乳腺癌细胞中mdm2调控p73α和erα相互作用-mdm 2 regulates the interaction between p73 α and er α in breast cancer cells.docxVIP

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2018-07-29 发布于上海
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乳腺癌细胞中mdm2调控p73α和erα相互作用-mdm 2 regulates the interaction between p73 α and er α in breast cancer cells.docx

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乳腺癌细胞中mdm2调控p73α和erα相互作用-mdm 2 regulates the interaction between p73 α and er α in breast cancer cells

ABSTRACTObjective:ToinvestigatetheregulationofMDM2ontheco-relationshipbetweenERαandP73αinbreastcancercellsMethods:ToobservetheeffectsoftransfectionandsmallinterferingRNA(siRNA)-targetingMurineDoubleMinute2(MDM2)ontheERα,p73αproteinexpressionandco-relationshipinbreastcancercells.ThebindingbetweenMDM2andp73α,ERαwastestedbyCo-Immunoprecipitation,theeffectbetweenMDM2andp73α,ERαinproteinexpressionwasexaminedbywesternblot.MCF-7cellswereculturedandpassagedbyusingDMEMcontaining10%offetalcalfserum.MCF-7cellsweretransfectedwithDNAsencodingERαandp73α.ThebindingbetweenMDM2andp73α,ERαwastestedbyCo-IP:TheexperimentgroupsincludeAgroup(PcDNAgroup),Bgroup(p73αgroup),Cgroup(ERαgroup),Dgroup(p73αandERαgroup),Egroup(p73αandMDM2group).CellsweretransfectedwithMDM2,ERαandp73αplasmidsfor18hours.TheproteasomeinhibitorMG-132wasaddedtopreventthedegradationofprotein.CelllysateswerecompletelymixedwithMDM2polyclonalantibodytopulldownMDM2antigen,theMDM2antigen-antibodycomplexwerecombinewithproteinAagarosebeads.ThenproteinAagarosebeadsimmunoprecipitateswereresolvedbysodiumdodecylsulfate-polyacrylamidegelelectrophoresis(SDS)totestthebindingbetweenMDM2andp73α,ERα.EffectbetweenMDM2andp73α,ERαinproteinexpressionwasexaminedbywesternblot:cellsweretransfectedwithMDM2,ERαandp73αplasmidsandMDM2siRNA,celllysateswereresolvedbysodiumdodecylsulfate-polyacrylamidegelelectrophoresis(SDS)totesttheirproteinexpression.Results:Co-IPindicatedthatMDM2andp73α,ERαbindedtoeachotherintheMCF-7breastcancercellWesternblotindicatedthatMDM2downregulatedp73αproteinexpressionandupregulatedERαproteinexpression.p73αproteinrelativegrayscalesinthegroup(p73αgroup)andthegroup(p73αandMDM2group)showed(1.02±0.01,0.00±0.00P0.01).ERαproteinrelativegrayscalesinthegroup(ERαgroup)andthegroup(ERαandMDM2group)showed(1.15±0.02,1.37±0.06P0.01).WesternblotindicatedthatMDM2downregulatedp73αproteinexpressionanddecreasedtheinhibitionbetweenP73αandERα,thenupregulatedERαproteinexpression.P73αandERαproteinrelativegrayscalesinthegroup(p73αandERαgroup)and