Downregulated plasma miR-92a levels have clinical impact on multiple myeloma and related disorders.

0.39 MB·English

by  Yoshizawa, S

Citation:BloodCancerJournal(2012)2,e53;doi:10.1038/bcj.2011.51
&2012MacmillanPublishersLimited Allrightsreserved2044-5385/12
www.nature.com/bcj
ORIGINAL ARTICLE
Downregulated plasma miR-92a levels have clinical impact
on multiple myeloma and related disorders
SYoshizawa1,JH Ohyashiki2, M Ohyashiki1,T Umezu3, KSuzuki4, AInagaki5, SIida5 and KOhyashiki1,3
Recent studieshave demonstrated that one-third ofknown microRNAs (miRNAs) arestably detectablein plasma. Therefore,we
assessedplasma miRNAs toinvestigate the dynamicsofoncomir 17-92a, which ishighlyexpressed inmultiple myeloma(MM)
patients. Theplasma miR-92alevel insymptomatic MM patientswas significantly downregulated compared withnormal
subjects (Po0.0001), regardlessofimmunoglobulin subtypes ordisease stage atdiagnosis. Incontrast, miR-92a levels in
peripheralbloodCD8þ orCD4þ cells fromMM patientswere lowerthanthoseof normalsubjects, andthe miR-92alevels of
thecellstendedto correlatewith plasma miR-92alevels. The plasma miR-92alevelin thecompleteremission group became
normalized,whereas thepartial response (PR)and very goodPR groups did notreach thenormal range.Insmoldering MM,
theplasmamiR-92a leveldid not showa significantdifference compared withnormal subjects. Our findingssuggest that
measurementof theplasma miR-92alevel inMMpatients could be useful forinitiation ofchemotherapy and monitoring
diseasestatus, and thelevelmay represent, inpart, theT-cellimmunity statusof these patients.
Blood CancerJournal (2012)2, e53;doi:10.1038/bcj.2011.51; publishedonline 20January 2012
Keywords: multiple myeloma; plasmamiR-92a; Tlymphocytes;
INTRODUCTION hematologicmalignancies.19Togainmoreinsightintotheclinical
MicroRNAs (miRNAs) consist of approximately 18--22-nucleotide relevance of plasma miR-92a expression, we evaluated plasma
non-coding RNAmoleculesthat regulateposttranscriptional gene miR-92a levels in MM patients at various phases and in patients
expression by degradation or repression of mRNA molecules.1--3 with related disorders. In addition, we examined cellular miR-92a
Individual miRNAs can target multiple mRNAs and control levels in circulating lymphocytes obtained from untreated MM
transcription in approximately one-third of human genes. Recent patients and compared them with the plasma miR-92a level
studies have shown that these miRNAs are closely involved in to ascertain the possible association between immunological
cell differentiation, proliferation, apoptosis or oncogenesis.1--3 condition andplasma miR-92a expression.
In various human cancer, there is evidence of the alteration of
tumor tissue-specific miRNA.4--6 A 2008 study demonstrated that
MATERIALSANDMETHODS
miRNAsstablyexistinserumandplasma,7,8andarecentadvance
Patientsand samples
revealed the presence of circulating miRNAs within lipoprotein
(known as microvesicles), which may help to protect the miRNAs We evaluated peripheral blood obtained from 168 patients with
from RNase-dependent degradation.9,10 Moreover, some extracel- monoclonalgammopathies:138withsymptomaticMM,8withsmoldering
lular circulating miRNAs in blood plasma are independent of MM (SMM) and 22 with monoclonal gammopathy of undetermined
exosomes and are bound to Ago2 protein, resulting in strong significance (MGUS). The diagnosis of monoclonal gammopathy was
nuclease/proteinaseresistance,11thusindicatingapossibleroleof basedonthedefinitionoftheInternationalMyelomaWorkingGroupusing
circulating miRNAs in healthy subjects and disease condition.7,8 thelevelofserumM-protein,proportionofplasmacellsinbonemarrow
The biological relevance of circulating miRNAs remains unclear, andthepresenceofend-organdamage.20Atthetimeofplasmacollection,
althoughtheymayhaveanimportantroleincancermetastasisor the disease status of the 138 patients with symptomatic MM was as
neo-angiogenesis. Therefore, circulating miRNAs are thought to follows(Table1):62newlydiagnosed,8completeremission(CR),11very
be possible diagnostic or prognostic biomarkers of human good partial response (VGPR), 15 partial response (PR), 14 stable
diseases.12--14 disease and 28 progressive disease. None of the 62 newly diagnosed
Itis well-knownthat multiplemyeloma(MM)cellshaveahigh MM patients had del(13q) anomaly, where the miR-17-92a located,
expression level of the miR-17-92a cluster,15 whereas plasma according to the standard cytogenetic technique. Of the eight SMM
miR-92a levels in acute lymphoid leukemia or non-Hodgkin’s patients, two had received cytotoxic therapy because of an increase
lymphomaareextremelydownregulated.16--18Downregulationof in M-protein (X5g/dl). No MGUS patients had received chemotherapy
miRNAs,let-7aandmiR-16inmyelodysplasticsyndromesalsohas beforeplasmacollection.Weanalyzedplasmafrom113normalindividuals
beenreported, andtheir levelsweresignificantly associatedwith and isolated lymphocytes from 21 healthy volunteers as the control.
progression-free survival and overall survival, suggesting that These samples were handled similarly to those obtained from MM
certain miRNAs in plasma can serve as noninvasive biomarker in patients.
1DepartmentofHematology,TokyoMedicalUniversity,Tokyo,Japan;2InstituteofMedicalScience,TokyoMedicalUniversity,Tokyo,Japan;3DepartmentofMolecularScience,
TokyoMedicalUniversity,Tokyo,Japan;4DepartmentofHematology,JapaneseRedCrossMedicalCenter,Tokyo,Japanand5DepartmentofMedicalOncologyandImmunology,
NagoyaCityUniversityGraduateSchoolofMedicalSciences,Nagoya,Japan.Correspondence:ProfessorKOhyashiki,DepartmentofHematology,TokyoMedicalUniversity,
6-7-1Nishi-shinjuku,Shinjuku-ku,Tokyo160-0023,Japan.
E-mail:[email protected]
Received6October2011;revised1December2011;accepted5December2011
PlasmamiR-92adownregulationinmultiplemyeloma
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Table1. PlasmamiR-92a/miR-638expressionlevelsinmonoclonalgammopathies
Numberofsubjects Median Mean Standarderror Minimum Maximam 95%CI
Normal 113 1.244 1.388 0.06993 0.2154 3.58 1.249--1.527
SymptomaticMMatdiagnosis 62 0.05202 0.1008 0.01899 0.000045 0.8586 0.06285--0.1388
Myelomasubtype
IgG 26 0.06107 0.121 0.03378 0.000045 0.8586 0.05146--0.1906
IgA 12 0.1508 0.05435 0.1901 0.006992 3.127 0.3020--1.089
IgD 3 0.1667 0.2353 0.1423 0.0304 0.5087 (cid:2)0.3769--0.8475
Bence--Jonesprotein 14 0.03866 0.0973 0.04252 0.004289 0.6177 0.005448--0.1892
Non-secretorymyeloma 4 0.06367 0.07089 0.02974 0.01013 0.1461 (cid:2)0.02376--0.1655
Plasmacellleukemia 3 0.02628 0.03341 0.01985 0.003151 0.07081 (cid:2)0.05201--0.1188
DiseasestatusofMM
Newlydiagnosed 62 0.05202 0.1008 0.01899 0.000045 0.8586 0.06285--0.1388
CR 8 1.494 1.602 0.354 0.2285 3.643 0.7653--2.439
VGPR 11 0.2176 0.3591 0.09528 0.04138 1.028 0.1468--0.5714
PR 15 0.8179 0.7967 0.2018 0.1216 1.979 0.5409--1.052
Stabledisease 14 0.3404 0.4251 0.2634 0.003508 0.9965 0.2479--0.6022
Progressivedisease 28 0.06335 0.2018 0.04978 0.003319 0.8586 0.09962--0.3039
SMM 8 0.7943 1.188 0.4791 0.09087 5.242 0.3002--1.348
MGUS 22 0.4055 0.6403 0.1309 0.007652 2.428 0.3681--0.9124
Abbreviations:CI,confidenceinterval;CR,completeremission;MGUS,monoclonalgammopathyofunderminedsignificance;MM,multiplemyeloma;
PR,partialresponse;SMM,smolderingMM;VGPR,verygoodpartialresponse.DiseasestatusofmultiplemyelomawascategorizedbyInternationalMyeloma
WorkshopConsensuscriteria.20
This study was approved by the institutional review board of Tokyo theexpressionlevelofmiR-92ainplasma,wecomparedplasmamiR-92a
Medical University (no. 930, approved 24 June 2008). Written informed expression using an external standard, cel-miR-39 (Hokkaido System
consentwas obtained from all the participants prior to the collectionof Science),aswellasaninternalstandard,miR-638,whichisstablydetected
specimensaccordingtotheDeclarationofHelsinki. inallsamples.TheresultsusingmiR-638arecompatiblewiththoseusing
cel-miR-39(datanotshown).WethereforeusedmiR-638asareferencein
eachplasmasample,aspreviouslyreported.17,18
TaqManlow-density array screening
Total RNA was isolated with the mirVana PARIS kit (Ambion, Austin, TX,
Lymphocyteseparation
USA).Plasmasamplesfromfive patientsor fivehealthyindividualswere
mixed evenly, and 500ml of mixed plasma was diluted with 500ml of TheCD4þ orCD8þ T-cellfractionswereseparatedwithanisolationkitfor
binding solution. After a 5-min incubation, 1ml of 1nM ath-miR-159 humans(MiltenyiBiotec,BergischGladbach,Germany)andAutoMACSPro
(Hokkaido System Science, Hokkaido, Japan) was added to each aliquot, Separator (Miltenyi Biotec), according to the supplier’s instruction, and
followed by vortexing for 30s and incubation on ice for 10min. storedat(cid:2)801Cuntiluse.
Subsequent phenol extraction and filter cartridge work was performed
according to the manufacturer’s instructions. In all, 3ml of RNA solution Statistical analysis
fromthe50-mlelutewasusedasaninputineachreversetranscription(RT)
GraphPad5.0software(GraphPadSoftwareInc,SanDiego,CA,USA)was
reaction.TheRTreactionandpre-amplificationstepweresetupaccording
usedforstatisticalanalysis.TheMann--Whitneytestwasusedtodetermine
tothemanufacturer’srecommendations.miRNAswerereversetranscribed
statisticalsignificancebetweentwogroupsandone-wayANOVAforthree
withtheMegaplexPrimerPools(HumanPoolsAv2.1;AppliedBiosystems,
or more groups. We also used the Chi-square test and Student’s t-test,
FosterCity,CA,USA).RTreactionproductsfromtheplasmasamplewere
whenappropriate.P-valueso0.05wereconsideredtoindicatestatistically
further amplified with Megaplex PreAmp Primers (Primers A v2.1). The
significantdifferences.
expressionprofileofthemiRNAswasdeterminedwiththeHumanTaqMan
miRNAArraysA(AppliedBiosystems).QuantitativeRT-PCRwasperformed
on an Applied Biosystems 7900HT thermocycler according to the
RESULTS
manufacturer’srecommendedprogram.Withtheuseof SDS2.2software
Identificationof differentially expressed plasmamiRNAs in
andDataAssist(AppliedBiosystems),theexpressionofplasmamiRNAswas
MMpatients andhealthy volunteers
calculatedbasedontheir Ct valuesnormalized bythose of ath-miR-159,
PlasmamiRNAexpressionwasinitiallyscreenedusingtheTaqMan
whichwasspikedineachplasmasample.
low-density arraysystem. Ofthe381 miRNAs represented on the
wellplates,331miRNAswerenotdetectedafterthe35PCRcycles.
miR-92aquantitative RT-PCR Among the remaining 50 miRNAs, upregulated and down-
Total RNA in cells was isolated with an miRNeasy Mini Kit (Qiagen, regulated miRNAs in MM plasma samples (expression level in
Germantown, MD, USA), and RNA in plasma was extracted as reported the sample was 4-fold greater or lower than that of healthy
previously.17,18 miRNAs were quantified with TaqMan MicroRNA Assays volunteers) were selected. We then evaluated the rank order of
(Applied Biosystems) with modification and miRNA-specific stem-loop expression of each miRNA by DCt value among all detected
primers(has-miR-92a,000431;has-miR-638,001582;AppliedBiosystems), miRNAs.
as we reported previously.17,18 The plasma miR-92a expression was Most of the miRNAs were downregulated in plasma samples
normalized to miR-638 expression, and the cellular miR-92a expression obtained from MM patients (Figure 1a). The most striking
levelswerenormalizedtoRNU6B(001093;AppliedBiosystems).Wehave difference of expression was found in miR-223. In addition,
neverdetectedU6Binaplasmasample,althoughU6Biscommonlyused members of the miR-17-92a cluster (miR-19b, miR-17, miR-92a,
asaninternalstandardformiRNAexpressionanalysisincells.Tonormalize miR-20a and miR-19a) were significantly decreased in MM
BloodCancerJournal &2012MacmillanPublishersLimited
PlasmamiR-92adownregulationinmultiplemyeloma
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a 35 a
10
30 Normal 38
6 1
25 Multiple Myeloma R-
Δ-2^t()C 112050 miR-92a/mi(log) 00.0.11
5 ma 0.001
0 as 0.0001
miR-223 miR-19b R1mi-06a miR-17 miR-92a miR-24 miR-648-5p R-4mi15 miR-484 miR-32-43p 2mi0R-a let7-b miR-39 i1Rm-2 2m-1R2i R2mi-b0 miR1-9a 2mi2R1- mi14R0--5p miR-210 Pl 0.00001Normal Dx IgG IgA IgD BJPNonsec PCL
b 5
Normal
b
100
4 Multiple Myeloma
Ct) 3 80
Δ
-2^( 2 %)
y ( 60
1 vit
nsiti 40
0 e
S
20
Figure1. IdentificationofexpressedplasmamiRNAsinMM.
(a)DownregulatedmiRNAsinMM(openbars).Theexpression 0
levelinthesamplewas4-foldlowerthanthatofnormalcontrols 0 20 40 60 80 100
(solidbars).ArrowsindicatemiR-17-92apolycistroniccluster.(b)
100% - Specificity%
UpregulatedmiRNAsinMM(openbars).Theexpressionlevelinthe
samplewas4-foldhigher thanthatofnormalcontrols(solidbars). Figure2. PlasmamiR-92avaluesinMMandreceiveroperating
characteristiccurve.(a)PlasmamiR-92alevel(miR-92a/miR-638)
inpatientswithMMatthetimeofdiagnosis.Asignificant
samples. Although we found several upregulated miRNAs in MM downregulatedplasmamiR-92alevelisnotableinMMatdiagnosis
samples, the Ct values were generally low in both MM samples (MM-Dx)(Po0.0001).NoparticulardifferenceinplasmamiR-92a
and normal controls (Figure 1b). We therefore focused on levelisevidentamongimmunoglobulinsubtypes,Bence--Jones
downregulation of the miR-17-92a cluster, rather than the single proteintypeornon-secretoryMMbyone-wayANOVA.Barsindicate
miRNA (that is, miR-223), because the expression of miR-17-92a minimumtomaximumplasmamiR-92alevelsandboxesindicate
95%confidenceinterval(CI).(b)Thecut-offlevelofplasmamiR-92a/
cluster is essential in the lymphoid ontogeny and measured the
miR-638inallMMatdiagnosisis0.2593.Thesensitivityis91.94%
expressionlevelof miR-92a inalargeseries ofpatients.
(95%CI:82.17--97.33%)andspecificityis99.12%(95%CI:95.17--
99.98%).
PlasmamiR-92a levelof symptomatic MM
PlasmamiR-92alevelsweresignificantlylowerinnewlydiagnosed sensitivity (95% confidence interval: 82.17--97.33%) and 99.12%
MM patients (n¼62) compared with healthy controls (Student’s specificity(95%confidenceinterval:95.17--99.98%)whenthecut-
t-test: Po0.0001); the level of plasma miR-92a in symptomatic offlevelsofplasmamiR-92ainMMpatientsatdiagnosiscouldbe
MM patients was o10% compared with normal subjects achieved(that is, 0.2593) (Figure 2b).
(mean±standard error 0.1008±0.01899 vs 1.388±0.06993;
Table 1). Among the MM patients, there were no significant
differences in the plasma miR-92a levels, irrespective of MM PlasmamiR-92a levelinthe variousstates ofMM
subtype (P¼0.4472) (Figure 2a), light chain type (P¼0.4413) We next compared the plasma miR-92a level in MM patients in
(Supplementary Figure 1A) or International Staging System various clinical states (Table 1). Patients in CR (n¼8) had a
staging (P¼0.1955) (Supplementary Figure 1B). The presence of significant increase in plasma miR-92a compared with those at
anemia (P¼0.0990), bone lesion (P¼0.6701), renal damage diagnosis (Po0.0001), and seven out of the eight patients were
(P¼0.4258) or hypercalcemia (P¼0.1989) did not affect plasma normalized (X0.2593 cut-off level). Of the PR patients (n¼15), a
miR-92a level at MM diagnosis (Supplementary Figure 2AD, significant increase in plasma miR-92a was notable (Po0.0001),
SupplementaryTable1).PlasmamiR-92alevelwasnotcorrelated but these patients still had low plasma miR-92a compared with
with beta2 microglobulin elevation (P¼0.3675), albumin level normal subjects (P¼0.0033). Although patients with stable
(P¼0.0693), elevated lactate dehydrogenase (P¼0.4863) or disease (n¼14) had a partially normalized plasma miR-92a level,
performance status(P¼0.9850) (SupplementaryTable1). three patients had an extremely low level, similar to those of
The receiver operating characteristic curve was generated by patients with newly diagnosed MM. In progressive disease
comparing the patients’ DCt values with those of healthy patients (n¼28), the plasma miR-92a level again downregulated
volunteers.TheanalysisshowedthatmiR-92awasagoodmarker and no significant difference was notable compared with newly
of newly diagnosed MM (AUC¼0.9810), indicating 91.94% diagnosedMM(Figure 3a).
&2012MacmillanPublishersLimited BloodCancerJournal
PlasmamiR-92adownregulationinmultiplemyeloma
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PlasmamiR-92a levels ofMGUSand SMM patients a P< 0.0001
WealsomeasuredplasmamiR-92alevelsin8patientswithSMM P< 0.0001
and 22 with MGUS (Table 1). Compared with normal subjects,
P= 0.4430
patients with SMM showed no significant differences in plasma 10
miR-92a levels (P¼0.4642), but they had elevated levels 8
compared with MM patients (P¼0.0496) (Figure 3b). The plasma 63 1
miR-92a level was significantly decreased in MGUS patients R-
mi 0.1
compared with normal subjects (Po0.0001), but they had a/
spnioagttnieeidfinctbasen(ttPwly¼eeh0ni.g0S0hM0e5rM).paNlanosdmdMaifGfemUreiSRn-pc9ea2taiiennleptsvlae(sPlms¼ac0om.m2i9Rp5-a99r2)e.adThlewevietphllsasMwmaMas miR-92(log) 0.01
miR-92alevelsdidnotcorrelatewithdurationbetweenthetimeof ma 0.001
diagnosis of MGUS and the measurement of levels in this study s
a 0.0001
(datanot shown). Pl
0.00001
mWeiR-t9h2eanlceovmelpianresdepmariRa-t9e2dalylemveplhsoincyTtelysmphocytesobtainedfrom Normal M M-Dx M M-CR M-VGPR M M-PR M M-SD M M-PD
healthy subjects (n¼21) with those of MM patients at diagnosis M
(n¼6)oratCR/VGPRstatus(n¼5).ThemiR-92alevelinCD4þ or
CD8þ TlymphocytesinMMpatientsatdiagnosiswassignificantly
b P = 0.4642
lower compared with that of healthy subjects (CD4þ: P¼0.0178;
CD8þ:P¼0.0092)(Figure4a).ComparisonofmiR-92aofCD4þ and
P = 0.0496
CD8þ Tlymphocytesshowedaroughlylinearcorrelation,andmost P = 0.0005
MMpatientsinCR/VGPRstatushadanincreasedlevel(Figure4b). 10
Although no apparent correlation was evident in normal subjects, 8
MM patients with low levels of plasma miR-92a had low levels of 63 1
miR-92aexpressioninCD4þ (Figure4c)andCD8þ Tlymphocytes R-
(Figure 4d), and three out of the five MM patients with CR/VGPR mi 0.1
a/
hadnormallevelsofplasmaandcellularmiR-92a. miR-92(log) 0.01
DISCUSSION a 0.001
m
The use of plasma miRNAs as potential biomarkers is a growing as 0.0001
research area,12--14 and this is the first study to look at plasma Pl
miRNAsinMM.Ourfindingsindicatethepossibleclinicalutilityof 0.00001
pnpelaatwsiemnaatgsmenwitRistNhAhaMlseMviem.2l1pinrIonMveMMdMpthatetrieenraettssmp.eoTnnhtse,ertehrcaeetendteaicnnidstrioosnduurctvotiivoasnltaoirntf Normal M M-Dx SM M M G US
chemotherapy usually depends on the presence of clinical
Figure3. PlasmamiR-92aexpressioninMMatvariousclinicalphases
symptoms with high evidence level, so-called CRAB (hypercalce-
andSMMorMGUS.(a)ThedownregulatedplasmamiR-92alevelat
mia,renalfailure,anemia,bonelesion)symptoms.22Moreover,the myelomadiagnosis(MM-Dx)wasnormalizedinCR(MM-CR)andwas
therapeutictargetpointinMMpatientshasbeenproposed,23and partiallynormalizedatVGPR(MM-VGPR)orPR(MM-PR)phase.(b)
practicalguidelinesforthetherapeuticstrategyhavebeenhelpful PatientswithSMMhadahigherplasmamiR-92alevelthanthatof
inclinicaldecision-makingforMMtreatment.24,25However,there symptomaticmyeloma(P¼0.0496),buttheyhadlowlevels
are some MM patients for whom the timing and combination of comparedwithnormalsubjects.AlthoughsomepatientswithMGUS
chemotherapy are difficult to decide,26 although high-risk MM hadlowlevelsofplasmamiR-92a,mostMGUSpatientshadplasma
patients can be classified.27,28 Because, decisions regarding miR-92alevelssimilartothosewithSMM.Barsindicateminimumto
maximumplasmamiR-92alevelsandboxesindicate95%CI.
chemotherapy are occasionally difficult in non-secretory or
asymptomaticMMpatients,anoveldiagnosticmarkerisurgently
required,inadditiontotheconventionaldiagnostictool.21Inthe
current study, we demonstrated the downregulation of plasma andsymptomaticmyeloma,thelevelofplasmamiR-92alevelcould
miR-92ainsymptomaticMMpatients,irrespectiveofthepresence reflectthepathologicalconditionofpatientsratherthanthetumor
or absence of renal damage or M-protein, suggesting that the burden of myeloma cells in the body. Moreover, normalization of
measurementoftheplasmamiR-92acouldbehelpfulfordeciding the plasma miR-92a level after obtaining CR suggests that the
whento initiatechemotherapy. plasma miR-92a level might serve as an indicator for therapeutic
Chromosomal abnormalities and molecular alterations in MM response. The number of treated patients studied was small,
cells have been extensively investigated, and some of them are however,andfurtherresearchisneededtoclarifythispossibility.
currently incorporated into the risk analysis.23,27,29 In addition, Studies have shown that some circulating miRNAs in cancer-
pathogenesis of extracellular circumstances, including angiogen- bearingpatientsaretumor-derived,7andthussuchmiRNAsmight
esis,30 in MM patients is an important issue not only for be used as biomarkers of cancer.12--14 The downregulation of
understanding the biology of myeloma but therapeutic strategies plasmamiR-92ahasbeenrecognizedinpatientswithhematologic
as well.21 In myeloma cells, overexpression of the miR-17-92a has neoplasia, including acute leukemia,16,17 non-Hodgkin’s lympho-
beennotedinthetransformationfromMGUStoMM.15Therefore, ma18 and hepatocellular carcinoma,31 as well as in patients with
miR-17-92aexpressionisthoughttocorrelatewithtumorburdenin cardiovascular diseases.32 Because miR-17-92a is essential in
MMpatients.Incontrast,wefoundnodifferenceinplasmamiR-92a the development and ontogeny of the lymphoid system,33,34
levelsbetweenMGUSandSMMpatients.BecausetheplasmamiR- weexaminedthecorrelationbetweenmiR-92alevelsinseparated
92a level was significantly different between patients with SMM lymphocytes and plasma. The plasma miR-92a levels was
BloodCancerJournal &2012MacmillanPublishersLimited
PlasmamiR-92adownregulationinmultiplemyeloma
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Normal
Myeloma
a 4 0.0178 b 3 Myeloma
CR/VGPR
B 0.0092
U6 3
a/ 2a 2
2 9
ar miR-9(ratio) 2 +D8 miR- 1
ul C
ell 1
C
0 0
4 4 8 8 0 1 2 3 4
D D D D
mal C ma C mal C ma C CD4+ miR-92a
Nor yelo Nor yelo
M M
Normal
c 2.5 d 2.5 Myeloma
Normal Myeloma
2.0 Myeloma 2.0 CR/VGPR
a a
2 Myeloma 2
9 9
R- 1.5 CR/VGPR R- 1.5
mi mi
a a
m 1.0 m 1.0
s s
a a
pl pl
0.5 0.5
0.0 0.0
0 1 2 3 4 0 1 2 3
CD4+ miR-92a CD8+ miR-92a
Figure4. CellularmiR-92ainCD8þ orCD4þ lymphocytesinMMandnormalsubjects.(a)TheCD4þ miR-92a(P¼0.0178)andCD8þ
miR-92a(P¼0.0092)aresignificantlydownregulated.(b)ComparisonbetweenmiR-92alevelsinCD4þ andCD8þ Tlymphocytesinnormal
subjects(closedcircles),MMatdiagnosis(opensquares)andmyelomainCR/VGPR(opentriangles),showingapositivecorrelation.
ComparisonbetweenplasmamiR-92aandthecellularmiR-92aofCD4þ Tlymphocytes(c)orCD8þ Tlymphocytes(d)inidenticalsubjects.
MyelomapatientswithlowlevelsofmiR-92ainCD4þ TlymphocytesclusteratlowlevelsofplasmamiR-92a,andsomeMMpatientswith
CR/VGPRshownormalizationofmiR-92alevelsinplasma,CD4þ andCD8þ Tlymphocytes.
correlated with the miR-92a level in T lymphocytes, especially downregulated plasma miR-92a level may be linked to the
CD8þ lymphocytes. Some MM patients in CR/VGPR showed expressionlevelofT-cell-derivedmiR-92a.Althoughweexamined
normalization of the plasma miR-92a level, in combination with asmallnumberofpatients,especiallyMMpatientswhohadbeen
upregulation of T-cell miR-92a. A recent study reported that the treated,theseinitialfindingssuggestthatplasmamiR-92alevelsin
miR-17-92a cluster was critical for optimal T-lymphocyte monoclonalgammopathypatientsmaybeapromisingparameter
co-stimulation via CD28 and was thereby essential for regulatory not only for determining disease status but also whether further
Tfunctionundercertainconditions.35Moreover,itisreportedthat treatment isrequired.
the type-1-skewing tumor microenvironment induces down-
regulation of miR-17-92a expression, with decreased levels of CONFLICTOFINTEREST
miR-17-92anotedinbothCD4þ andCD8þ cellsintumor-bearing Theauthorsdeclarenoconflictofinterest.
mice and in CD4þ cells in patients with glioblastoma multi-
forme.36 Together, these findings suggest that the low level of ACKNOWLEDGEMENTS
miR-92a in T lymphocytes may represent dysregulation of WethankCKobayashi,RSoya-HamamuraandAHirotafortheirtechnicalassistance.
immunity and the downregulated plasma miR-92a level in MM ThisworkwassupportedbythePrivateUniversityStrategicResearchBasedSupport
patients may reflect the dysfunction of T lymphocytes in vivo. In Project:EpigeneticsResearchProjectAimedatGeneralCancerCureUsingEpigenetic
addition to miR-17-92a downregulation, the TaqMan low-density Targets from the Ministry of Education, Culture, Sports, Science and Technology,
array screening revealed downregulation of miR-223, a regulator Tokyo,Japan.
of neutrophil proliferation and activation.37 The ultimate down-
regulation of plasma miR-223 in MM patients might also be AUTHORCONTRIBUTIONS
related withgranulocyte dysfunction. SY, KO and JHO designed the study, analyzed the data, interpreted the data
In conclusion, the plasma miR-92a level may serve as a and wrote the manuscript; TU and MO performed quantitative PCR analysis of
biomarker for monitoring therapeutic response in MM patients plasma miRNA; KO performed statistical analysis; SY KO, KS, AI, and SI provided
and disease progression in asymptomatic MM patients. The patientsamples.
&2012MacmillanPublishersLimited BloodCancerJournal
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