CRISPR-Cas9介導(dǎo)的miR-155敲除在FLT3-ITD+急性髓系白血病中對(duì)TKI敏感性及糖代謝影響的研究

CRISPR-Cas9介導(dǎo)的miR-155敲除在FLT3-ITD+急性髓系白血病中對(duì)TKI敏感性及糖代謝影響的研究摘要：背景：FLT3-ITD+急性髓系白血病（AML）患者中，F(xiàn)LT3驅(qū)動(dòng)的信號(hào)通路對(duì)細(xì)胞增殖和存活至關(guān)重要。miR-155已被證明是FLT3信號(hào)通路的下游分子，其調(diào)控與AML中細(xì)胞增殖和生存相關(guān)的基因有關(guān)。本研究的目的是研究CRISPR/Cas9介導(dǎo)的miR-155敲除對(duì)FLT3-ITD+AML中TKI敏感性及糖代謝的影響。

方法：在DBML雙陰性細(xì)胞株和FLT3-ITD+AML細(xì)胞系MV4-11中，使用CRISPR/Cas9系統(tǒng)敲除miR-155。使用MTT和AnnexinV/PI雙染色體分析評(píng)估細(xì)胞增殖和存活率。使用代謝物分析和實(shí)時(shí)熒光定量PCR檢測(cè)糖代謝和基因表達(dá)。

結(jié)果：miR-155敲除是有效的，導(dǎo)致FLT3-ITD+AML細(xì)胞的生長(zhǎng)和增殖減緩。miR-155敲除可增強(qiáng)FLT3抑制劑對(duì)AML細(xì)胞的毒性。糖酵解途徑的代謝物分析結(jié)果顯示，miR-155敲除引起的糖酵解通路的改變與細(xì)胞凋亡有關(guān)。實(shí)時(shí)熒光定量PCR結(jié)果表明，miR-155敲除能夠抑制AMPK和PI3K-Akt信號(hào)通路，并通過增強(qiáng)p53靶基因誘導(dǎo)細(xì)胞凋亡。

結(jié)論：本研究表明，miR-155敲除可以減緩FLT3-ITD+AML細(xì)胞的生長(zhǎng)和增殖，并增強(qiáng)FLT3抑制劑對(duì)這些細(xì)胞的毒性。此外，這個(gè)研究也表明，miR-155敲除對(duì)糖代謝通路的影響可能是通過AMPK和PI3K-Akt信號(hào)通路來調(diào)節(jié)。這些發(fā)現(xiàn)為實(shí)現(xiàn)miR-155作為潛在的TKI治療響應(yīng)預(yù)測(cè)因子提供了新支持。

關(guān)鍵詞：FLT3-ITD+急性髓系白血病，miR-155，CRISPR/Cas9，TKI，糖代謝。

Abstract:Background:Inacutemyeloidleukemia(AML)patientswithFLT3-ITD+mutation,FLT3-drivensignalingpathwayiscriticalforcellproliferationandsurvival.MiR-155hasbeenprovedtobeadownstreammoleculeofFLT3signalingpathway,anditsregulationisassociatedwithgenesrelatedtocellproliferationandsurvivalinAML.ThepurposeofthisstudywastoinvestigatetheeffectofCRISPR/Cas9-mediatedmiR-155knockoutonTKIsensitivityandglucosemetabolisminFLT3-ITD+AML.

Methods:MiR-155wasknockedoutusingCRISPR/Cas9systeminDBMLbi-nucleatedcelllineandFLT3-ITD+AMLcelllineMV4-11.CellproliferationandsurvivalratewereevaluatedusingMTTandAnnexinV/PIdoublestaininganalysis.Metaboliteanalysisandreal-timefluorescentquantitativePCRwereusedtodetectglucosemetabolismandgeneexpression.

Results:MiR-155knockoutwaseffective,leadingtoslowgrowthofAMLcellswithFLT3-ITD+mutation.MiR-155knockoutenhancedthetoxicityofFLT3inhibitortoAMLcells.ThemetaboliteanalysisofglycolysispathwayshowedthatthechangesofglycolysispathwaycausedbymiR-155knockoutwererelatedtocellapoptosis.Real-timefluorescentquantitativePCRresultsshowedthatmiR-155knockoutcouldinhibitAMPKandPI3K-Aktsignalingpathway,andinducecellapoptosisbyenhancingp53targetgenes.

Conclusion:ThisstudysuggeststhatmiR-155knockoutcanslowthegrowthandproliferationofFLT3-ITD+AMLcells,andenhancethetoxicityofFLT3inhibitortothesecells.Inaddition,thisstudyalsosuggeststhattheeffectofmiR-155knockoutonglucosemetabolismmayberegulatedthroughAMPKandPI3K-Aktsignalingpathways.ThesefindingsprovidenewsupportformiR-155asapotentialTKItreatmentresponsepredictionfactor.

KeyWords:FLT3-ITD+acutemyeloidleukemia,miR-155,CRISPR/Cas9,TKI,glucosemetabolismFLT3-ITD+acutemyeloidleukemia(AML)isasubtypeofAMLcharacterizedbyamutationintheFLT3gene,whichencodesforareceptortyrosinekinasethatplaysacriticalroleinregulatinghematopoieticstemcellproliferationanddifferentiation.TargetedtherapywithFLT3inhibitors(TKIs)hasshownpromiseintreatingFLT3-ITD+AML,buttheresponsetoTKIscanvarywidelybetweenpatients.

MiR-155isamicroRNAthathasbeenimplicatedinthedevelopmentandprogressionofvariouscancers,includingAML.RecentstudieshavesuggestedthatmiR-155mayplayaroleinregulatingglucosemetabolismincancercells,whichisanimportantpathwayfortumorgrowthandproliferation.However,theroleofmiR-155inTKIresponseandglucosemetabolisminFLT3-ITD+AMLhasnotbeenwellstudied.

Inthisstudy,weusedCRISPR/Cas9geneeditingtechnologytoknockoutmiR-155expressioninFLT3-ITD+AMLcells.WefoundthatmiR-155knockoutslowedthegrowthandproliferationofthesecells,andenhancedthetoxicityofaFLT3inhibitortothesecells.ThesefindingssuggestthatmiR-155mayplayaroleinmediatingTKIresponseinFLT3-ITD+AML.

Furthermore,weexploredthepotentialmechanismsunderlyingtheeffectofmiR-155onglucosemetabolism.WefoundthatmiR-155knockoutledtodecreasedglucoseuptakeandlactateproductioninFLT3-ITD+AMLcells.Inaddition,wefoundthattheeffectofmiR-155onglucosemetabolismmayberegulatedthroughtheAMPKandPI3K-Aktsignalingpathways.Thesepathwaysareknowntoplayimportantrolesinregulatingglucosemetabolismandcancercellsurvival.

Overall,ourfindingssuggestthatmiR-155maybeapotentialTKItreatmentresponsepredictionfactorinFLT3-ITD+AML.Inaddition,ourstudyprovidesnewinsightsintotheroleofmiR-155inregulatingglucosemetabolismincancercells,andmayhaveimplicationsforthedevelopmentofnoveltherapiestargetingthispathwayInconclusion,ourstudyhighlightstheimportanceofunderstandingthemolecularmechanismsunderlyingTKIresistanceinFLT3-ITD+AML.ThroughtheidentificationofmiR-155asapotentialbiomarkerforpredictingTKItreatmentresponse,wehaveprovidedapotentialavenueforthedevelopmentofpersonalizedtreatmentstrategiesforpatientswithFLT3-ITD+AML.Furthermore,ourstudyprovidesnewinsightsintotheroleofmiR-155inregulatingglucosemetabolismandcancercellsurvival,suggestingthattargetingthispathwaymayhavetherapeuticpotentialinarangeofmalignancies.

However,furthervalidationofourfindingsinlargerpatientcohortsandfunctionalstudiesareneededtofullyunderstandtheclinicalrelevanceofmiR-155inTKIresistanceandcancermetabolism.Additionally,thedevelopmentofnoveltherapiestargetingthispathwaywillrequireextensivepreclinicaltestingandevaluationofsafetyandefficacy,andwilllikelyrequirecollaborationbetweenacademia,industry,andregulatoryagencies.

Inconclusion,ourstudyprovidesnewinsightsintothemolecularmechanismsunderlyingTKIresistanceinFLT3-ITD+AML,andhighlightsthepotentialofmiR-155asabiomarkerforpredictingtreatmentresponse.OurfindingsmayhaveimportantimplicationsforthedevelopmentofpersonalizedtreatmentstrategiesandnoveltherapiestargetingcancermetabolisminarangeofmalignanciesFurthermore,ourstudyalsoemphasizestheneedforfurtherresearchanddevelopmentofnewtherapeuticagentsthatcantargetmetabolicpathwaysincancercells.Giventhecomplexityanddiversityofcancermetabolism,amulti-targetedapproachthatcombinesdifferentmetabolicinhibitorswithconventionalchemotherapyortargetedtherapymayberequiredtoachieveoptimaltherapeuticefficacy.

Inadditiontothedevelopmentofnewtherapies,thereisaneedforinnovativediagnostictoolsthatcanaccuratelypredicttreatmentresponseandidentifypatientswhoaremorelikelytobenefitfromspecifictherapies.OurstudysuggeststhatmiR-155mayhavepotentialasabiomarkerforpredictingTKIresistanceinFLT3-ITD+AML,