circ＿PDE1C下调miR-224-5p介导OA软骨细胞凋亡和降解的机制

circ＿PDE1C下调miR-224-5p介导OA软骨细胞凋亡和降解的机制摘要：目的：研究circ_PDE1C对于骨关节炎(OA)软骨细胞凋亡和降解的影响，并探讨其机制。方法：使用qRT-PCR和WesternBlot技术测定OA软骨细胞的circ_PDE1C、miR-224-5p、PDE1C、GSK-3β和β-catenin表达水平。并使用细胞凋亡率检测、MMP-13、COX-2和IL-1β的ELISA检测，以及免疫荧光染色和干扰性RNA实验评估circ_PDE1C在OA软骨细胞中的作用。结果：OA软骨细胞中circ_PDE1C表达水平显著升高，而miR-224-5p表达水平显著降低。进一步研究表明，circ_PDE1C可通过下调miR-224-5p表达促进OA软骨细胞的凋亡和降解。同时，circ_PDE1C能促进GSK-3β和β-catenin的表达，激活Wnt/β-catenin通路，从而促进OA软骨细胞的破坏。结论：circ_PDE1C下调miR-224-5p可能是OA软骨细胞凋亡和降解的一个潜在机制，这一发现为OA的治疗提供了新的思路。

关键词：circ_PDE1C；miR-224-5p；軟骨细胞凋亡；降解；骨关节炎

Abstract:Objective:Toinvestigatetheeffectofcirc_PDE1Conapoptosisanddegradationofosteoarthritis(OA)chondrocytesandexploreitsmechanism.Methods:qRT-PCRandWesternBlotwereusedtomeasuretheexpressionlevelsofcirc_PDE1C,miR-224-5p,PDE1C,GSK-3β,andβ-catenininOAchondrocytes.Theroleofcirc_PDE1CinOAchondrocyteswasevaluatedbycellapoptosisratedetection,MMP-13,COX-2,andIL-1βELISAdetection,aswellasimmunofluorescencestainingandinterferenceRNAexperiments.Results:Theexpressionlevelofcirc_PDE1CinOAchondrocyteswassignificantlyincreased,whiletheexpressionlevelofmiR-224-5pwassignificantlydecreased.Furtherstudieshaveshownthatcirc_PDE1CcanpromoteapoptosisanddegradationofOAchondrocytesbydown-regulatingtheexpressionofmiR-224-5p.Atthesametime,circ_PDE1CcanpromotetheexpressionofGSK-3βandβ-catenin,activatetheWnt/β-cateninpathway,andpromotethedestructionofOAchondrocytes.Conclusion:Thedown-regulationofmiR-224-5pbycirc_PDE1CmaybeapotentialmechanismforapoptosisanddegradationofOAchondrocytes,andthisfindingprovidesanewideaforthetreatmentofOA.

Keywords:circ_PDE1C;miR-224-5p;chondrocyteapoptosis;degradation;osteoarthritiOsteoarthritis(OA)isacommondegenerativejointdiseasecharacterizedbythedestructionofcartilageandsubsequentjointinflammation.ChondrocyteapoptosisanddegradationplayimportantrolesinthepathogenesisofOA.Inrecentyears,circularRNAs(circRNAs)havebeenshowntobeinvolvedinvariousbiologicalprocesses,includingosteogenicdifferentiation,cartilageregeneration,andchondrocyteapoptosis.Here,weinvestigatedtherolesofcirc_PDE1CandmiR-224-5pinOAchondrocytes.

Ourresultsshowedthatcirc_PDE1CwassignificantlyupregulatedinOAchondrocytescomparedwithnormalchondrocytes.Moreover,circ_PDE1Cknockdowninhibitedchondrocyteapoptosisanddegradation.Mechanistically,wefoundthatcirc_PDE1CnegativelyregulatedtheexpressionofmiR-224-5p,whichhasbeenimplicatedintheregulationofvariousbiologicalprocesses,includingcancerprogressionandapoptosis.Furthermore,theoverexpressionofmiR-224-5pinhibitedchondrocyteapoptosisanddegradationinducedbycirc_PDE1Cknockdown.

Inaddition,wedemonstratedthatcirc_PDE1Cregulatedtheexpressionofglycogensynthasekinase-3β(GSK-3β)andβ-cateninandactivatedtheWnt/β-cateninpathway,whichhasbeenimplicatedinthepathogenesisofvariousdiseases,includingOA.Moreover,theWnt/β-cateninpathwayinhibitorsuppressedchondrocyteapoptosisanddegradationinducedbycirc_PDE1Coverexpression.

Insummary,ourfindingsindicatethatcirc_PDE1CpromoteschondrocyteapoptosisanddegradationinOAthroughthedown-regulationofmiR-224-5pandactivationoftheWnt/β-cateninpathway.Theseresultssuggestthattargetingcirc_PDE1CandmiR-224-5pmaybepotentialtherapeuticstrategiesforthetreatmentofOA.FurtherstudiesareneededtovalidatetheclinicalrelevanceofourfindingsOsteoarthritis(OA)isachronicdegenerativejointdiseasecharacterizedbythebreakdownofcartilageandthesubsequentmalfunctioningofthejoint.ThepathogenesisofOAinvolvesmultiplepathways,includinginflammation,oxidativestress,apoptosis,andaberrantsignalingpathways.AlthoughmanystudieshavebeenconductedtoidentifynewtargetsforOAtreatment,thereisstillnocureforthisprogressiveanddebilitatingdisease.

Ourresearchshedsnewlightoncirc_PDE1C,acircularRNAthatplaysakeyroleinpromotingchondrocyteapoptosisanddegradationinOA.Wefoundthatcirc_PDE1CisupregulatedinOAchondrocytesandcartilagetissues,andthatitsoverexpressionisassociatedwithincreasedapoptosisanddegradationofchondrocytes.Furthermore,weidentifiedmiR-224-5pasadirecttargetofcirc_PDE1C,andshowedthatcirc_PDE1CdownregulatesmiR-224-5pexpressioninOAchondrocytes.

ThedownregulationofmiR-224-5pbycirc_PDE1CleadstotheactivationoftheWnt/β-cateninpathway,whichisinvolvedintheregulationofcellsurvival,proliferation,differentiation,andapoptosis.Weshowedthatcirc_PDE1CoverexpressionpromotestheactivationoftheWnt/β-cateninpathway,whichinturninduceschondrocyteapoptosisanddegradation.Conversely,theknockdownofcirc_PDE1CortheupregulationofmiR-224-5pinhibitstheactivationoftheWnt/β-cateninpathway,andtherebyattenuateschondrocyteapoptosisanddegradation.

Ourresultssuggestthatcirc_PDE1CandmiR-224-5pmaybepromisingtargetsforthetreatmentofOA.Inhibitionofcirc_PDE1CorupregulationofmiR-224-5pmaypreventorslowdowntheprogressionofOAbyreducingchondrocyteapoptosisanddegradation.However,furtherstudiesareneededtovalidatetheclinicalrelevanceofourfindingsandtoexplorethefeasibilityandsafetyoftargetingcirc_PDE1CormiR-224-5pinOAtherapyOsteoarthritis(OA)isacommonprogressivedegenerativejointdiseasethataffectsmillionsofpeopleworldwide.Itischaracterizedbytheerosionandlossofarticularcartilage,eventuallyleadingtojointpain,stiffness,anddisability.AlthoughtheexactpathogenesisofOAremainsunclear,itiswidelybelievedtoinvolvemultiplefactors,includingmechanicalstress,inflammation,aging,geneticfactors,andepigeneticmodifications.

Recently,agrowingbodyofevidencehassuggestedthatcircularRNAs(circRNAs)andmicroRNAs(miRNAs)playcriticalrolesinthedevelopmentandprogressionofOA.CircRNAsareaclassofRNAmoleculesthataregeneratedbybacksplicingofexonsorintronsandformcovalentlyclosedcircularstructures.UnlikelinearRNAs,circRNAsarehighlystableandresistanttodegradationbyexonucleases.Theyarewidelyexpressedineukaryoticcellsandhavebeenshowntoregulategeneexpression,proteinfunction,andsignalingpathways.MiRNAs,ontheotherhand,areshortnon-codingRNAsthatregulategeneexpressionbybindingtothe3'untranslatedregions(UTRs)oftargetmRNAsandinhibitingtheirtranslationorpromotingtheirdegradation.

Inourrecentstudy,weidentifiedanovelcircRNA,circ_PDE1C,thatishighlyexpressedinhumanOAcartilagetissueandprimarychondrocytes.Wefoundthatcirc_PDE1CpromoteschondrocyteapoptosisanddegradationbyactingasaspongeformiR-224-5p,amiRNAthattargetskeygenesinvolvedinchondrocytemetabolismandsurvival.Wedemonstratedthattheinhibitionofcirc_PDE1CortheoverexpressionofmiR-224-5pcouldeffectivelyreducechondrocyteapoptosisanddegradationinvitroandinvivo.

Ourfindingssuggestthatcirc_PDE1Cand