CRISPR-Cas9介導(dǎo)的microRNA-21敲除增加慢性髓性白血病細胞的TKIs敏感性

CRISPR-Cas9介導(dǎo)的microRNA-21敲除增加慢性髓性白血病細胞的TKIs敏感性摘要：越來越多的證據(jù)表明microRNA在腫瘤的發(fā)生和發(fā)展中發(fā)揮著重要作用。microRNA-21在許多種癌癥中高表達，與癌細胞的增殖、凋亡和藥物耐藥等過程密切相關(guān)。本研究采用CRISPR/Cas9技術(shù)靶向敲除microRNA-21基因，發(fā)現(xiàn)敲除后慢性髓性白血病細胞的生長明顯受到抑制，并且對常用的靶向治療藥物特比替尼、伊馬替尼等的敏感性明顯增強。進一步研究發(fā)現(xiàn)，microRNA-21的敲除可以通過降低其目標(biāo)基因PTEN和PDCD4的表達來促進細胞凋亡和增加藥物敏感性。本研究揭示了microRNA-21在慢性髓性白血病中的作用機制，并為將其作為治療靶點提供了理論基礎(chǔ)。

關(guān)鍵詞：CRISPR/Cas9；microRNA-21；慢性髓性白血病；TKIs敏感性；PTEN；PDCD4

Introduction

慢性髓性白血病（CML）是一種由于白細胞系統(tǒng)異常增殖引起的骨髓性疾病，常見于中年人。盡管伊馬替尼和其他靶向治療藥物已經(jīng)被用于治療CML多年，但癌癥復(fù)發(fā)和耐藥是治療失敗的主要原因之一。因此，尋找新的治療策略來提高TKIs治療的有效性顯得非常關(guān)鍵。

microRNA是一類長度約為22nt的非編碼RNA，通過對靶基因的調(diào)節(jié)來影響細胞的生長、增殖、凋亡等過程。在癌癥中，許多種類的microRNA都被發(fā)現(xiàn)存在異常表達，其中microRNA-21的高表達在許多種癌癥中都得到了證實。

在CML中，microRNA-21也被發(fā)現(xiàn)高表達，并與伊馬替尼等TKIs藥物的耐藥性相關(guān)。與此同時，許多研究表明microRNA-21具有促進細胞增殖和凋亡抑制的功能，但其在CML中的確切作用機制還不完全清楚。

Methods

本研究采用CRISPR/Cas9技術(shù)定向敲除microRNA-21基因，利用qRT-PCR檢測細胞內(nèi)microRNA-21的表達。通過MTT實驗、流式細胞術(shù)和Westernblot等手段檢測細胞生長、凋亡和相關(guān)蛋白的表達變化。重點關(guān)注microRNA-21是否會影響CML細胞對TKIs等藥物的敏感性，并進一步挖掘其作用機制。

Results

本研究成功靶向敲除了microRNA-21基因，并發(fā)現(xiàn)敲除后CML細胞的生長明顯受到抑制。進一步研究發(fā)現(xiàn)，CML細胞中microRNA-21的表達水平與PTEN和PDCD4基因的表達水平呈負相關(guān)，說明microRNA-21敲除后降低了這些基因的抑制作用。Westernblot結(jié)果表明，敲除microRNA-21后，CML細胞中凋亡相關(guān)蛋白capase-3和Bax的表達顯著增加，細胞抗TKIs藥物的能力明顯減弱。

Conclusion

本研究揭示了microRNA-21在CML細胞中的作用機制，表明其通過抑制PTEN和PDCD4基因的表達來促進細胞增殖和凋亡的抑制，并影響細胞對TKIs的敏感性。因此，這些發(fā)現(xiàn)為開發(fā)新的治療靶點提供了理論基礎(chǔ)，具有重要的臨床應(yīng)用價值Introduction

Chronicmyeloidleukemia(CML)isahematopoieticstemcelldisordercharacterizedbythePhiladelphiachromosome,whichleadstothefusionoftheBCRandABLgenesandtheproductionoftheBCR-ABLfusionprotein.TheBCR-ABLproteinhastyrosinekinaseactivity,whichisresponsiblefortheabnormalproliferationandsurvivalofCMLcells.Tyrosinekinaseinhibitors(TKIs),suchasimatinib,haverevolutionizedthetreatmentofCMLbyinhibitingBCR-ABLactivity.However,someCMLpatientsdevelopresistancetoTKIs,whichisamajorclinicalchallenge.microRNAsaresmallnon-codingRNAsthatregulategeneexpressionpost-transcriptionally.microRNA-21isoverexpressedinvariouscancersandhasbeenimplicatedintumorgrowth,angiogenesis,anddrugresistance.However,theroleofmicroRNA-21inCMLisnotfullyunderstood.

Methods

WeknockedoutmicroRNA-21inCMLcellsusingCRISPR/Cas9technologyandconfirmedtheknockdownefficiencybyqRT-PCR.WeassessedtheeffectsofmicroRNA-21knockoutoncellproliferation,apoptosis,andTKIsensitivityusingMTTassay,flowcytometry,andWesternblot.WealsoexaminedthecorrelationbetweenmicroRNA-21expressionandtheexpressionofPTENandPDCD4,whicharetwoknowntargetsofmicroRNA-21thatareinvolvedincellgrowthandapoptosis.

Results

WefoundthatknockoutofmicroRNA-21significantlyinhibitedCMLcellproliferationandinducedapoptosis.Westernblotanalysisrevealedthattheexpressionofcaspase-3andBax,twoapoptoticmarkers,wasincreasedinmicroRNA-21knockoutcells,whereastheexpressionofBcl-2,ananti-apoptoticprotein,wasdecreased.Moreover,microRNA-21knockoutsensitizedCMLcellstoimatinibanddasatinib,twowidelyusedTKIs.WealsoobservedanegativecorrelationbetweenmicroRNA-21expressionandtheexpressionofPTENandPDCD4inCMLcells,suggestingthatmicroRNA-21mayplayaroleinregulatingthesegenes.

Conclusion

OurstudyprovidesnovelinsightsintotheroleofmicroRNA-21inCML.WedemonstratethatmicroRNA-21promotescellproliferationandinhibitsapoptosisbysuppressingtheexpressionofPTENandPDCD4,andthatmicroRNA-21isinvolvedinTKIresistanceinCMLcells.OurfindingssuggestthattargetingmicroRNA-21couldbeapromisingstrategytoimprovetheefficacyofTKIsandovercomedrugresistanceinCML.FurtherstudiesarewarrantedtoexplorethetherapeuticpotentialofmicroRNA-21inhibitioninCMLChronicmyeloidleukemia(CML)isahematopoieticstemcelldisordercharacterizedbythepresenceofthePhiladelphiachromosome(Ph+),whichencodesfortheconstitutivelyactivetyrosinekinaseBCR-ABL.Treatmentwithtyrosinekinaseinhibitors(TKIs)hasrevolutionizedthemanagementofCML,leadingtohighratesofcompletecytogeneticandmolecularresponses.However,asignificantproportionofpatientsdevelopresistancetoTKIs,whichcanleadtodiseaseprogressionandpooreroutcomes.ThemechanismsunderlyingTKIresistancearemultifactorialandcomplex,involvingbothgeneticandepigeneticalterations.

MicroRNAs(miRNAs)aresmallnon-codingRNAsthatregulategeneexpressionbytargetingmessengerRNAs(mRNAs)fordegradationortranslationalrepression.DysregulationofmiRNAshasbeenimplicatedinthepathogenesisofvariouscancers,includingCML.miRNA-21isoneofthemostextensivelystudiedmiRNAsandhasbeenshowntopromotecancercellproliferationandinhibitapoptosisinvariousmalignancies.

InCML,severalstudieshavereportedanupregulationofmiRNA-21expressioninPh+cellscomparedtonormalcells.OurgroupfurtherinvestigatedtheroleofmiRNA-21inCMLandfoundthatmiRNA-21promotescellproliferationandinhibitsapoptosisthroughthesuppressionoftwotumorsuppressorgenes,PTENandPDCD4.PTENisanegativeregulatorofthePI3K/AKTpathway,whichisknowntobeactivatedinCMLandpromotescellsurvivalandgrowth.PDCD4isalsoinvolvedintheregulationofcellapoptosisandhasbeenshowntobedownregulatedinCML.

WealsoexploredtheinvolvementofmiRNA-21inTKIresistanceinCMLcells.WefoundthatmiRNA-21expressionwassignificantlyincreasedinTKI-resistantCMLcellscomparedtoTKI-sensitivecells.Furthermore,inhibitionofmiRNA-21expressionsensitizedTKI-resistantcellstoTKIs,suggestingthatmiRNA-21isinvolvedinthedevelopmentofTKIresistance.

Takentogether,ourfindingssuggestthatmiRNA-21playsanimportantroleinthepathogenesisofCMLbypromotingcellproliferationandinhibitingapoptosisthroughthemodulationofPTENandPDCD4.Moreover,miRNA-21isinvolvedinTKIresistanceinCMLcells.TargetingmiRNA-21couldbeapromisingstrategytoimprovetheefficacyofTKIsandovercomedrugresistanceinCML.FurtherstudiesarewarrantedtoexplorethetherapeuticpotentialofmiRNA-21inhibitioninCMLInadditiontomiRNA-21,othermiRNAshavealsobeenimplicatedinthepathogenesisanddrugresistanceofCML.MiRNA-155hasbeenshowntobeupregulatedinCMLcellsandcontributestoleukemogenesisbytargetingvariouspathways,suchasthePI3K-AKTsignalingpathwayandthetumorsuppressorSOCS1(suppressorofcytokinesignaling1)(25,26).MiRNA-203hasbeenreportedtobedownregulatedinCMLpatientsandcelllines,anditsoverexpressioncaninhibitcellproliferationandinduceapoptosisthroughtargetingBCR-ABLandBCL2(27).MiRNA-31,miRNA-34a,andmiRNA-126havealsobeenshowntocontributetoCMLpathogenesisanddrugresistance(28-30).

TargetingmiRNAscouldbeapromisingtherapeuticapproachforCMLtreatment.SeveralpreclinicalstudieshaveshownthatmiRNAinhibitorsormimicscansuppressleukemogenesisandsensitizeCMLcellstoTKIs(31-34).Forexample,arecentstudyhasdemonstratedthatacombinationofamiRNA-21inhibitorandimatinibcaneffectivelyinhibitcellproliferationandinduceapoptosisinCMLcelllinesandpatient-derivedcells(35).AnotherstudyhasreportedthatmiRNA