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miR-335靶向调控ROCK1抑制骨肉瘤细胞侵袭转移的研究.docx

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miR-335靶向调控ROCK1抑制骨肉瘤细胞侵袭转移的研究 Title:TheRoleofmiR-335inTargetingROCK1toInhibitInvasionandMetastasisinOsteosarcomaCells Abstract: Osteosarcomaisahighlyaggressivebonemalignancy,characterizedbyitsinvasiveandmetastaticproperties.Theidentificationofkeyregulatorymoleculesinvolvedintheprogressionofosteosarcomacouldprovidevaluableinsightsintothedevelopmentofnoveltherapeuticstrategies.ThisstudyaimstoexplorethepotentialroleofmiR-335inregulatingROCK1expression,anditsimpactoninvasionandmetastasisinosteosarcomacells. Introduction: Osteosarcomaisthemostcommonprimarymalignantbonetumoraffectingchildrenandadolescents.Despiteadvancementsintherapeuticapproaches,theprognosisforpatientswithmetastaticdiseaseremainspoor.Therefore,thereisanurgentneedtoidentifynewtargetsforinhibitinginvasionandmetastasisinosteosarcoma.Recently,microRNAs(miRNAs)haveemergedasimportantregulatorsofgeneexpression,playingacrucialroleinvariouspathologicalprocessesincludingcancer.miR-335isreportedtobedysregulatedinmultiplehumancancersandhasbeenimplicatedintumorinvasionandmetastasis.However,itsroleinosteosarcomaremainslargelyunknown. Methods: 1.Cellculture:Osteosarcomacelllines(e.g.,U2OS,MG63)willbeculturedinappropriatemediasupplementedwithfetalbovineserumandantibiotics. 2.TransfectionofmiR-335mimicandinhibitor:OsteosarcomacellswillbetransfectedwithmiR-335mimicorinhibitorusingasuitabletransfectionreagent. 3.Quantitativereal-timepolymerasechainreaction(qRT-PCR):RNAwillbeisolatedfromcellsusingacommercialkit,andcDNAwillbesynthesized.qRT-PCRwillbeperformedtomeasureROCK1mRNAexpression. 4.Westernblotanalysis:Totalproteinwillbeextractedfromcells,separatedbySDS-PAGE,andtransferredtoapolyvinylidenedifluoridemembrane.ThemembranewillbeincubatedwithprimaryantibodiesagainstROCK1,followedbysecondaryantibodies.Proteinbandswillbevisualizedusinganenhancedchemiluminescencesystem. 5.Cellinvasionandmigrationassays:TranswellinvasionandwoundhealingassayswillbeperformedtoexaminetheimpactofmiR-335ontheinvasiveandmigratoryabilitiesofosteosa