levels in women with breast cancer undergoing hormone therapy.

12

Original article

3

Effect of a high-intensity interval training on serum microRNA

4

levels in women with breast cancer undergoing hormone therapy.

5

A single-blind randomized trial

6 ^Q1

Shaban Alizadeh

^a

, Amin Isanejad

^b,c

, Sanambar Sadighi

^d

, Solmaz Khalighfard

^e,f

,

7

Ali Mohammad Alizadeh

^e,g,

*

8 ^aDepartmentofHematology,AlliedMedicalSchool,TehranUniversityofMedicalSciences,Tehran,Iran 9 ^bImmunoregulationResearchCenter,ShahedUniversity,Tehran,Iran

10 ^cPhysicalEducationDepartment,ShahedUniversity,Tehran,Iran

11 ^dMedicalOncologyandHematologyDepartment,CancerInstituteofIran,TehranUniversityofMedicalSciences,Tehran,Iran 12 ^eCancerResearchCenter,TehranUniversityofMedicalSciences,Tehran,Iran

13 ^fDepartmentofBiology,ScienceandResearchBranch,IslamicAzadUniversity,Tehran,Iran 14 ^gBreastDiseaseResearchCenter,TehranUniversityofMedicalSciences,Tehran,Iran

15

16 1. Introduction

17 Breastcancer isthe mostcommon diagnosedcancer among 18 women and the second leading cause of gynecological cancer

19 deathsintheUnitedStates.MicroRNAs(miRs)areagroupofshort

20 non-coding RNAsthat regulate protein-coding geneexpression.

21 Several studieshave shown that theexpressionofmiRs differs

22 betweennormaltissueandtumourtissue[1].MiRscanbecancer

23 miRs (oncomiRs)and/ortumoursuppressor miRs (TSmiRs).The

24 expressionofabnormalmiRsresultsinachangeinexpressionof

25 the protein-encoding genes that can play a central role in

26 tumorigenesisand/ortumoursuppression[2].Theinteractionof

27 miRs with target genes can confirm their role in cell growth, ARTICLE INFO

Articlehistory:

Received17November2018 Accepted7July2019

Keywords:

microRNAs Breastcancer Hormonetherapy Training

ABSTRACT

Background:TheroleofmicroRNAs(miRs)inhormonetherapy(HT)isofkeeninterestindeveloping biomarkersand treatmentsforindividuals withbreast cancer. AlthoughmiRsare oftenmoderate regulatorsunderhomeostaticconditions,theirfunctionischangedmoreinresponsetophysicalactivity.

Objective:This single-blind randomizedtrial aimedto exploretheeffect ofhigh-intensity interval training(HIIT)onserumlevelsofmiRsinindividualswithearly-stagebreastcancerundergoingHT.

Methods:Hormonereceptor-positivewomenwithbreastcancerandhealthywomenwererandomly assignedtoahealthycontrolgroup(n=15),healthygroupwithHIIT(n=15),breastcancergroupwith HT(HT,n=26),and breastcancergroupwithHTandHIIT(HT+HIIT,n=26).Theexercisegroups underwentintervaluphillwalkingtrainingonatreadmill3timesaweekfor12weeks.Attheendofthe study,weanalyzedchangesinlevelsofcancer-relatedmiRs(oncomiRs)andtumoursuppressormiRs (TSmiRs)inresponsetotheHTandHIIT.

Results:Inwomenwithbreastcancerversushealthycontrols,theexpressionofsomeoncomiRswas significantly increased — miR-21 (P<0.001), miR-155 (P=0.001), miR-221 (P=0.008), miR-27a (P<0.001),andmiR-10b(P=0.007)—andthatofsomeTSmiRswassignificantlydecreased—miR-206 (P=0.048),miR-145(P=0.011),miR-143(P=0.008),miR-9(P=0.020),andlet-7a(P=0.005).Moreover, HT considerably downregulated oncomiRs and upregulated TSmiRs. HIIT for 12weeks with HT significantlydecreasedtheexpressionoftheoncomiRsandsignificantlyincreasedthatoftheTSmiRsas comparedwithHTalone.

Conclusions:HITTcouldamplifythedecreaseand/orincreaseinexpressionofmiRsassociatedwithHTin womenwithbreastcancer.AprospectivetrialcoulddeterminewhethertheuseofcirculatingmiRsfor monitoringtreatmentcanbeusefulintherapydecisions.

Trialregistration:IranianRegistryofClinicalTrials(No.:IRCT201202289171N1).

C 2019PublishedbyElsevierMassonSAS.

* Correspondingauthorat:CancerResearchCenter,TehranUniversityofMedical Sciences,P.O:1419733141,Tehran,Iran.

E-mailaddresses:aalizadeh@sina.tums.ac.ir,alizadehtums92@gmail.com (A.M.Alizadeh).

Availableonlineat

ScienceDirect

www.sciencedirect.com

https://doi.org/10.1016/j.rehab.2019.07.001 1877-0657/^C 2019PublishedbyElsevierMassonSAS.

28 apoptosis,anddifferentiation[3].Thecirculatorylevelsofsome 29 miRsarehigherinindividualswithcancerthaninhealthypeople 30 [1].Heneghanetal.demonstratedthatthelevelsofmiR-195and 31 Let-7aasoncomiRscouldbesignificantlyincreasedinthebloodof 32 individuals with breast cancer as compared with disease-free 33 controls[1].

34 Allovertheworld,morethan70%ofbreastcancercasesexpress 35 ahormonereceptor,eitherestrogenand/orprogesteronereceptor.

36 Hormonetherapy (HT)is a formofsystemic treatment recom- 37 mendedforwomenwithhormonereceptor-positivebreastcancer 38 [4].Inthiscontext,HTcanleadtodecreasedexpressionofsome 39 miRsinindividualswithbreastcancer[2].Likewise,miR-21,miR- 40 155,andmiR-10bmayactasoncomiRswithconsistentdecreasein 41 expressionwithHT[5].Becauseoftheeasyidentificationandhigh 42 stabilityofthesemiRs,theymaybesuitableasbiomarkers[6].The 43 identificationofcirculatingmiRsandtheirregulationafterexercise 44 suggest that they may be useful biomarkers of health and 45 adaptationto treatment interventions. There is someevidence 46 ofanassociationbetweencardiorespiratoryand physicalfitness 47 withcirculatorymiRs[7,8].Byeetal.showedthatlevelsofmiR- 48 210,miR-21,andmiR-222wereincreasedinhealthyindividuals 49 withlowmaximaloxygenuptake(VO2max)[7].Regularphysical 50 activitycandownregulatemiR-21 expressioninhealthy people 51 [9]. Exercise may also affect the levels of circulating anti- 52 angiogenicmiRs suchas miR-20a,miR-210, miR-221,miR-222, 53 and miR-328 [10]. Recently, in preclinical studies, our team 54 showedthatexercisecouldaffecttumourgrowthviachangesin 55 levelsofmiRssuchasmiR21,miR206,andlet-7a[11,12].

56 Typically, studies have examined the effect of light and 57 moderateintensityexerciseonphysicalandpsychologicalindices 58 inindividualswithcancer [13].High-intensityexercisetraining 59 (HIIT)haslongbeendemonstratedtohelpimprovecardiorespira- 60 toryfitnessandcorrespondingphysiologicalvariablesinhealthy 61 individuals[14].Thetraininginvolvesrepeatedshorttolongbouts 62 of relatively high-intensity exercise alternating with recovery 63 periodsof low-intensity activityor passive rest[15].HIIT may 64 improvesurvivalrateand reduce therisk of cancer recurrence 65 [16].However,consideringthebenefitsofHIIT,thisusefultraining 66 methodhasnotbeenstudiedinindividualswithbreastcancer.

67 Inthisstudy,wehypothesizedthatmiRssecretedfrommuscle 68 tissue or taken up by muscles or other organs can mediate 69 transientandadaptiveresponsestotheexercise.Inthiscontext, 70 2mainchallengesforthesuccessfultreatmentofbreastcancerare 71 the development of more specific biomarkers that predict 72 therapeuticresponsetoendocrinetherapiesandtheidentification 73 of new therapeutic targets for endocrine-resistant disease.

74 However,theidentificationoftheexpressionpatternofthemiRs 75 characterizing exercise training may be useful for monitoring 76 physical fatigue and recovery and even to evaluate physical 77 performance capacity. Therefore, the present study aimed to 78 investigatetheeffectof HIITwithorwithoutHTonserummiR 79 levelsinwomenwithearly-stagebreastcancer.

80 2. Methods 81 2.1. Studydesign

82 This single-blind randomized trialincluded 4 arms: healthy 83 controlandhealthycontrolwomenwithHIITandwomen with 84 Q2breastcancerwithHTorHTwithHIIT(HT+HIIT)(Table1).All 85 womenwithbreastcancer[estrogenreceptor-positive(ER+)and 86 progesterone receptor-positive (PR+)and/or only ER+] received 87 letrozole or tamoxifen as an HT regimen. Participants were 88 recruited from the Cancer Institute of Iran (Hospital Imam 89 Khomeini, Tehran, Iran) from March2013 to March2014. The

90 trial was registeredwith the IranianRegistry of Clinical Trials

91 (IRCT201202289171N1).

92 Participantswhogavetheir signedwritteninformedconsent

93 weredeemedeligibleforthestudyiftheywerebetween30and

94 60years old;hadinsufficientphysicalactivitylevel(<150min/

95 week);hadhormonereceptor-positivebreastcancer;performed

96 no strenuous exercise such as running, cycling, swimming or

97 resistance training; completed adjuvant chemotherapy and

98 radiotherapyinthelastmonth;andtookletrozoleortamoxifen

99 (HT). Non-inclusion criteria werecurrent smoking; evidence of

100 metastatic breast cancer; planning to receive any additional

101 adjuvant chemotherapy or surgery; pregnant or breastfeeding;

102 cardiovascular comorbidities such as myocardial infarction or

103 coronary artery disease; uncontrolled hypertension defined as

104 systolic blood pressure180mmHg or diastolic blood pressu-

105 re100mmHg; high-risk or uncontrolled heart arrhythmias;

106 decompensated heart failure; known aortic aneurysm; chronic

107 obstructivepulmonarydisease;oranyotherconditionthatmay

108 impedetestingofthestudyhypothesisormakeitunsafetoengage

109 intheexerciseprogram.

110 2.2. Randomizationandblinding

111 Thephysicalactivitylevelofallparticipantswasmeasuredby

112 International Physical ActivityQuestionnaire. Participants were

113 randomizedandstratifiedtothehealthy controlgroup(n=15),

114 healthygroupwithHIIT(n=15),breastcancergroupwithHT(HT,

115 n=26), and breast cancer group with HT and HIIT (HT+HIIT,

116 n=26) accordingtoa computerized randomnumber generator

117 (1998-2017RANDOM.ORG).Therandomizationcodewasdevel-

118 opedtoselectrandomizedpermutedblocks.Aninvestigatorwho

119 was not involved in the assessment, treatment or statistical

120 analysisperformedtherandomization.

121 2.3. Exerciseintervention

122 WomenwithbreastcancerwhowereassignedtoHIITreceived

123 usualcare(routinedailyactivity)andalsoattendedasupervised

124 high-intensityaerobicintervalexerciseprogram3timesaweekfor

125 12weeks. The HIIT groups participated in 3 familiarization

126 sessionsbeforestartingthemainexerciseprogram.

127 WeusedtheHIITprotocolthatwaspreviouslyreportedasasafe

128 trainingregimenforindividualswithheartfailureandcoronary

129 artery diseases [17]. The training intensity was determined

130 accordingtothepredictedmaximalheartrate(HR),despitethe

131 long-standinglimitationofthisformulaascomparedwithactual

132 measurementofmaximalHR(HRmax)byamaximumstresstest

Table1

Generalcharacteristicsofwomenwithbreastcancerinthestudywhoreceived hormonetherapy(HT)orHTplushigh-intensityintervaltraining(HIIT).

Variables HT

n=26

HT+HIIT n=24

Age,years,mean(SD) 48.42(7.54) 49.2(9.7)

Meantumorsize,mean(SD) 3.63(2.07) 3.04(1.7) Tumorgrade

Well-differentiated 5 4

Moderatelydifferentiated 17 16

Poorlydifferentiated 3 3

Tumorstaging

T1 9 6

T2 10 9

T3 6 8

T4 10 8

N0 13 14

N1 2 1

133 [18].Eachparticipantunderwenttheexercisesessionindividually 134 withsupervision byanexercisephysiologist. Themainexercise 135 intervalsconsistedof44minofuphillwalkingat90%to95%

136 HRmax(exercise)and 43minofuphillwalkingat50% to70%

137 HRmax(activerecovery)onamotorizedtreadmill(Impulse,USA) 138 [17].Theoveralltime ofeachsession was38min,consistingof 139 5minwarm-up,5mincooldown,16minHIIT,and12minactive 140 recovery between intervals. The HR of participants was fully 141 monitoredduringeverytrainingsession[19].Allparticipantsused 142 a HR monitor (Polar Electro, Kempele, Finland) to obtain the 143 assigned exercise intensity. The speed and inclination of the 144 treadmillwerecontinuouslyadjustedtoensurethateverytraining 145 sessionwascarriedoutattheassignedHRthroughoutthetraining 146 period.Duringthetrainingsessions,participantswereadvisedto 147 respecttheirphysical limitations.The moderatorsmanagedthe 148 participants’dailyadherencetoexercisecourses.Womeninthe 149 generalcaregroupwereinstructedtocontinuewiththeirroutine 150 activities [17,19]. The general care was to maintain baseline 151 physical activity levels of participants during 12weeks of the 152 intervention. Theexercise trainersmonitored adherence to the 153 intervention.

154 2.4. Bloodsampling

155 Bloodsampleswereobtainedfromtheantecubitalveininthe 156 morning after a 12-hr fast. The serum was collected and 157 centrifugedat 12,000gfor 15min toremove cell debris. Post- 158 intervention blood collection was3days after the last training 159 session toavoid theacuteeffect ofthe exercise.Sampleswere 160 aliquotedandstoredat80CuntilmiRdetection.

161 2.5. Identificationofcancer-relatedbreastmiRs

162 TheGeneExpressionOmnibusdatabase(GEO,http://www.ncbi.

163 nlm.nih.gov/geo/) in the National Center for Biotechnology 164 Information (NCBI) is the public gene expression resource and 165 includes214,268samplesand4500platforms[20,21].Theselected 166 miRsweresearchedinthehumanmiRdatabase(HMDD;http://

167 cmbi.bjmu.edu.cn/hmdd and http://202.38.126.151/hmdd/tools/

168 hmdd2.html) tofurther select the differentially expressed miRs 169 relatedtobreastcancer.Asadatabaseforexperimentallysupported 170 humanmiRsanddiseaseassociations,HMDDisavaluableresource 171 forstudyingtherolesofmiRsinhumandiseases[22].Furthermore, 172 the target genes of the differentially expressed breast cancer- 173 relatedmiRswerepredictedbyfivedatabases, namelyMiRanda 174 (http://microrna.sanger.ac.uk/) [23], MirTarget2 (http://nar.

175 oxfordjournals.org/cgi/content/abstract/34/5/1646) [24], PicTar 176 (http://pictar.bio.nyu.edu/) [25], PITA (http://genie.weizmann.ac.

177 il/pubs/mir07)[26],andTargetScan(http://targetscan.org/)[27].As 178 well,thepublishedoncomiRsandTSmiRsforbreastcancerwere 179 selected from TSGene (http://bioinfo.mc.vanderbilt.edu/

180 TSGene/)[28]andTumor-AssociatedGene(TAG;http://www.

181 binfo.ncku.edu.tw/TAG/)databases[29]. Inthepresentstudy, 182 weusedKyotoEncyclopediaofGenesandGenomes(KEGG)and 183 enrichmentanalysesofthegenesfortheidentifiedtargetgenes 184 [30,31].

185 2.6. QuantitativeRT-PCR

186 Total RNAwas extracted from100

m

L plasma samplesby 187 using 1ml Trizol reagent according to the manufacturer’s 188 instructions(Sinagene,Tehran,Iran).Qualitativeandquantita- 189 tiveassessmentsofisolatedRNAinvolvedelectrophoresisand 190 spectrometrymethods[31,32].TheRNAwasstoredat808C.

191 ForquantificationofmiRsbyRT-PCRinallsamples,10

m

Ltotal

192 RNAwasreverse-transcribedina20-

m

Lreactionmixbyusingthe

193 BONmiR1st-strand cDNA synthesis kit (Bonyakhteh, Tehran, Iran)

194 followingthemanufacturer’srecommendations.Then,cDNAwasused

195 intheRT-PCRassayswiththeBONmiRqPCRKit(Bonyakhteh,Tehran,

196 Iran)basedonthemanufacturer’sinstructions. RT-PCRanalysesof

197 miRswereperformedintriplicate.ThemiRlevelswerenormalizedto

198 SNORD RNA level (an internal control). MiRgene expression was

199 analyzedbyusingtheStep-Onesystem(ABI,Massachusetts,USA).

200 RelativeexpressionofmiRswascalculatedbythe2^(DDct)method.

D

CTwascalculated bysubtractingtheCT valuesforSNORDfrom 201 202 those of the target miRs [31].

DD

CT was then determined by

203 subtractingthemean

D

CTofthecontrolsamplefromthatofthecase

204 samples.ThefoldchangeinlevelsofcandidatemiRswascalculatedby

205 theequation2^(DDct)[31,33].Sequencesoftheforwardprimersareas

206 follows:MiR-21forwardprimer:ACGTGTTAGCTTATCAGACTG;MiR-

207 155 forward primer: CCGTTAATGCTAATCGTG; MiR-10b forward

208 primer: GGTTAATAAAGCCGCCATCC; Let-7a forward primer:

209 GGCTGAGGTAGTAGGTTGTATAG; Mir-9 forward primer: AGG-

210 CATCTTTGGTTATCTAG;Mir-27aforwardprimer:CCGTTCACAGTGGC-

211 TAAG;Mir-143forwardprimer:CTGTTGAGATGAAGCACTGT;Mir-145

212 forward primer:GTCCAGTTTGCCCAGGA;Mir-221 forwardprimer:

213 AGCCGAGCTACATTGTCT;Mir-206 Forwardprimer:GGAATGTAAG-

214 GAAGTGTGTG; Snord forward primer: ATCACTGTAAAACCGTTCCA.

215 Universal Reverse Primers were obtained from Bonyakhteh Co.

216 (Tehran,Iran).

217 2.7. Statisticalanalysis

218 The primaryand secondaryoutcomeswerephysical activity

219 andmiRlevel,respectively,atbaselineandaftertheintervention.

220 Samplesize calculationwasbasedon theeffectsof exerciseon

221 cardiorespiratoryfitnessinbreastcancersurvivorsintheliterature

222 [34].Withapowerof0.80,two-tailedalpha<0.05,andlargeeffect

223 size(d)=0.80,weneeded26participantsineachcancergroup.All

224 data are presented as mean (SD). All statistical analyses were

225 performed withSPSS16.0(SPSS Inc.,Chicago,IL).Kolmogorov–

226 Smirnov test was used to evaluate the distribution of data.

227 UnivariateANOVAwasusedtocomparemeans,andTukeytestwas

228 usedtodeterminepairwisedifferences.P<0.05wasconsidered

229 statisticallysignificant.

230 2.8. Ethics

231 ThestudywasapprovedbytheMedicalEthics Committeeof

232 Tehran University of Medical Sciences (No.: 113825) and the

233 Iranian Randomized Control Trial (IRCT) ethical board (No.:

234 IRCT2014081018745N1).

235 3. Results

236 Fig.1 reflectstheparticipants’ distributioninthestudy.We

237 included 52 non-metastatic and hormone receptor-positive

238 women with breast cancer aged 31 to 69years and healthy

239 women aged 30 to 60years. Two women with breast cancer

240 droppedoutofthestudyforpersonalreasons.Therefore,datafor

241 50womenwithbreastcancer(26eachgroup)wereanalyzed.The

242 physicalactivityamountofparticipantswas<600MET/minute/

243 week:healthycontrols(mean[SD]400[125]),HIIT(426[96]),HT

244 (325[113]),andHT+HIIT(345[116]).Inthepost-test,mean(SD)

245 physicalactivityamountdidnotsignificantlychangein healthy

246 controls (450 [165], P=0.097) and HT (330 [130], P=0.086)

247 groups.Weobservedasignificantincreaseinmean(SD)physical

248 activitylevelinHIIT(705[99],P=0.032)andHT+HIIT(650[48],

249 P=0.023)groupsafterexercisetraining.

250 3.1. ExpressionofoncomiRs

251 Mean (SD)levelsof someoncomiRswerehigherforwomen 252 withbreast cancer than healthycontrols: miR-21 (5.5 [0.6] vs.

253 0.47 [0.01], P<0.001), miR-155 (5.2 [0.4] vs. 0.33 [0.07];

254 P=0.001),miR-221(4.8[0.5]vs.0.33[0.01],P=0.008),miR-27a 255 (5.2[0.06]vs.0.13[0.01],P<0.001),andmiR-10b(4.2[0.8]vs.

256 0.15[0.01],P=0.007)(Fig.2).HTconsiderablydownregulatedthe 257 expressionoftheoncomiRs(4.2[0.4];P=0.004,3.2[0.3];P=0.01, 258 3.1[0.6];P=0.035,3.1[0.1];P=0.002,and3.05[0.12];P=0.008, 259 respectively)(Fig.2).HIITfor12weekswithHTconferredagreater 260 reduction in expression of the oncomiRs (2.5 [0.5]; P=0.018,

261 1.3[0.4];P=0.005,1.5[0.02];P=0.037,and1.95[0.6];P=0.031,

262 respectively)ascomparedwithHTalone(Fig.2).However,miR-

263 221 was not further downregulated as with HIIT (2.8 [0.4];

264 P=0.137)wasnotsignificant(Fig.2C).Ofnote,theexpressionof

265 theoncomiRsbeforeandaftertraininginhealthycontrolsdidnot

266 changeascomparedwithhealthycontrolswithHIIT(Fig.2).

267 3.2. ExpressionofTSmiRs

268 Mean(SD)levelsofsomeTSmiRswerelowerinwomenwith

269 breast cancer than healthy controls: miR-206 (1.1 [0.8] vs.

270 2.8 [0.02], P=0.048), miR-145 (1.8 [0.12] vs. 5.8 [0.07],

Fig.1.Flowdiagramfordistributionofparticipants.HT:hormonetherapy;HIIT:high-intensityintervaltraining.

Fig.2.EffectofHIITontheexpressionofmiR-21(A),miR-155(B),miR-221(C),miR-27a(D),andmiR-10b(E)inwomenwithbreastcancerundergoingHT.Dataaremean(SD).

*P<0.05comparedtocontrol,^#P<0.05comparedtopre-HT,^$P<0.05comparedtopre-HT+HIIT,^yP<0.05comparedtopost-HT.CON:control;HIIT:high-intensityinterval training;HT:hormonetherapy.ValuesarerelativetoSNORDasaninternalcontrol.

271 P=0.011), miR-143(2.08 [0.5] vs. 5.08[0.02], P=0.008), let-7a 272 (0.90 [0.5] vs. 4.6 [0.01], P=0.005), and miR-9 (2.2 [0.27] vs.

273 2.14[0.01],P=0.0207)(Fig.3).TheTSmiRswereupregulatedby 274 HT(2.33[0.05];P=0.009,5.1[0.01];P=0.001,3.8[0.1];P=0.012, 275 1.8 [0.81]; P=0.005, and 4.04 [0.3]; P=0.001, respectively).

276 Moreover, HIIT for 12weeks withHT significantly upregulated 277 the levels (3.01 [0.5]; P=0.008, 6.9 [0.2]; P=0.001, 6.1 [0.5];

278 P=0.023,and2.6[0.25];P=0.036,respectively)ascomparedwith 279 HTalone (Fig.3). However, miR-9 wasnotfurther upregulated 280 withHIIT(4.3[0.5];P=0.566)(Fig.3D).Aswell,theexpressionof 281 theTSmiRsdidnotdifferbeforeandaftertheexerciseprotocolin 282 healthy controls as compared with health controls with HIIT 283 (Fig.3).

284 4. Discussion

285 OurstudyshowedachangeinexpressionofseveraloncomiRs 286 andTSmiRsinresponseto12weeksofHIITandHTinwomenwith 287 breastcancer.TheexpressionofthemiRsdidnotchangeinhealthy 288 women after 12weeks of HIIT. HIIT together with HT had a 289 remarkableeffectontheexpressionofsomemeasuredmiRs.

290 Recently,DiLevaetal.showedthatoverexpressionofoncomiRs 291 inER-positiveindividualswithbreastcancermightaccountforthe 292 developmentof a moreinvasiveand deadly tumourphenotype 293 [35].Inthiscontext,alteredlevelsofsomecirculatingmiRsmay 294 reflect the dysregulation of cell growthand the impact of the 295 therapy [36]. Arecent study showedupregulationofoncomiRs 296 suchasmiR-221intherapy-resistantbreastcancercells[37].MiR- 297 221isoverexpressedinER-

a

-associated(ER-

a

)breastcancercells 298 andtumours[38,39].Ithasalsobeenidentifiedasanindependent 299 predictoroftheresponsetotamoxifen[40],whichhasbeenshown

300 tohavearoleinincreasingprogression-freesurvival[41].MiR-221

301 isanegativeregulatorofp27kip1,acellcycleinhibitorandtumour

302 suppressor[42,43],andtheupregulationofmiR-221andsignifi-

303 cantreductioninp27kip1levelhavebeenreportedintamoxifen-

304 resistantbreast cancer cells.Therefore,miR-221might regulate

305 tamoxifensensitivityviathedirecttargetingofp27kip1[38,44].

306 Inthisstudy,wehavedemonstratedthatHIITcoulddecrease

307 miR-221 level in women with breast cancer. A differential

308 expression pattern of circulatory miR-221 may be involved in

309 angiogenesisdependingon typeanddurationoftheexercise.In

310 thisregard,Baggishetal.observedthatmiR-21andmiR-221were

311 immediately upregulated after peak acute exercise in trained

312 rowers.Conversely,theirlevelsweresignificantlydecreasedafter

313 1h of rest following acute exercise, which suggests that these

314 dynamic changes may reflect a real response to exercise

315 [45].Moreover,Wardleetal.observedadifferingpatternofmiRs

316 bytypeofexercise:upregulationanddownregulationofmiR-21

317 and miR-221 in endurance and strength training athletes,

318 respectively[46].Thus,levelsofparticular miRsmaychangein

319 opposite directions after endurance versus resistance exercise.

320 However, other studies have reported conflicting results.Some

321 reported lower circulating levels of miR-21 and miR-222 in

322 individualswithhighversuslowVO2max[7]orareductioninmiR-

323 21levelintrainedmenafter12weeksofenduranceexercise[9]in

324 athletesorhealthypeople.However,inthepresentstudy,forthe

325 firsttime,weshowedalowerlevelofmiR-221after12weeksof

326 HIITinwomenwithbreastcancerundergoingHT.

327 Likewise,Jungetal.[47]suggestedthatthecirculatingmiR-21

328 level is usefulfor predictingand/ormonitoring thetherapeutic

329 response totreatment. Upregulation of miR-21 expressionhas

330 beenfoundassociatedwithresistancetotrastuzumabinhuman

Fig.3.EffectofHIITontheexpressionofmiR-206a(A),miR-145(B),miR-143(C),miR-9(D),andlet-7a(E)inwomenwithbreastcancerundergoingHT.Dataaremean(SD).

*P<0.05comparedtocontrol,^#P<0.05comparedtopre-HT,^$P<0.05comparedtopre-HT+HIIT,^yP<0.05comparedtopost-HT.CON:control;HIIT:high-intensityinterval training;HT:hormonetherapy.ValuesarerelativetoSNORDasaninternalcontrol.

331 epidermal growth factor receptor 2-positive breast cancer 332 [48]. Besides, there is some evidence that exercise can affect 333 miR-21expressioninin vitroand invivo studies.Forexample, 334 Fernandes et al. observed decreased expression of miR-21 335 associatedwithimprovedmicro-vascularizationinanimalswith 336 aerobic swimming training [49]. In this context, we have 337 previously shown downregulated miR-21 in exercise-trained 338 animals,anER-positivebreastcancer model[12].Similarly,Bye 339 etal.reported aninverseassociationbetween cardiopulmonary 340 fitness (VO2max) and miR-21 expression [7]. Exercise could 341 decrease the miR-21 level in blood circulation in healthy 342 individuals [9]. Therefore, ER-

a

-associated tumours may be 343 associatedwithincreasedmiR-21expression[50].According to 344 ourresults, HT as an ERpathway suppressor can decrease the 345 expressionofmiR-21.Therefore,forthefirsttime,weshoweda 346 lowexpressionofmiR-21inindividualswithbreastcancerafter 347 HIIT.

348 However, Adams etal. showedmiR-206expressionstrongly 349 inhibitedbyER-

a

agonists[2].Isanejadetal.recentlyfoundhigh 350 expressionofmiR-206aftertheHIITprotocolwithHTinamouse 351 modelofbreasttumour[12].TheauthorsconcludedthatmiR-206 352 might have anti-angiogenic effects in breast tumours and thus 353 decreased tumour size in trained animals. Yan et al. [51]also 354 providedinsightsinto theroleof themiR-143/145clusteras a 355 tumoursuppressor inbreasttumours.MiRsincludingmiR-221/

356 222,miR-342-3p [52], miR-873[53], and Let7b/Let-7i[54]can 357 downregulateER-

a

protein expression, which agrees with our 358 data.However, studiesconcerning thedirect effects of specific 359 circulating miRs on exercise performance and physiological 360 responsesarefew.Inthiscontext,itseemsthatthealteredlevels 361 oftheabove-mentionedmiRsarerelatedtotheadaptabilityofthe 362 HIITtoendurance/aerobiccapacityandmodulationofangiogene- 363 sis, inflammation, muscle damage, skeletal muscle and heart 364 functionsinresponsetoHIIT.Thus,byincreasingordecreasingthe 365 expression of specific circulating miRs, the interval exercise 366 protocolmaymodulatethegeneexpressionprofileinmanycells 367 andtissues,inducingrelatedphysiologicaladaptationsbydifferent 368 mechanisms[55].Inthiscontext,Nielsenetal.[56]demonstrated 369 thatmuscle-specificmiRssuchasmiR-1,miR-133a,miR-133b,and 370 miR-206 are negatively modulated in human muscles after 371 12weeks of endurance exercise. Several of these miRs are 372 associatedwithoxygentension,angiogenesisandadevelopmental 373 tissuenetworkforadaptationtoaerobictraining[57].Moreover, 374 theexercisecanrevealthemechanismofbenefitsbyhavingan 375 impactondifferentimmunecells,includingneutrophils,macro- 376 phages,andlymphocytes.Inthiscontext,miR-155seemsessential 377 tothebalancebetweenpro-inflammatoryM1macrophagesand 378 anti-inflammatory M2 macrophages during skeletal muscle 379 regeneration and could be a target molecule for degenerative 380 musclediseases[58].

381 Although the HIIT in our study could protect women with 382 breastcancer,futureresearchisneededtodefinetheidealrate, 383 duration,andintensityofthephysicalactivitythatcanattenuate 384 cancerprogress.Furthermore,thesefindingsmaysupportfurther 385 evaluationofthepredictivepotentialoftheHIITforHTincancer 386 andtheycansuggestthatthesemiRsmayalsomarkthepotential 387 therapeuticaimsinclinicalapplications.Moreover,theresultsmay 388 informdecisionsonHTcombinedwiththeexercise.

389 5. Conclusion

390 Ourresultsindicatethatadecreaseorincreaseinexpressionof 391 somemiRscouldplayavitalroleinestrogen-dependentfunctions 392 inwomenwithbreastcancer.Alteredpatternsoftheexpressionof 393 thecirculatingmiRscanindicatetheimpactoftreatmentoncancer

394 andthuscouldprovideanearlydecisionfortreatmentcontinua-

395 tion.Aprospectiverandomizedcontrolledtrialisneededtoclarify

396 theeffectsoftheHIITinthiscondition.AlthoughfindingnewmiRs

397 interveninginHTisimperative,futureresearchisneededtodefine

398 themechanismsoftheidentifiedmiRswithunknownrolesinHT

399 and develop ‘‘targeted’’ therapeutics to miR dysregulation and

400 enhancehormonalsensitivity.

401 Funding

402 This trial was supported by Tehran University of MedicalQ3

403 Sciences(grantNo.:24928).Thefundingsourcesdidnothaveany

404 roleinthestudydesign,collection,analysisorinterpretation of

405 data,writingofthereport,orthedecisiontosubmitthearticlefor

406 publication.

407 Authors’contributions

408 SA:studyconceptionanddesign.AI:samplecollection,sample

409 processing, and article revision. SK: data analysis and sample

410 processing. SS: clinicalannotation. AMA: studyconception and

411 designandmanuscriptpreparation.

412 Ethicalapproval

413 Allproceduresperformedinstudiesinvolvinghumanpartici-

414 pantsfollowed theethicalstandardsof theinstitutionaland/or

415 national researchcommitteeand the1964 Helsinkideclaration

416 anditslateramendmentsorcomparableethicalstandards.

417 Disclosureofinterest

418 Theauthorsdeclarethattheyhavenocompetinginterest.

419 Acknowledgements

420 We thank the patients, families, and study staff for their

421 participationinthestudy.TheauthorsthankDrs.Elahi,Kalaghchi,

422 andShahiforhelpingrecruitpatientsforthestudy.

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