functionalcomplementation.

Recent studies by us (14,15) and others(16) have demonstrated that CLL patientsubgroups defined by specific molecularcharacteristics of their clonotypic BcRshave distinct patterns of TLR pathwaygene expression, function and/or toler-ance. These findings indicate that specificmodalities of BcR/TLR collaborationand/or regulation may eventually affectthe biological behavior of the malignantclones.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expres-sion by binding to specific mRNA tar-gets and promoting their degradationand/or translational inhibition (17).miRNAs participate in the regulation ofdiverse cellular processes, from develop-ment, differentiation and cell cycle regu-lation to senescence and metabolism (18)and the modulation of immune signal-ing (19,20). Moreover, an intertwinedconnection between epigenetics andmiRNAs has been supported by theidentification of a specific subgroup ofmiRNAs called “epi-miRNAs” that canmodulate the activity of the epigeneticmachinery. The complexity of this con-nection is enhanced by the epigeneticregulation of miRNA expression thatgenerates a fine regulatory feedbackloop (21).

Taking all the above into considera-tion, it is no surprise that miRNAs havebeen found to be instrumental for CLL

physiology (22). In particular, certainmiRNAs are deregulated in CLL com-pared with normal B cells (23–26),whereas distinct microRNA signaturesare associated with prognostic factorsand disease progression (24–29), re-sponse to treatment (30,31) and variousother disease characteristics (26,32,33).

Prompted by these findings, we inves-tigated whether miRNAs could be impli-cated in shaping the biological and, bylogical inference, clinical behavior of sub-sets of patients with CLL with particularBcR molecular features and distinct BcRand/or TLR molecular and functionalprofiles. We report that specific miRNAsdifferentially expressed among differentCLL subsets can affect their cellularphysiology, at least under certain ex vivoconditions. We also provide for the firsttime molecular and functional evidencelinking overexpression of the enhancer ofzeste homolog 2 (EZH2) protein in clini-cally aggressive CLL with downregula-tion of miR-101. Altogether, these find-ings argue for the operation ofsubset-specific miRNA-regulatedprocesses that may eventually affect theevolution of CLL clones with distinctBcRs and immune signaling signatures.

MATERIALS AND METHODS

Patient GroupBlood samples were collected from 79

patients diagnosed with CLL accordingto the recently revised guidelines of theInternational Workshop Chronic Lym-phocytic Leukemia/National Cancer In-stitute (34). All patients were either un-treated or off therapy for at least 6months before the study. The study wasapproved by the local ethics review com-mittee of the participating institutions.Demographic, clinical and biological

data for the patient cohort are given inTable 1 and Supplementary Table S1.

Polymerase Chain ReactionAmplification and Sequence Analysisof IGHV-IGHD-IGHJ Rearrangements

Reverse transcriptase–polymerasechain reaction (RT-PCR) amplificationand sequence analysis of IGHV-IGHD-IGHJ gene rearrangements was per-formed as previously described (8).

Cell PurificationCD19+ B cells were negatively selected

from whole blood using the RosetteSepB-cell enrichment kit (StemCell Technolo-gies, Vancouver, BC, Canada) followingthe manufacturer’s instructions. The pu-rity of all cell preparations always ex-ceeded 95% for CD19+ cells.

miRNA Isolation and ReverseTranscription of Mature miRNAs

Small RNA fractions (<200 nucleotides)were extracted with the TRIsure Reagent(Bioline, London, UK) and the RT2 qPCR-Grade miRNA Isolation Kit (SABio-sciences, Valencia, CA, USA). Then 100ng purified RNA was used for the syn-thesis of cDNA with the RT2 miRNA FirstStrand Kit (SABiosciences).

Identification of miRNAs PotentiallyImplicated in the Regulation ofImmune Signaling Pathways in CLL

On the basis of published work, weidentified a series of miRNAs reported asrelevant for CLL biology: (a) miRNAsderegulated in CLL cells compared withnormal B cells (24,25,35) and (b) miRNAsdifferentially expressed in subgroups ofCLL patients with distinct profiles of bio-logical prognostic markers (24,25,28,33)(Supplementary Table S2). Through theuse of our purpose-built computational

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m i R N A P R O F I L I N G O F C L L S U B S E T S

Table 1. Clinical and biological data of the patient cohort.

Parameter n

Sex (M/F) 52/27Binet stage at diagnosis (A/B/C) 67/9/3CD38 expression (positive/negative) 28/50IGHV gene mutational status (mutated/unmutated) 45/34

algorithms and tools (DIANA-microTv3.0 algorithm and DIANA-mirPath toolv1.0; both freely available at http://mi-crorna.gr), we investigated which ofthese miRNAs are potentially implicatedin the regulation of biological processesrelevant for B cells, with a focus on im-mune signaling. In particular, we evalu-ated the BcR and TLR pathways as wellas the mitogen-activated protein kinase(MAPK) pathway, which integrates withboth of the above. We selected for bioin-formatics analysis those molecules in theaforementioned pathways for whichgene expression data were available fromour previous work (14). Through this ap-proach, we identified 33 miRNAs withpredicted targets in the examined path-ways (Supplementary Tables S3 and S4).Details about the bioinformatics analyti-cal strategy are given in the Supplemen-tary Methods.

Quantification of miRNA ExpressionExpression profiling of all 33 mature

miRNAs listed in Supplementary Table S3was performed by real-time quantitativePCR (RQ-PCR) on customized PCR arrays (SABiosciences), consisting of apanel of 41 primer sets used for the am-plification of the target microRNAs plusfour reference small noncoding RNAs(snRNA U6, SNORD44, SNORD47 andSNORD48), two reverse transcriptioncontrols and two PCR quality controls.Further details are provided in the Sup-plementary Methods.

Quantification of EZH2 mRNAExpression

Total RNA was extracted from cellswith the use of TRIsure (Bioline, London,UK). Quantification of EZH2 mRNA lev-els was achieved by RQ-PCR, by usingthe RT2 qPCR Primer Assay for thehuman EZH2 gene (SABiosciences), ac-cording to the manufacturer’s instruc-tions and as detailed in the Supplemen-tary Methods.

Western Blot AnalysisTotal cellular protein was isolated from

purified B cells, run on 10% NuPAGE Bis-

Tris gel (Invitrogen/Life Technologies,Carlsbad, CA, USA) and transferred topolyvinylidene fluoride membranes (Invitrogen/Life Technologies) as re-ported previously (15). Immunoblot anal-ysis was performed for certain proteinsidentified through bioinformatics analy-sis as potential targets of differentially expressed miRNAs (namely EZH2, mitogen-activated protein kinase 8[MAPK8] and cellular FBJ murine osteosarcoma viral oncogene homolog [c-FOS]) by using appropriate mousemonoclonal anti-human antibodies(EZH2; BD Biosciences, San Jose, CA,USA; MAPK8 and FOS, Santa CruzBiotechnology, Santa Cruz, CA, USA).Further details are given in the Supple-mentary Methods.

Nucleofection of CLL CellsPurified CD19+ B cells from one subset

1 and one subset 4 case were transfectedwith specific miRNA mimic and miRNAinhibitor, respectively, by using theAmaxa Human B Cell Nucleofector Kit(Lonza, Colonge, Germany). In detail, 5 ×106 CD19+ B cells of each case were resus-pended in 100 μL Human B Cell Nucleo-fector Solution and mixed with 40 nmol/LSyn-hsa-miR-101-3p miScript miRNAMimic (Qiagen, Hilden, Germany) in thecase of subset 1 and 100 nmol/L Anti-hsa-miR-101-3p miScript miRNA Inhibitor(Qiagen) in the case of subset 4.

The 5 × 106 CD19+ B cells of each casewere also transfected with the appropri-ate negative control. In the subset 1 case,40 nmol/L AllStars Negative ControlsiRNA (Qiagen) was used, whereas 100 nmol/L miScript Inhibitor NegativeControl (Qiagen) was used in the subset4 case. The same amount of cells was cul-tured as untranfected control. Nucleofec-tions were performed by using theAmaxa Nucleofector device with the U-15 program (Lonza). Transfection effi-ciency was measured with the Block-iTAlexa Fluor Red Fluorescent Oligo (Invitrogen/Life Technologies). CLL cellswere collected and processed for im-munoblotting analysis at 24 and 40 hafter nucleofection.

Statistical AnalysisDescriptive statistics were performed

as previously described (14,15). Correla-tion analysis was performed by using thePearson correlation coefficient. For allcomparisons, a significance level of p ≤0.05 was set. All statistical analyses wereperformed with the use of the GraphPadPrism 5 software (GraphPad Software,La Jolla, CA, USA).

All supplementary materials are availableonline at www.molmed.org.

RESULTS

IGHV Repertoire and MutationalStatus

IGHV-IGHD-IGHJ sequences wereavailable for all studied cases (Supple-mentary Table S5). Following the 98%germline identity cutoff value (4,5), 45 of79 sequences (57%) were defined as mu-tated, whereas the remainder (34 of 79sequences, 43%) had unmutated IGHVgenes; in the latter group, 25 cases car-ried IGHV genes with 100% identity tothe germline.

The study was intentionally biased tocases expressing stereotyped BcRs as-signed to subset 1 [stereotypedIGHV1/5/7-IGKV1(D)-39 BcRs, sevencases] and subset 4 [stereotyped IGHV4-34/IGKV2-30 BcRs, eight cases] (Supple-mentary Table S5) (8). These subsets maybe seen as prototypes of “unmutated/ adverse prognosis” and “mutated/goodprognosis” subsets, respectively. Further-more (perhaps more importantly for thepurposes of the present study), thesesubsets also exhibit distinct immune sig-naling profiles (14,15). All subset 1 casescarried unmutated IGHV genes, whereas,in contrast, all subset 4 cases carried mu-tated IGHV genes.

miRNA Expression Profiling: Analysis atthe Cohort Level

A total of 33 miRNAs predicted to tar-get the BcR and/or TLR pathways wereevaluated. Significant differences wereidentified with regards to miRNA ex-pression levels, which ranged from high

R E S E A R C H A R T I C L E


to undetectable (Supplementary Table S6;Supplementary Figure S1). Additionally,for most studied miRNAs, significant in-terpatient variation was observed. Statis-tical analysis of miRNA expression pro-files with various clinical parameters,including stage at diagnosis and time tofirst treatment, did not identify signifi-cant associations, likely because of (a) therelatively small size of the cohort and (b)case selection.

miRNA Expression Profiles in Relationto BcR Molecular Features

We sought for differences in miRNAexpression in subgroups of cases definedby BcR molecular features, such as themutational status of the clonotypic IGHVgenes and the expression of stereotypedBcRs.

The comparison between CLL casescarrying mutated versus unmutatedIGHV genes (mutated [M]-CLL and un-mutated [U]-CLL: 45 and 34 cases, re-spectively) revealed differential expres-sion of nine miRNAs (SupplementaryTable S7A). Amongst these, the mostpronounced differences (p < 0.05) were

seen for miR-150, miR-15a, miR-29c,miR-223 and miR-143. The greatest dif-ference overall was recorded for miR-15a, which was the only downregulatedmiRNA in M-CLL (Figure 1).

We explored whether the observed dif-ferences regarding miR-15a expressionbetween M-CLL versus U-CLL could re-late to the genomic status at chromosome13q14, which harbors the miR-15a locus(22). Among 59 cases with available in-terphase fluorescence in situ hybridiza-tion (FISH) data, 7 (11.9%) and 19 (32.2%)carried biallelic or monoallelic del(13q),respectively, whereas the remainder (33cases, 55.9%) did not exhibit aberrationsof chromosome 13q. The majority ofcases carrying del(13q) [5 of 7 cases withbiallelic del(13q) and 13 of 19 cases withmonoallelic del(13q)] carried mutatedIGHV genes.

Cases with biallelic del(13q) exhibitedsignificantly downregulated miR-15a lev-els compared with cases with eithermonoallelic del(13q) (p = 0.002) or no de-tectable chromosome 13q aberration (p <0.001) (Supplementary Figure S2).Monoallelic del(13q) cases also exhibited

downregulated miR-15a levels comparedwith cases with no detectable chromo-some 13q aberration, although not reach-ing statistical significance (p = 0.07).

We next evaluated differences inmiRNA expression between clinically ag-gressive stereotyped subset 1 (U-CLL)versus clinically indolent stereotypedsubset 4 (M-CLL). We found 12 differen-tially expressed miRNAs, all downregu-lated in subset 1, with miR-150, miR-29b,miR-29c and miR-101 showing statisti-cally significant difference between thetwo subsets (p ≤ 0.05) (Figure 2, Supple-mentary Table S7B). However, miR-150and miR-29c appeared as differentiallyexpressed between M-CLL versus U-CLL,whereas miR-29b and miR-101 showedsubset-biased differential expression irre-spectively of mutational status.

Impact of Differentially ExpressedmiRNAs on Immune Signaling in CLL

Given that CLL cases with distinct im-munogenetic features also exhibit distinctresponses to immune triggering(15,16,36), we next investigated if andhow the differentially expressed miRNAsmight be implicated in regulating thefunction of immune signaling pathways.We focused on miRNAs, showing thehighest fold difference between thegroups under comparison, namely miR-15a for M-CLL versus U-CLL and miR-101for subset 1 versus 4. Among all predictedtargets of these two miRNAs in the exam-ined pathways, we selected for analysisthose for which gene expression data wasavailable from our recent profiling studyof immune signaling in CLL (14).

In the case of miR-15a, potential tar-gets identified through bioinformaticsanalysis included MAPK8, IRAK2, CD80and IKBKB, with MAPK8 having thehighest prediction score (SupplementaryTable S8A). A significant negative corre-lation (r = –0.3, p < 0.01) was identifiedbetween miR-15a and MAPK8 mRNAlevels (Figure 3A). In contrast, no correla-tion existed between miR-15a and IRAK2,CD80 or IKBKB mRNA levels. Contraryto expectations, comparison of MAPK8protein expression in seven M-CLL ver-



Figure 1. Differential miRNA expression in M-CLL versus UM-CLL. Only miRNAs with fold dif-ference in expression of ≥1.50 and significantly different expression in M-CLL versus U-CLL(p ≤ 0.05) are represented in the graphs. Among them, miR-15a, downregulated in M-CLL,has the highest fold difference. The y axis depicts the relative expression (2–ΔCt). Error barsrepresent standard deviation (SD). *p ≤ 0.05 compared with U-CLL.

sus seven U-CLL cases revealed signifi-cantly (p < 0.05) increased levels in U-CLL (Figure 3B). Given that U-CLLalso show elevated levels of miR-15a,this result indicates additional modes ofregulation of MAPK8 expression.

In the case of miR-101, FOS andMAPK8 were identified as potential tar-gets, with FOS having the highest predic-tion score (Supplementary Table S8B) andbeing reported to be regulated by miR-101 in other types of cancer (37,38). Wefound a negative correlation (r = –0.8, p <0.01) between miR-101 and FOS mRNAlevels (Figure 3C), but not with MAPK8mRNA levels. Western blot analysis re-vealed similar FOS protein levels be-tween subset 1 and 4 cases (Figure 3D).

Next we investigated the impact ofmiR-101 forced overexpression in pri-mary CLL cells. We transfected CLL cellsfrom one subset 1 case with miR-101mimic, and, after 40 h in culture, we ob-served downregulation of FOS proteinlevels compared with untransfected ells or cells transfected with negativecontrol (Figure 3E). Furthermore, wefound that transfecting subset 4 cells

with miR-101 inhibitor induced FOSdownregulation, yet to a lesser extentcompared with miR-101 forced overex-pression (Figure 3F). These results indi-cate that miR-101 may be implicated inthe regulation of FOS expression in CLL.

miR-101 Modulates the Expression ofthe EZH2 Methyltransferase in DifferentCLL Subsets

miR-101 is considered an “epi-miRNA,” since it targets EZH2, a histonemethyltransferase that mediates tran-scriptional repression acting in concertwith DNA methyltransferases (21). Inboth solid tumors and hematopoietic ma-lignancies, more aggressive subtypeswere associated with overexpression ofEZH2 caused by different mechanisms,including genomic loss of the miR-101locus (39–41). Relevant to the latter ob-servation, induced expression of miR-101in cancer cell lines inhibits the expressionand function of EZH2 (42).

With this in mind, and also promptedby our finding of significant downregu-lation of miR-101 in subsets 1 versus 4,we investigated the potential role of

miR-101 in regulating EZH2 expressionin these paradigmatic CLL subsets. Wedetected significantly higher levels ofEZH2 mRNA in subset 1 (p < 0.05) (Fig-ure 4A) that negatively correlated (r =–0.75, p < 0.05) with miR-101 expression(Figure 4B). We also found significantlyhigher EZH2 protein expression in sub-set 1 versus subset 4 (p < 0.05) (Figure4C), again showing significant negativecorrelation (r = –0.7, p < 0.05) with miR-101 expression levels (Figure 4D).

Finally, we investigated the impact of(a) forced overexpression and (b) down-regulation of miR-101 in primary CLLcells. In subset 1 CLL cells, the miR-101mimic induced downregulation of EZH2protein levels after 40 h in culture com-pared with untransfected cells or cellstransfected with negative control (Fig-ure 4E). In subset 4 CLL cells transfectedwith miR-101 inhibitor, upregulation ofEZH2 protein levels was seen after 24 hcompared with untransfected cells orcells transfected with negative control(Figure 4F). Hence, not only is miR-101differentially expressed in different CLLsubsets, but also (at least partly) regu-lates EZH2 expression, potentially lead-ing to differential biological effects.

DISCUSSIONImmune-mediated pathways are im-

portant in the pathogenesis of CLL(14–16,36,43). Indeed, the in vivo accu-mulation of CLL clonal cells is promotedby classic receptor-ligand interactionswith other cells and soluble factors oc-curring within the tumor microenviron-ment (2). However, CLL cells from dif-ferent patients exhibit heterogeneousfunctional responses when stimulatedthrough immune receptors, that is, theBcR and/or the TLRs (14–16,36,43),prompting the investigation into the un-derlying mechanisms.

Recent evidence suggests that miRNAstarget key molecules of the B-cell im-mune pathways (20,44,45). With this inmind and also taking into account thatseveral miRNAs are differentially ex-pressed in CLL, we investigated whethermiRNAs could also be implicated in the



Figure 2. Differentially expressed miRNAs in subset 1 versus 4 CLL cases. Only miRNAs withfold difference in expression of ≥1.50 and significantly different expression (p ≤ 0.05) arerepresented in the graphs. Among them, miR-101, downregulated in subset 1, shows thehighest fold difference. The y axis depicts the relative expression (2–ΔCt). Error bars repre-sent SD. *p ≤ 0.05 compared with subset 4.

distinct immune signaling profiles ofCLL subgroups with different clinicalpresentation and outcome. In particular,we profiled a series of well-characterizedCLL patients for the expression of 33miRNAs verified as relevant for CLL bi-ology and also identified as potentialmodulators of immune signaling path-ways in B cells.

Comparison of miRNA expression be-tween M-CLL and U-CLL cases of thepresent study confirms and extends pre-vious reports. In particular, our finding ofoverexpression of miR-150, miR-29c, miR-223 in M-CLL and miR-15a in U-CLL is inaccordance with the literature (24,25,28),whereas the relative upregulation of miR-143 in M-CLL versus U-CLL is reported

for the first time. Among the differen-tially expressed miRNAs in M-CLL ver-sus U-CLL, miR-15a showed the highestfold difference; miR-15a levels were alsostrongly associated with deletion of chro-mosome 13q14, in line with previous ob-servations (22,46).

CLL subgroups defined by IGHV genemutational status are not homogeneous.Rather, within each mutational sub-group, cases assigned to subsets express-ing distinct stereotyped BcR have beenshown to share distinctive clinicobiologi-cal profiles and outcome (47). On thisbasis, we narrowed down our compar-isons to cases assigned to different sub-sets with stereotyped BcR with a specialfocus on subsets 1 and 4, which were

overrepresented in our cohort for bothpractical and scientific reasons.

In particular, because even the mostpopulated stereotyped subsets accountfor only a minor fraction of the cases withavailable IGHV-IGHD-IGHJ sequence in-formation, the choice to study subsets 1and 4, the most populated subsets amongU-CLL and M-CLL, respectively (6,8,47),offered us the possibility to obtain a size-able collection of samples for meaningfulexperimental analysis and statistical eval-uation. Admittedly, numbers are still rela-tively low, as in all previous studies ana-lyzing stereotyped subsets comparedwith studying CLL in general (47). Thatbeing said, the subsets selected for analy-sis are opposites in terms of clinical evo-lution and outcome (subset 1: aggressivedisease; subset 4: remarkably indolentdisease) (8,47). Furthermore, they exhibitconsistent yet distinct immune signalingprofiles, even when compared with non-subset cases of similar IGHV gene muta-tional status (14,15). Thus, the subsets canbe considered as prototypes of distinct bi-ological and clinical setting.

Comparison of these two subsets re-vealed 12 differentially expressed miRNAs, all downregulated in subset 1,with miR-101 showing the most pro-nounced difference in expression be-tween subsets 1 (low) versus 4 (high). In-terestingly, no differences in miR-101expression were identified when we com-pared M-CLL versus U-CLL; hence, thedifferential profiles in subsets 1 and 4 arenot a mere reflection of differential IGHVgene mutational status; rather, they implya role for miR-101 in shaping the biologi-cal behavior of the respective clones.

miR-101 is one of the so-called epi- miRNAs, a group of miRNAs that regulatethe expression of components of the epige-netic machinery. Aberrant expression ofepi-miRNAs has been linked to the devel-opment or progression of human cancer(21). miR-101 regulates EZH2, the enzy-matic subunit of the polycomb repressivecomplex 2 (PRC2), which induces gene re-pression through trimethylation of histoneH3 at lysine 27. EZH2 overexpressioncaused by different mechanisms, includ-



Figure 3. miR-15a/MAPK8 and miR-101/FOS are negatively correlated at mRNA but not atprotein level. MAPK8 (JNK) is the target with the highest prediction score for miR-15a. (A,C) Correlation and linear regression between miR-15a/MAPK8 and miR-101/FOS mRNAlevels, respectively. ΔCt values were used and the correlation is statistically significant (p <0.01). The slope in both cases is negative in both cases, which means that when miRNAexpression is upregulated, mRNA expression is downregulated. (B, D) Protein levels ofMAPK8 and FOS in representative M/U-CLL and subset 1 and 4 cases, respectively. MAPK8is overexpressed in U-CLL. (E) miR-101 forced overexpression in one subset 1 case by usingmiR-101 mimic induced FOS protein downregulation compared with untransfected cellsor cells transfected with negative control. (F) Transfection of subset 4 cells with miR-101 in-hibitor induced FOS downregulation, yet to a lesser extent compared with miR-101–forcedoverexpression.

ing activating mutations, was reported ina broad range of both hematopoietic andsolid human malignancies and associatedwith poor prognosis (48,49).

Here, we provide for the first time evi-dence that EZH2 might be implicated inshaping the distinct behavior of CLLsubsets 1 versus 4 and that its expressionis regulated, at least in part, by miR-101.In particular, we report a significant in-verse correlation between EZH2 mRNAand protein with miR-101 expression,suggesting that increased EZH2 expres-sion in clinically aggressive subset 1 islinked to downregulation of miR-101, al-luding to the operation of a regulatory

loop. This scenario is also supported byour finding that induced overexpressionor downregulation of miR-101 affectedEZH2 protein expression in primary CLLcells from subsets 1 and 4.

With this evidence that miRNAs withdistinct expression profiles among differ-ent CLL subgroups with distinct BcR IGscan be linked to the behavior of the re-spective CLL cases, we next sought to in-vestigate the possible impact of differen-tially expressed miRNAs on theexpression of key molecules of immunesignaling pathways.

In particular, we demonstrate for thefirst time a significant inverse correlation

of miR-15a with MAPK8 mRNA expres-sion in M-CLL versus U-CLL, albeit withno correlation between miRNA levelsand protein levels. A similar situationwas identified for miR-101 and FOS ex-pression in subsets 1 versus 4 (namelyassociation between miRNA and mRNAyet not with protein levels). These obser-vations are in line with previous reportsthat miR-101 regulates FOS mRNA ex-pression in gastric cancer (38), whereasthe available data about protein regula-tion by miR-101 overexpression in cancercell lines (40) and human hepatocellularcarcinoma (37) are inconclusive. Interest-ingly, we found that forced overexpres-sion of miR-101 downregulated FOS pro-tein levels in primary CLL cells.

miRNAs cause inhibition of translationor degradation of the target mRNA.However, as recently reported, they pre-dominantly affect mRNA rather thanprotein levels (50). Of note, the degree ofthe protein downregulation imposed bymiRNAs often tends to be quantitativelymodest (even an overexpressed miRNAtypically downregulates most of its en-dogenous targets by <50%) (51). Further-more, as already suggested, miRNAsmight fully control the expression of im-mune signaling proteins through collabo-ration with multiple other mechanisms,including physical interactions, confor-mational changes, posttranslational mod-ifications and proteasome-mediated deg-radation. These findings may be relevantfor the observed inconsistencies betweenmiRNA, mRNA and protein expressionof MAPK8 and FOS in the studied CLLcases, indicating the need for further in-vestigation. All that notwithstanding, itis worth mentioning that our analyseswere conducted at basal level withoutany form of stimulation, which is rele-vant in view of recent reports by us (52)and others (44) that the triggering of im-mune receptors modulates miRNA ex-pression in CLL, alluding to autoregula-tory control.

CONCLUSIONWe demonstrate that subgroups of CLL

cases with distinct BcR IGs are character-



Figure 4. miR-101 differentially regulates EZH2 at mRNA and protein levels in subset 1 versus4 CLL cases. (A) EZH2 mRNA levels in subsets 1 and 4 (four and four cases, respectively). They axis represents relative expression (2–ΔCt). The box-and-whisker plots repesent median withminimum and maximum values. EZH2 is upregulated in subset 1 cases (fold difference inexpression of 2.3; p ≤ 0.05). (B) Correlation and linear regression between miR-101 andEZH2 mRNA levels. ΔCt values were used and the correlation is statistically significant (p ≤ 0.05). The slope is negative, which means that when miR-101 expression is upregulated,EZH2 expression is downregulated (r = –0.75). (C) Protein levels of EZH2 in subsets 1 and 4(six and eight cases, respectively). EZH2 protein levels are higher in subset 1 (p < 0.005). (D) Correlation and linear regression between miR-101 relative expression (2–ΔCt) and EZH2protein levels (r = –0.7, p < 0.05). (E) In subset 1 CLL cells, miR-101 mimic induces downregu-lation of EZH2 protein levels compared with untransfected cells and cells transfected withnegative control. (F) In subset 4 CLL cells, miR-101 inhibitor induces EZH2 protein upregula-tion compared with untransfected cells or cells transfected with negative control.

ized by distinct miRNAs profiles that canpotentially be implicated in the regulationof immune signaling (49). Furthermore,we provide for the first time evidence thatmiR-101 regulates EZH2 expression levelsin CLL and that EZH2 overexpression is afeature of aggressive CLL.

ACKNOWLEDGMENTSThis project was supported by the

ENosAI project (code 09SYN-13-880) andcofunded by the European Union (EU)and the Hellenic General Secretariat forResearch and Technology; Cariplo Foun-dation (Milan, Italy); and the ProgramMolecular Clinical Oncology-5 per millenumber 9965, Associazione Italiana perla Ricerca sul Cancro (Italy).

DISCLOSUREThe authors declare that they have no

competing interests as defined by Molec-ular Medicine, or other interests thatmight be perceived to influence the re-sults and discussion reported in thispaper.

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Differential microRNA Profiles and Their Functional ...•ργασίες... · Chronic lymphocytic leukemia (CLL) was considered a homogeneous disease of naive, immune-incompetent,