| Objective:ToinvestigateNYD-SP14proteinandthoseproteinswhichcaninteractwithNYD-SP14,furtherexploretheeffectsofNYD-SP14oncelldivision,growthandthemechanismoftheseeffectsonthespermatogenesisandtumorigenesis.Methods:ThehomologydifferencesofNYD-SP14indifferentspecieswereviewedthroughbioinformaticmethods.TheexpressionprofilesofNYD-SP14wereviewedthroughRT-PCRindifferenttissuesofmouseanddifferentspermsamples,theexpressionprofilesofNYD-SP14andrelatedproteins----thescaffoldsubunitAαofPP2A(PP2R1A),heatshockprotein70(HSP70)andHIV-1revbindingprotein2(HRB2),wereviewedindifferentcelllines.DifferentplasmidsofNYD-SP14andtheinteractingproteinsthatwerebasedonretroviralandlentiviralvectorswereconstructed,thesteadyexpressionHEKTERcelllineswereobtained,thesubcellularlocationsoffusionproteinswereobserved,andtheinteractionofNYD-SP14andPP2R1Awasidentifiedthroughimmunoprecipitation.ThefunctionofNYD-SP14andtheinteractingproteinsoncellgrowth,proliferationandcellcyclewereinvestigatedandthepotentialbiologicalfunctionsofNYD-SP14weresuspected.Forfurtherresearch,NYD-SP14conditionalknockoutmousemodelwasgeneratedtostudytheconcretefunctiononembryogenesisandontogenesis.Resultsls:NYD-SP14iswidelyexpressedandhighlyconservedinvariousspeciesfromprokaryotetoeukaryote.TheexpressionprofileofNYD-SP14showedthatNYD-SP14iswidelyexpressedinmurinetissuesandhumanspermsamplesbuthighlyexpressedinmurinetesticle,brainandallfertilespermspecimens;furtheranalysisshowedthewideexpressionofNYD-SP14andrelatedproteins,suchasHSP90Aα,HSP90Aβ,HSP90B,HSP70andHRB2indifferentcelllines,includingHEK293T,immortalHEKTER,breastcancercelllineMCF-7andHELA,indicatingapotentialroleforNYD-SP14intestisandcancerdevelopmentand spermatogenesis.ThesubcellularlocalizationsoftheGFP-fusionproteinsrevealedNYD-SP14,PP2R1A,andHSP70werelocatedincytoplasmmostly,whilethemitosiscycle-dependentprotein,HRB2wasexpressedinthecytoplasmbeforecelldivision,butinthenucleusaftercelldivision.WesternBlottingidentifiedthestableinfectedcelllinesthatexpressedNYD-SP14anditsrelatedproteinssteadilyandImmunoprecipitationprovedtheinteractionbetweenNYD-SP14andPP2R1A.ThedetectiononcellcycleandcellgrowthcurveindicatedNYD-SP14couldpromoteG1toSphase,blockmotosisinSphaseandinduceapoptosis,whiletheoverexpressionofPP2R1AcouldsuppressthegrowthofHEKTERandenhancetheblockingeffectofNYD-SP14.TheNYD-SP14flox/+andflox/floxmiceaswellastheNYD-SP14+/–mouseweregeneratedandconfirmedwithspecificprimersbyPCR.Conclusion:MurineNYD-SP14,whichwaswidelyexpressedinmulti-tissuesandhighlyexpressedinmurinetestis,brain,fertilehumanspermatozoaspecimenandcancercells,hasaC-terminalthreecontinuousTPRdomains.SubcellularlocationanalysisrevealsthatNYD-SP14isco-locatedwithPP2R1AandHSP70.TheresultsthatNYD-SP14couldblockmitosisinSphase,inducetheapoptosisinimmortalHEKTERcellandPP2R1Acouldenhancetheeffectsuggestingtheinteractionasacellcycleregulator.TheseresultsconducetorevealthefunctionofNYD-SP14onspermatogenesis,cellcycleandtumorigenesis,andlayafoundationforthefunctionalstudyofNYD-SP14.TheCKOmousealsoprovidesnewwaysforthefurtherstudiesofNYD-SP14onembryogenesisandontogenesis. |
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