Asian Indian Phenotype

14 Pages·2015·0.78 MB·English

RESEARCHARTICLE
‘
Circulating MiRNAs of Asian Indian
’
Phenotype Identified in Subjects with
Impaired Glucose Tolerance and Patients
with Type 2 Diabetes
ParamasivamPrabu1,SophieRome2,ChandrakumarSathishkumar1,
SankaramoorthyAravind1,BalakumarMahalingam1,Coimbatore
SubramanianShanthirani1,CarolineGastebois2,AudreyVillard2,ViswanathanMohan1,
MuthuswamyBalasubramanyam1*
1 MadrasDiabetesResearchFoundationandDr.Mohan’sDiabetesSpecialitiesCentre,WHOCollaborating
CentreforNon-CommunicableDiseasesPreventionandControl&IDFCentreofEducation,Gopalapuram,
Chennai-600086,India,2 CarMeNLaboratory(INSERM1060,INRA1397,INSA),UniversityofLyon,
FacultédeMédecineLyon-Sud,CheminduGrandRevoyet,69600,Oullins,France
* [email protected]
OPENACCESS
Citation:PrabuP,RomeS,SathishkumarC,Aravind
Abstract
S,MahalingamB,ShanthiraniCS,etal.(2015)
CirculatingMiRNAsof‘AsianIndianPhenotype’
IdentifiedinSubjectswithImpairedGlucose Severalomicstechnologiesareunderwayworldwidewithanaimtounravelthepathophysi-
ToleranceandPatientswithType2Diabetes.PLoS ologyofacomplexphenotypesuchastype2diabetesmellitus(T2DM).Whilerecentstud-
ONE10(5):e0128372.doi:10.1371/journal.
iesimplyaclinicallyrelevantandpotentialbiomarkerroleofcirculatorymiRNAsinthe
pone.0128372
etiologyofT2DM,thereislackofdataonthisaspectinIndians—anethnicpopulationchar-
AcademicEditor:RatnaB.Ray,SAINTLOUIS acterizedtorepresent‘AsianIndianphenotype’knowntobemorepronetodevelopT2DM
UNIVERSITY,UNITEDSTATES
andcardiovasculardiseasethanEuropeans.WeperformedglobalserummiRNAprofiling
Received:February14,2015
andthevalidationofcandidatemiRNAsbyqRT-PCRinacohortofsubjectscomprisedof
Accepted:April26,2015 normalglucosetolerance(NGT),impairedglucosetolerance(IGT)andpatientswithT2DM.
Published:May28,2015 Ourstudyrevealed4differentiallyexpressedmiRNAs(miR-128,miR-130b-3p,miR-374a-
5p,miR-423-5p)insubjectswithIGTandT2DMpatientscomparedtocontrolsubjects.
Copyright:©2015Prabuetal.Thisisanopen
accessarticledistributedunderthetermsofthe Theywerepositivelyornegativelycorrelatedtocholesterollevels,HbA1C,HOMA-IRand
CreativeCommonsAttributionLicense,whichpermits fastinginsulin.Interestingly,circulatinglevelofmiR-128andmiR-130b-3pwerealsoaltered
unrestricteduse,distribution,andreproductioninany
inserumofdiet-induceddiabeticmicecomparedtocontrolanimals.Amongthealteredcir-
medium,providedtheoriginalauthorandsourceare
culatingmiRNAs,miR-128hadneverbeendescribedinpreviousstudies/populationsand
credited.
appearedtobea‘NewLead’inIndians.Itwaspositivelycorrelatedwithcholesterolbothin
DataAvailabilityStatement:Allrelevantdataare
prediabeticsubjectsandindiet-induceddiabeticmice,suggestingthatitsincreasedlevel
withinthepaperanditsSupportingInformationfiles.
mightbeassociatedwiththedevelopmentofdyslipedemiaassociatedwithT2DM.Ourfind-
Funding:Authorsacknowledgethefinancialsupport
ingsimplydirectionalitytowardsbiomarkerpotentialofmiRNAsintheprevention/diagnosis/
fromtheDepartmentofBiotechnology(DBT),
GovernmentofIndia.Thisworkwasalsosupported treatmentoutcomesofdiabetes.
bygrantsfromtheFrenchFondationpourla
RechercheMédicale(FRM-20101220456)andis
supportedbytheFrench—Indianmedicalresearch
cooperationprogrambetweenINSERMandICMR.
Theauthorsthankthestudyparticipants,andthe
clinicalassistantsandadministrativepersonnelatthe
PLOSONE|DOI:10.1371/journal.pone.0128372 May28,2015 1/14
CirculatoryMiRNAsofAsianIndianPhenotype
MadrasDiabetesResearchFoundation,Chennai, Introduction
India.
SouthAsiaformsoneoftheepicentersoftheglobaldiabetesepidemic.Overthepastcoupleof
CompetingInterests:Theauthorshavedeclared
decades,therehasbeenaworryingincreaseintheprevalenceratesoftype2diabetesmellitus
thatnocompetinginterestsexist.
(T2DM)intheregionandIndiainparticular.ThehigherprevalenceofdiabetesinSouth
Asiansisleastexplainedbytraditionallymeasuredriskfactors.Insulinresistanceandabnor-
malitiesofinsulinsecretioninpancreaticß-cellsarethemaindefectsthatleadtoT2DM.The
socalled“AsianIndianPhenotype”referstocertainuniqueclinicalandbiochemicalabnormal-
itiesinIndianswhichincludeincreasedinsulinresistance,greaterabdominaladiposityi.e.,
higherwaistcircumferencedespitelowerbodymassindexandT2DMoccurringatayounger
agethanCaucasians[1,2].SouthAsiansarealsocharacterizedbyauniquemetabolicprofile
withhigherinsulinlevels[3],agreaterdegreeofinsulinresistance[4]andahigherprevalence
ofdiabetes[5].InsulinresistancehasbeendemonstratedinAsianIndiansevenduringadoles-
cence[6]andhyperinsulinemiaseemstobepresentinAsianIndiansevenatbirth[7].Morere-
centstudiesdoimplythatSouthAsiansmayhaveanearlydeclineinß-cellfunctionsaswell
[8,9,2].
GiventhepredictedexplosioninthenumberofcasesofprediabetesandT2DMworldwide
andinIndia,continuedresearchisessential,particularlyinthenewerareassuchasapplication
ofmicroRNA(miRNA)technologies.MiRNAsareaclassofevolutionallyconservednon-cod-
ingRNAsof19–22nucleotidesandfunctionasnegativeregulatorsofgeneexpression[10].Im-
portantrolesofmiRNAshaveemergedinthecontrolofmetabolicpathwaysinvolvedinlipid
metabolism,adipocytedifferentiationandpancreasdevelopment,energyhomeostasis,glucose-
stimulatedinsulinsecretionandinflammation[11–16].Recently,significantamountsofmiR-
NAshavebeenfoundnotonlyintracellularly,butinextracellularhumanbodyfluids(e.g.;
serum,plasma,saliva,urine,tears,amnioticfluidsandmilk)[17–19].Theyareremarkablysta-
bledespitehighextracellularRNAseactivities.ExtracellularmiRNAsareenclosedinsmall
membranousvesicles(e.g.;inexosomes,sheddingvesicles,apoptoticbodies)orassociated
with,orpackagedwithinhigh-densitylipoprotein,orassociatedwithRNA-bindingproteins(e.
g.;high-densitylipoprotein,Argonaute2andnucleophosmin1)[20–22].LevelsofmiRNAsin
theserumofhumanshavebeenshowntobestable,reproducible,consistentamongsthealthy
individualsandchangeduringpathophysiology,allowingthemtobeofpotentialvalueasbio-
markersofdisease[23].Recently,novelcirculatorymiRNAsignatureshavebeenstudiedand
shownassociatednotonlywiththediseasebutalsotheseverityofdiseasessuchasvariouscan-
cers,liverandcardiovasculardiseases[24,25].Whereasseveralstudieshaveevaluatedtherole
ofmiRNAsincancer,muchlessisknowninthefieldofdiabetes.Differentiallyexpressedcircu-
latorymiRNAsignaturecharacteristicofT2DMhavebeenreportedfromrecentstudiesout-
sidetheIndiabasedontheanalysisofsmallgroupofsubjects[26–33].Fromthesestudies,itis
expectedthattheidentificationofpotential-specificbiomarkersofmiRNAsmayhelppredict
ordetectthedevelopmentandprogressionofdiabetesanditscomplicationsatanearlystage,
andthereforeallowtimelyintervention.However,thereislackofclinicallyrelevantdataonthe
potentialofcirculatorymiRNAsinIndians,whoarehighlyinsulinresistantandmoreproneto
developT2DMandcardiovasculardiseasethanEuropeans[5].Whilethehealthburdenof
T2DMissomehowassociatedwithobesityasaco-morbidityinCaucasians,metabolicriskfac-
torsareeitherhigherinAsianIndiansindependentofobesityoroperateatalowerBMIthresh-
old.Therefore,thisstudyisaimedtoidentifycirculatingmiRNAsthatcouldbedifferentially
expressedinsubjectswithprediabetesandpatientswithT2DMandtodissectouttheirpoten-
tialbiomarkersroleinassociationwithclinicalparameters/conventionalriskfactorsofT2DM.
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CirculatoryMiRNAsofAsianIndianPhenotype
ResearchDesignandMethods
RecruitmentoftheStudySubjects
SubjectswithNGT(NormalGlucoseTolerance,n=49),subjectswithIGT(ImpairedGlucose
Tolerance,n=47)andpatientswithType2DiabetesMellitus(T2DM,n=49)wererecruited
fromDr.Mohans’DiabetesSpecialitiesCentre,Chennaiandfromtheon-goingepidemilogical
studies.ThestudywasconductedaccordingtotheprinciplesofDeclarationofHelsinkiandap-
propriateapprovalbytheInstitutionalEthicsCommitteeoftheMadrasDiabetesResearch
Foundation.EthicsCommitteeapprovedwritteninformedconsentwasobtainedfromall
studysubjects.NGT,IGTandT2DMweredefinedusingWorldHealthOrganizationconsult-
inggroupcriteria.Thosewhowereconfirmedbyoralglucosetolerancetesttohave2-hour
plasmaglucosevalue11.1mmol/L(200mg/dL)ormorebasedonWorldHealthOrganization
consultinggroupcriteriawerediagnosedasdiabeticpatients.Thosewith2-hourpostglucose
value(cid:1)7.8mmol/L(140mg/dL)and<11.1mmol/L(200mg/dL)werediagnosedassubjects
withIGT,andthosewith2-hourpostglucosevalueoflessthan7.8mmol/L(140mg/dL)as
subjectswithNGT.Toruleoutthemedicationeffects,allourT2DMsubjectsareofnewly
diagnosednature.
AnthropometricMeasurements
Anthropometricmeasurementsincludingheight,weightandwaistcircumstancewereobtained
usingstandardizedtechniques.Heightwasnotedwithatapemeasuredtothenearestcentime-
ter.Weightwasmeasuredwithtraditionalspringbalancethatwaskeptonafirmhorizontal
surface.Thebodymassindex(BMI)wascalculatedusingtheformula,weight(kg)/height(m)2.
Waistcircumferencewasmeasuredusinganonstretchablefibermeasuringtape.Bloodpres-
surewasrecordedfromtherightarmofstudysubjectswhentheywererelaxedandinsitting
positiontothenearest2mmHgwithamercurysphygmomanometer(DiamondDeluxeBPap-
paratus,Pune,India).Tworeadingweretaken5minapartandthemeanofthetwowastaken
asthebloodpressure.
BiochemicalParameterInvestigations
Fastingplasmaglucose(glucoseoxidase-peroxidasemethod),serumcholesterol(cholesterol
oxidase-peroxidase-amidopyrinemethod),serumtriglycerides(glycerolphosphateoxidase-
peroxidase-amidopyrinemethod)andHDLcholesterol(directmethod-polyethyleneglycol-
pretreatedenzymes)weremeasuredusingHitachi-912Autoanalyser(Hitachi,Mannheim,
Germany).Theintraandinterassayco-efficientofvariationforthebiochemicalassayswere
<5%.Low-densitylipoprotein(LDL)cholesterolwascalculatedusingFriedewaldformula.
Glycatedhemoglobin(HbAlc)wasestimatedbyhigh-pressureliquidchromatographyusing
thevariantanalyzer(Bio-Rad,Hercules,Calif.,USA).SerumInsulinwasestimatedusingen-
zyme-linkedimmunosorbentassay(Calbiotech,CA).Theintra-assayandtheinter-assaycoef-
ficientsofvariationforinsulinassaywas<10%.Insulinresistancewascalculatedusingthe
homeostasisassessmentmodel(HOMA-IR)usingtheformula:{fastinginsulin(μIU/mL)x
fastingglucose(mmol/L)}/22.5.
CirculatingRNAExtractionandPurification
Fastingserumsample(from5mlofblood)wasobtainedbystandardvenepunctureusing
VacutainerPlusPlasticSerumandSSTTubes(Becton-Dickinson,Franklin,lakes,NJ).Thesep-
arationoftheserumwasperformedbycentrifugationat4,000rpmfor10min,followedby
12000rpmfor15mintocompletelyremovecelldebris.TotalRNAwasisolatedfrom0.25ml
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CirculatoryMiRNAsofAsianIndianPhenotype
Table1. Clinicalandbiochemicalcharacteristicsofthesubjectsinvolvedinthestudy.
Wholecohort Subgroupofsubjectsusedtoselectcandidate
miRNAs
Parameters NGT IGT T2DM NGT IGT T2DM
(n=49) (n=47) (n=49) (n=12) (n=12) (n=12)
Males/females (26/23) (23/24) (25/24) (6/6) (6/6) (5/7)
Age(years) 44.3±6.9 44.1±7.0 44.4±8.1 38.5±3.0 41.2±4.0 40.2±5.0
BMI(kg/m2) 24.5±2.6 24.9±2.9 25.7±3.5 23.1±2.0 23.9±1.0 26.7±1.0
Fastingplasmaglucose(mg/dl) 85±11 105±18* 146±34* 83±7.0 101±7.0* 150±27*
2hrplasmaglucose(mg/dl) 96±21 166±18* 269±63* 89±24 160±20* 264±61*
HbA1c(%) 5.6±0.4 6.3±0.8* 7.8±1.6* 5.2±0.5 6.0±0.5 7.9±±1.0*
HOMA-IR 2.0±1.1 3.5±1.4* 6.3±1.8* 1.5±0.4 2.7±1.0 5.0±3.0*
SerumCholesterol(mg/dl) 170±24 187±35 185±52 168±29 185±39 185±52
SerumTriglycerides(mg/dl) 97±38 148±76 136±62 119±65 134±69 148±67
HDLcholesterol(mg/dl) 41.2±9.0 40±5.0 37±7.0 41±9.0 41±10 40±8.0
LDLcholesterol(mg/dl) 111±22 117±32 121±46 107±26 117±33 115±44
VLDL(mg/dl) 19±7.6 30±16 25±13 24±13 27±14 29±13
FastingInsulin(μIU/ml) 7.6±2.0 11±4.0 13±6.7 9.6±4.6 13.4±4.6* 17.8±4.0*
Systolicbloodpressure(mmHg) 117±9.0 121±12 124±12 123±20 124±12 126±16
Diastolicbloodpressure(mmHg) 78±10 82±5.0 81±7.0 80±10 80±8.0 84±9
Waistcircumference(cm) 87.1±1.21 93.8±1.60* 92.3±1.38* 87.3±8.4 93.5±10.9* 92.3±9.6*
Allthevaluerepresentsmeanand±standarddeviation
*p<0.05comparedcontrol
ND,notdetermined
doi:10.1371/journal.pone.0128372.t001
ofserumusingQiagenmiRNeasyMinikit(Qiagen,Valencia,CA)forRNAcollectionandpu-
rificationaccordingtothemanufacturer'sprotocol.TotalRNAwascontrolledusingNanodrop
2000UVspectrophotometerandstoredat-80°Cuntiluse.
GlobalSerummicroRNAProfiling
GlobalserummiRNAprofilingwasdoneinadiscoverycohortcomprisedof12individuals
eachofNGT,IGTandT2DM(Table1).ToassesshemolysistwomicroRNAswereused.One
thatisexpressedinredbloodcells(miRNA-451),andonethatisrelativelystableinserumand
plasmaandnotaffectedbyhemolysis(miRNA-23a).TheratiobetweenthesetwomiRNAscor-
relatestodegreeofhemolysis.Inourexperiencesampleswithratiosabove8.0willhaveanin-
creasedriskofbeingaffectedbyhemolysis.Wevalidatedthatoursampleshadlowerratiosand
werenotaffectedbyhemolysis.DuetothelowlevelsofmicroRNAsandpotentiallyhighlevels
ofinhibitorsinsamplesderivedfromserum/plasmawhichalsodiffersfromsampletosample,
Exiqonprotocol(www.exiqon.com/serum-plasma-guidelines)recommendsusingRNA
amountsbasedonstartingvolumeratherthanRNAquantity.8μLofelutedhumanplasma/
serumRNAina40μLRTreactiongenerallygiveagoodsignalwithmaximalmiRNAdetection
inmiRNAPCRpanels[34].Thereforeinourstudy,8μlRNAwasreversetranscribedin40μl
reactionsusingthemiRCURYLNAUniversalRTmicroRNAPCRcDNAsynthesiskit.An
RNAspike-incontrol(Sp6)wasaddedtothereversetranscriptionstep.Thiscontrolisusedto
confirmthatthereversetranscriptionandamplificationoccurswithequalefficiencyinallsam-
ples.cDNAwasdiluted50xandassayedin10μlPCRreactionsaccordingtotheprotocolfor
miRCURYLNAUniversalRTmicroRNAPCR;eachmicroRNAwasassayedoncebyqPCRon
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CirculatoryMiRNAsofAsianIndianPhenotype
themicroRNAReady-to-UsePCR(miRCURYLNAmicroRNAHumanpanelIExiqon).Neg-
ativecontrolsexcludingtemplatefromthereversetranscriptionreactionwasperformedand
profiledlikethesamples.TheamplificationwasperformedinaLightCycler480Real-Time
PCRSystem(Roche)in384wellplates.Theamplificationcurveswereanalyzedusingthe
RocheLCsoftware,bothfordeterminationofCp(bythe2ndderivativemethod)andformelt-
ingcurveanalysis.AmplificationefficiencywascalculatedusingalgorithmssimilartotheLin-
Regsoftware.AllassayswereinspectedfordistinctmeltingcurvesandtheTmwascheckedto
bewithinknownspecificationsfortheassay.Furthermoreassaysmustbedetectedwith5Cp’s
lessthanthenegativecontrol,andwithCp<37tobeincludedinthedataanalysis.Datathat
didnotpassthesecriteriawereomittedfromanyfurtheranalysis.UsingNormFinderthebest
normalizationprocedurewasfoundtobetheaverageofassaysdetectedinallsamples.Alldata
werenormalizedtotheaverageofassaysdetectedinallsamples(average—assayCp).Compari-
sonbetweengroupsweremadebyusingthestudentt-test(p<0.05)onnormalizeddata,tose-
lectthedifferentiallyexpressedmiRNAs.
ValidationofcandidatemiRNAsbyindividualqRT-PCR
miRNAsshortlistedasdifferentiallyexpressedfromthediscoverysetwererevalidatedina
largercohortcomprisingof145individuals(50%men,50%women)withNGT(n=49),IGT
(n=47)andT2DM(n=49)byindividualPCRassays.TotalRNAfromserumwasextracted
asdescribedaboveandusedforqRT-PCRbyusingtheUniversalmiRCURYLNAmicroRNA
PCR,PolyadenylationandcDNAsynthesiskitIIandthemiRCURYLNAmicroRNAPCRsys-
tem,ExilentSYBRgreenmastermix,fromExiqon,onABI7000AppliedBiosystemsthermo-
cycler.RelativeexpressionofmicroRNAexpressionwascalculatedby2ˆ(-"DeltaCt")method.We
evaluatedasuitablenumberofreferencemiRNAs,basedontheincreasedexpressionstability
[28].However,wewereunabletoidentifystablemiRNAsthatdidnotdiscriminatebetween
maleandfemaleorthatwerenotcorrelatedwithmetabolicparameters,orthatwasnotprevi-
ouslyidentifiedasrelevantforthepathologyinpreviousstudies.Thiscouldbeexplainedpartly
bythefactthatinsidethesamegroup,womenandmenmightdifferedintheirlevelofcholes-
terolandtriglycerides(S1Table).Thus,inthisstudy,datawerenormalisedonthevolumeof
totalRNAthatwasusedforqRT-PCR(i.e.;afixedvolumeof8μl).
StatisticalMethods
StatisticalanalyseswereperformedwiththeSPSSstatisticalsoftware(SPSSV12.0,Inc.,Chi-
cago,IL),andtheRStatisticalSoftware(http://www.r-project.org/).ANOVAand/orpairedt-
testswereperformedtostudydifferencesonquantitativevariablesbetweengroups.
BioinformaticAnalysis
CellularpathwaydeterminationforeachmiRNAwasdonebyusingDIANA-miRPath(http://
diana.imis.athena-innovation.gr/DianaTools/index.php?r=site/index)whichpredicted
targetgenes.
AnimalsMaintenance,PreclinicalCharacterizationandmiRNAProfiling
MaleC57BL/6Jmice,10–12weeksold(bodyweight,18–22g)wereobtainedfromSriVenka-
teswaraEnterprises(Bangalore)andmaintainedat22±1°Cundera12-hlight-darkcycle(lights
onfrom6:00AMto6:00PM).Allexperimentswereperformedinaccordancewithregulations
specifiedbytheCommitteeforthePurposeofControlandSupervisiononExperimentsonAn-
imals(CPCSEA),GovernmentofIndiaandapprovalbytheInstitutionalAnimalEthical
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CirculatoryMiRNAsofAsianIndianPhenotype
Committee(IAEC)oftheMadrasDiabetesResearchFoundationChennai.Food(Nutrilab,
Bangalore)andwaterweregivenadlibitumtotheanimals.Highfatdiet(57%)wasprocured
fromtheNationalInstituteofNutrition,Hyderabad,India.Afteracclimatization,basalfasting
plasma/serumbiochemicalmarkerswereestimated.Micewererandomlydividedintotwo
groupsi.e.,micefedwithnormalpelletdiet(NPD;n=5)andmicefedwithhighfatdiet(HFD;
n=6),withsimilaraveragebodyweightandplasmaglucoseonday0.Allgroupswerefedwith
respectivedietandwateradlibitumfor6months.Allbiochemicalandpreclinicalmeasure-
mentsweredoneasperthestandardprocedures(S2Table).Attheendoftheprotocol,HFD
fedanimalswerecharacterizedasglucose-intolerantandinsulin-resistantbyOGTT(OralGlu-
coseToleranceTest)andITT(InsulinToleranceTest),respectively.Serumsamples(0.25ml)
wereusedtoquantifycandidatemiRNAbyqRT-PCRaspreviouslydescribed.
Results
Inthisstudy,wehaveanalysedtheexpressionofcirculatingmicroRNAsinserumof145non
obesesubjects(50%malesand50%females)sufferingfromT2DM(n=49)comparedwith
controls(n=49)orpre-diabeticsubjects(n=47).AsshownonTable1,bothprediabetics
(IGT)andpatientswithtype2diabetes(T2DM)hadpoorglycemiccontrolasreflectedbysig-
nificanthigherfastingplasmaglucoseand2hrplasmaglucoseandhigherHbA valuescom-
1c
paredwithcontrols.Subjectswithprediabetesandpatientswithtype2diabetesexhibited
significantlyhigherinsulinresistanceasrevealedbyHOMA-IRestimation.Thelipidlevelsand
bloodpressuredidnotsignificantlydifferamongthe3groups.
ProfilingofCirculatingmiRNA
SerummiRNAprofilingwasfirstperformedonasubgroupof36subjectsfromthe145subjects
(50%malesand50%females).T2DMandIGTpatientsofthisidentificationsampleonlydif-
feredclinicallyfromcontrolsubjectsinfastingglucoseand2hrplasmaglucoseandinfasting
insulin.BothT2DMandIGTpatientshadsignificantlyhigherwaistcircumferencethanthe
controlgroupofsubjects(Table1).TheglobalserummiRNAprofiling(miRCURYLNA
microRNAHumanpanelIV3-Exiqon)detectedanaverageof~159miRNAspersampleof
which112miRNAsweredetectedinallgroups.AsshownonS1Fig,themajorityofthe112
miRNAswereexpressedinasimilarwayandhierarchicalclusteringofthedatadidnotpermit
todiscriminatebetweenthe3groups.Statisticaldataanalysisrevealedthatthemeanexpres-
sionlevelof9miRNAs(i.e.;miR-128,miR-99b-5p,miR-130b-3p,miR-142-3p,miR-374a-5p,
miR-423-5p,miR-484,miR-629-5p,let-7d-3p)wassignificantlydifferent(studentt-test
p<0.05)acrossthestudiedgroups(Table2).TwomiRNAs(i.e.;miR-128andmiR-99b-5p)
hadincreasedcirculatingconcentrationsbothinprediabeticsubjectsandpatientswithtype2
diabetescomparedtocontrolsubjects(Table2).
Allthe9miRNAsdifferentiallyexpressedamongthediscoverygroupswereanalysedby
quantitativeRT-PCRusingthewholecohortof145individuals(Fig1)containing50%men
and50%women.AsshownonFig1,alteredexpressionsofmiR-128andmiR-423-5pinpre-
diabeticpatientscomparedtocontrolswereconfirmedwithhighsignificance(p<0.05).Inad-
dition,miR-374a-5pandmiR-130b-3pwerealteredinthesamedirectioninthewholegroup
ofpatientssufferingfromT2DMthanindiscoverygroup,comparedtothecontrolgroupof
subjects,andmiR-130b-3pwasonlyaffectedintheT2DMgroup.AlteredexpressionsofmiR-
629a-5p,let-7d-3p,miR-142-3pandmiR-484werenotconfirmedinthewholecohort
(p<0.05).
ThenmiRNAexpressionswereanalysedinwomenandmenindependently(Fig2).Altered
expressionsofmiR-128inpre-diabeticstatevscontrolwasfoundsignificantinthegroupof
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Table2. ListofdifferentiallyexpressedcirculatingmiRNAsintheidentificationgroup.
Groups miRNAs Foldchanges pvalue
NGTvsIGT hsa-miR-128 1.42 0.041
hsa-miR-99b-5p 1.41 0.028
IGTvsT2DM hsa-miR-130b-3p 1.41 0.035
hsa-miR-142-3p 0.57 0.0041
hsa-miR-374a-5p 0.58 0.039
hsa-miR-423-5p 1.54 0.011
hsa-miR-484 1.37 0.004
hsa-miR-629-5p 1.84 0.030
NGTvsT2DM hsa-let-7d-3p 1.26 0.029
hsa-miR-128 1.50 0.047
hsa-miR-130b-3p 1.46 0.049
hsa-miR-142-3p 0.65 0.039
Ctvalueswereusedtoevaluatedifferencesamongtheidentificationgroupandtoselectcandidate
miRNAs
doi:10.1371/journal.pone.0128372.t002
womenonly.Onthecontrary,alteredexpressionofmiR-423-5pwassignificantlydecreased
inthegroupofmen(NGTvsIGT)only.TheincreaselevelofmiR-374a-5pinthediabetic
Fig1.CirculatingmiRNAconcentrationofthe9selectedmiRNAs,inthewholecohortof145
individuals.**=pvalues<0.05.Dataareexpressedasarbitraryunits(AU).
doi:10.1371/journal.pone.0128372.g001
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CirculatoryMiRNAsofAsianIndianPhenotype
Fig2.CirculatingmiRNAconcentrationsofthe9selectedmiRNAs,inthewholecohortof145
individuals,butconsideringmen(M)andwomen(F)independently.**=pvalues<0.05.Dataare
expressedasarbitraryunits(AU).Black,controlsubjects;Grey,pre-diabeticsubjects;White,
diabeticpatients.
doi:10.1371/journal.pone.0128372.g002
subjectsvscontrolswasfoundsignificantinthegroupofwomenonly.Finally,miR-142-3p
whichwasnotdifferentiallyexpressedconsideringthewholecohort(Fig1),wasfounddiffer-
entlyexpressedwhenthegroupofwomenwasanalysedindependently(NGTvsIGT,Fig2).
CorrelationofmiRNAswithMetabolicParameters
CorrelationsbetweenthelevelsofthevalidatedmiRNAswiththemetabolicparameterswere
calculatedinthewholecohortofsubjects(Table3).Theywerepositivelyornegativelycorrelat-
edtocholesterollevels,HbA ,insulinresistanceandhyperinsulinemia.MiR-128,whichwas
1C
increasedinprediabeticpatients(Fig1),waspositivelycorrelatedwithserumcholesterol.On
thecontrary,miR-423-5pdecreasedintheserumofprediabeticpatients(Fig1)wasnegatively
correlatedtoHDL-cholesterol.
miRNATargetGenesAnalysis
Targetgenesofthe4validatedmiRNAswerepredictedandsignificantcellularpathwaysaffect-
edbythesegeneswereretrievedforeachmiRNA.SignificantKEGGpathwaysareshownon
Fig3.Therearerelatedtocellcycle,signalingpathways,lipidmetabolism,glycanbiosynthesis,
brainfunctionsandimmunesystem.
CirculatorymiRNAlevelsinHFDmicevsNPDfedmice
Tofurthercorroboratetheassociationsassessedinhuman,thepresentstudyalsoanalyzedby
meansofqRT-PCRcirculatingmiRNAconcentrationsintheplasmaofdiet-induceddiabetic
mice(HFD).Inthecontextofthewell-knowninsulin-resistanceeffects,high-fatdietledto
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Table3. CorrelationsbetweencirculatingmiRNAconcentrationsandmetabolicparametersinthestudysubjects.
miRNAs Metabolicparameters Estimate StdErr tValue Probt
Correlationsnotadjustedforsex miR-128 Serum-cholesterol 0.003482 0.001333 2.61 0.0099
miR-423-5p HDL-cholesterol -0.3067 0.1363 -2.25 0.0260
miR-130b-3p HbA1C 0.1902 0.04745 4.01 <.0001
miR-374a-5p HbA1C 0.1745 0.07149 2.44 0.0159
miR-374a-5p HOMA-IR 0.1093 0.04364 2.50 0.0134
miR-374a-5p Fasting-Insulin 0.04148 0.01844 2.25 0.0260
miR-128 Serum-cholesterol 0.003594 0.001323 2.72 0.0074
miR-423-5p HDL-cholesterol -0.3052 0.1266 -2.41 0.0172
Correlationsadjustedforsex miR-130b-3p HbA1C 0.1903 0.04728 4.03 <.0001
miR-374a-5p HbA1C 0.1763 0.07174 2.46 0.0152
miR-374a-5p HOMA-IR 0.1082 0.04383 2.47 0.0147
miR-374a-5p Fasting-Insulin 0.04138 0.01852 2.23 0.0270
Tocalculatecorrelations,Ctvalueswereconvertedintocopynumbers(10^((Ct-25)/-3,3)*100)totakeintoaccountthelogarithmicscaleofthedata.
doi:10.1371/journal.pone.0128372.t003
Fig3.PredictedcellularpathwaysofthetargetgenesofthesignificantlyalteredcirculatingmiRNAs
inserumofpre-and/ordiabeticpatientsvscontrols.OnlyvalidatedmiRNAsinthewholecohort
wereconsidered.
doi:10.1371/journal.pone.0128372.g003
PLOSONE|DOI:10.1371/journal.pone.0128372 May28,2015 9/14
CirculatoryMiRNAsofAsianIndianPhenotype
Fig4.CirculatingmiRNAconcentrationsofthe9selectedmiRNAsidentifiedinthediscoverygroup
quantifiedintheserumofmicefedwithnormalpelletdiet(NPD;n=5)orwithhighfatdiet(HFD;
n=6).Dataareexpressedasarbitraryunits(AU).White,NPDmice;Black,HFD.
doi:10.1371/journal.pone.0128372.g004
increasedcirculatingconcentrationsofmiR-128,miR-130b-3p,miR-99b-5p,miR-629a-5pand
miR-let-7d-3pexpressioninHFDmicecomparedwithNPDfedmice(p<0.05).Conversely,
miR-142-3pwassignificantlylowerinHFDanimalscomparedtocontrolmice(Fig4)Correla-
tionsbetweentheexpressionsofalteredmiRs(HFDvsNPD)withthemetabolicparameters
werecalculatedandareshowninTable4.Asinhumanpre-diabeticsubjects,miR-128waspos-
itivelycorrelatedwithcholesterol.
Discussion
SouthAsiansarecharacterizedbyauniquemetabolicprofilewithhigherinsulinlevels[3],a
greaterdegreeofinsulinresistance[4],greaterabdominaladiposityi.e.,higherwaistcircumfer-
encedespitelowerbodymassindex[2]andahigherprevalenceofdiabetes[5].Insulinresis-
tancehasbeendemonstratedinAsianIndiansevenduringadolescence[6]and
hyperinsulinemiaseemstobepresentinAsianIndiansevenatbirth[7].Itappearsthatsome
Table4. CorrelationsbetweencirculatingmiRNAconcentrationsandmetabolicparametersinmice.
miRNAs metabolicparameters Estimate StdErr tValue Probt
miR-128 cholesterol -0.0292 0.005164 -5.65 0.0005
miR-484 Glucose -0.01151 0.00478 -2.41 0.04
miR-629-5p Glucose 0,04474 0,01286 3,48 0,007
miR-128 Glucose 0,02141 0,00837 2,56 0,031
miR-130b-3p HbA1c -1.2578 0.4202 -2.99 0.019
miR-99b-5p HbA1c -2.4481 0.463 -5.29 0.001
miR-130b-3p HDL -0.03028 0.00876 -3.46 0.011
miR-99b-5p HOMA_IR 0.1122 0.04224 2.66 0.026
miR-let-7d-3p HOMA_IR 0.02725 0.01108 2.46 0.036
miR-99b-5p TGL 0.02065 0.00898 2.3 0.047
miR-let-7d-3p TGL 0.005752 0.00214 2.69 0.025
miR-99b-5p VLDL 0.1033 0.04491 2.3 0.047
miR-let-7d-3p VLDL 0.02876 0.0107 2.69 0.025
doi:10.1371/journal.pone.0128372.t004
PLOSONE|DOI:10.1371/journal.pone.0128372 May28,2015 10/14