3-67 MiR-449a Overexpression Enhances the Radiosensitivity in Prostate Cancer Cells

ThisstudycouldprovidescientificbasisforsafetyofCORM-3appliedtotheclinic.Reference

[1]

RMotterlini,J.EClark,RForesti,etal.,CirculationResearch,90(2002)17.

Foundationitem:KeyProgramofNationalNaturalScienceFoundationofChina(U1432248),NationalNaturalScienceFoundationofChina(11305226,11175222).

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3-67MiR-449aOverexpressionEnhancestheRadiosensitivity

inProstateCancerCells∗

MaoAihong,LiuYangandZhangHong

MultiplelinksbetweenmiRNAactivityandcancerhavebeenestablished.SeveralmiRNAshavebeendescribedasoncogeneswhileothersactastumoursuppressors[1].MiR-449aisamemberofmiR-34familywhichlocatesonhumanchromosome5q11.2,aregionidentifiedasasusceptibilitylocusinavarietyofmalignancies,includingprostatecancer[2].Inlinewiththetumor-suppressiveroleofmiR-34,miR-449awasshowntobesignificantlydown-regulatedinprostatecancercelllinesandtissuerelativetonormaltissuesandplaysacriticalroleingrowthofprostatecancercells[3,4].

Inthepresentstudy,thepre-miR-449aanditsflanking200bpsequencewasclonedtothemulti-clonesite(MCS)ofH1-MCS-CMV-GFP-SV40-Hygrcmycin,andtheplasmidwastransfectedinthehumanprostatecancercell(LNCaP),theexpressionofmiR-449awasdetectedbyReal-TimePCR.ThebiologicactivityofmaturemiR-449awasdeterminedbyfluorescencequenchmethod.TheresultsshowedthattheexpressionofLNCaPwasmuchhigherthancontrolgroup.H1-MCS-CMV-GFP-SV40-HygrcmycincouldefficientlyexpressmaturemiR-449awithbiologicactivity.Inthefurtherstudy,wealsofoundthatoverexpressionofmiR-449ainLNCaPcellsinhibitedproliferationandenhancedapoptosisinducedbyX-rays(asshowninFigs.1and2).

Fig.1(coloronline)LNCaPcellstransfectedwithmiR-449aoverexpressionvector.(a)NormalLNCaPcellsbefore

transfected.(b)LNCaPcellstransfectedwith5µgmiR-449aovere-xpressionvectorplasmidDNAormiRcon31controlplasmidDNA(H1-MCS-CMV-GFP-SV40-Hygrcmycin)for48h.

Fig.2(coloronline)miR-449aoverexpressionenhancesapoptosisinducedbyX-raysinprostatecancerLNCaPcells.LNCaP

cellswerestainedbyHoechest333258post-irradiation4GyX-raysat48h.LNCaPweretransfectedwithmicron(a),(b)andmiR-449a(c),(d).

2014IMP&HIRFLAnnualReport·161·

References

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Y.L.Wang,S.Wu,B.Jiang,etal.,ClinGenitourinCancer(2015)S1558.

Y.Pan,WO.Lui,N.Nupponen,etal.,GenesChromosomesandCancer,30(2001)187.E.J.Noonan,R.F.Place,D.Pookot,etal.,Oncogene28(2009)1714.E.J.Noonan,R.F.Place,SBasak,etal.,Oncotarget1(2010)249.

Foundationitem:KeyProgramofNationalNaturalScienceFoundationofChina(U1432248),NationalNaturalScienceFoundationofChina(10835011,11205219),WesternTalentProgramofChineseAcademyofSciences(Y260230XB0).

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3-68

LossofNrf2EnhancestheRadiosensitivityin

HumanLungCancerCells∗

ZhaoQiuyueandZhangHong

Nuclearfactorerythroid2-relatedfactor2(Nrf2)isacrucialtranscriptionfactorregulatingtheexpressionofantioxidantgenes.Underoxidativestressconditionsorotherstimulus,Nrf2translocatingfromthecytoplasmintothenucleus,bindstoantioxidantresponseelements,andincreasestheexpressionofantioxidantenzymes[1,2].ConstitutiveNrf2activationinmanytumorsenhancescellsurvivalandresistance.Forinstance,highlevelofNrf2isobservedinnon-smallcelllungcancerA549cells[3,4].ThegainofNrf2functionhasbeenimplicatedintheresistanceofcancercellstoradiationtherapy.

Underionizingradiation,ithasbeenconfirmedthatROSplaysmainroleinthecytotoxicaction.Inthiswork,cellswereirradiatedwithX-raysatadoseof4Gyand4groupswerestudied:Negativecontrolgroup(NC),thecellstransfected48hwithNrf2siRNAgroup(siRNA),irradiatedgroup(IR),andirradiatedcellsaftertransfectiongroup(siRNA+IR).OurresultsshowedthecellstransfectedwithNrf2siRNAincreasedthelevelofROSwithouttheradiationexposurecomparedwithnegativegroup.KnockingdownNrf2canincreasedROSaccumulationinirradiatedcellscomparedwithcellsexposedtoradiationalone(Fig.1).IncreasingthelevelofROSmaychangeredoxstateofthecell,andthenaffectcellsurvivalandincreaseradiosensitivity.

Fig.1(coloronline)Nrf2siRNApromotesROSaccumulationinA549cells.A549cellswereincubatedwith10µM

H2DCFH-DAfor30minbeforeIR.ROSwasmeasuredbyfluorescencemicroscopy.

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E.J.Moon,A.Giaccia,Free.Radic.Biol.Med,79(2015)292.

C.M.Mahaffey,H.Zhang,A.Rinna,etal.,Free.Radic.Biol.Med,46(2009)1650.X.Tang,H.Wang,L.Fan,etal.,Free.Radic.Biol.Med,50(2011)1599.S.Lee,M.J.Lim,M.H.Kim,etal.,Free.Radic.Biol.Med,53(2012)1650.

Foundationitem:KeyProgramofNationalNaturalScienceFoundationofChina(U1432248),NationalNaturalScienceFoundationofChina(11205219),WesternTalentProgramofChineseAcademyofSciences(Y260230XB0).

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