hnRNPB-1siRNA对肺腺癌细胞A549p53表达的影响-韩娟

hnRNPB1siRNAA549p53*韩　娟1,李为民1,2△,唐凤鸣1,蒲　丹1,陈文彬11.(610041);2.　　【】　　(hnRNP)B1A549p53。　hnRNPB1RNA(siRNA)(AD)A549,A549A549。10μmol/L24h。RT-PCRp53mRNA,WesternblotP53P53。　p53mRNA,hnRNPB1siRNAA549p53mRNA。hnRNPB1siRNAP530.87±0.06、0.75±0.06,(0.30±0.08)(0.21±0.04)(P0.01)。　hnRNPB1siRNAA549P53,。【】　　p53　B1　RNA【】　R734.2,R73-37EffectsofhnRNPB1siRNAontheExpressionofp53inHumanLungAdenocarcinomaCellLineA549　HANJuan,LIWei-min△,TANGFeng-ming,PUDan,CHENWen-bin.　DepartmentofRespiratoryMedicine,WestChinaHospital,SichuanUniversity,Chengdu610041,China【Abstract】　Objective　ToexploretheeffectsofhnRNPB1ontheexpressionofp53inhumanlungadenocarcinomacelllineA549.Methods　TwolinesofA549cellstransfectedbyhnRNPB1specificsiRNA(recombinantplasmidAandD),whichhadahightransfectionefficiency,wereestablishedasexperimentalgroups.OnelineofA549cellsandonelineofA549cellstransfectedbypureplasmidservedascontrols.Allofthecellswerecollectedaftertreatedwith10μmol/Lcisplatinfor24h.Theexpressionsofp53mRNAandproteinweredetectedbyreal-timeRT-PCRandWesternblotanalysis.TheexpressionofPhospho-P53proteinwasdetectedbywesternblotanalysis.Results　Nosignificantdifferencesintheexpressionofp53mRNAandproteinamongthegroupswerefound.TheexpressionratioofPhospho-P53proteininthecellstransfectedbyhnRNPB1siRNAwere0.87±0.06and0.75±0.06,whichweresignificantlygreaterthanthoseintheA549cell(0.21±0.04)andintheA549cellstransfectedbypureplasmid(0.30±0.08)(P0.01).Conclusion　hnRNPB1specificsiRNAcanupregulatetheactivityofp53inA549cells.【Keywords】　　Lungadenocarcinoma　　p53　　HeterogeneousnuclearribonucleproteinB1　　siRNA　　p53,p53,p53,。(heterogeneousnuclearribonucleprotein,hnRNP)B1,,,mRNA*(30270587)(20050610056)△Correspondingauthor,E-mail:[e-mail],RNA、,RNA、PremRNA[1],。4hnRNPB1RNA(siRNA)A549,A549hnRNPB1。hnRNPB1siRNAA549p53mRNAP53(P-P53),hnRNPB1p53,hnRNPB1、()JSichuanUniv(MedSciEdi)　2008;39(5):812-814　　　　　　　　　　　　　　　　　　　　　　　　　　　　　。1　1.1　A549。hnRNPB1siRNA(AD)A549[2],A549A549,EF。10%DMEM、37℃、5%CO2。A、D、EG418(200μg/mL)。1.2　RT-PCRp53mRNAPrimerpremier5.0。p53PCR:5′-TTTCCGTCTGGGCTTCTT-3′;5′-GCCTCACAACCTCCGTCA-3′。200bp。GAPDH,PCR:5′-CCTCAAGATCATCAGCAAT-3′;5′-CCATCCACAGTCTTCTGGGT-3′。141bp。RNA,。RT-PCR()。PCR:cDNA5μL,10mmol/L1μL,10mmol/L1μL,2×TaqPCRMAsTerMix12.5μL,30μL。FTC2000(FUNGLYN)PCR。94℃5min,94℃,53℃(p53)/54℃(GAPDH),20s(p53)/30s(GAPDH),72℃,30s,45(p53)/25(GAPDH),72℃10min。DNA,,RNA,1μLRNAse(1μg/μL)37℃20min。PCR15g/L。1.3　WesternblotP53P-P5310μmol/L24h,PBS,200μL,BCA,15～20μg,4∶1,100℃5min,25μL5μLMarkerSDS-PAGE,80V,2hPVDF,5%1～2h,〔P53(SantaCruz)1∶500,Phospho-P53(Ser15)(CellSignaling)1∶1000,β-actin(SantaCruz)1∶1000〕,4℃,〔IgG(CellSignaling)1∶10000〕1h。QuantityOneWesternblot。,P53/β-actin、P-P53/β-actin。1.4　x-±s。,α=0.05。2　2.1　A549p53mRNA(2-ΔΔCt)p53mRNA(P0.05),1。hnRNPB1siRNAA549p53mRNA。1　p53mRNATable1　Relativeexpressionquantityofp53mRNAindifferentgroupsGroupn△△Ct2-ΔΔCtA6-1.17±0.712.39±1.13D6-0.84±0.761.97±1.08E6-0.84±0.291.81±0.38F6-0.92±0.461.92±0.47　　A:TransfectedbyAplasmid;D:TransfectedbyDplasmid;E:Transfectedbypureplasmid;F:Untransfected　P53(A)P-P53(B)Fig　ProteinexpressionofP53(A)andP-P53(B)indifferentgroupsA,D,E,F:Denotesthesameasthoseintable1813　JSichuanUniv(MedSciEdi)Vol.39No.520082.2　A549P53P-P53Westernblot(),P53,hnRNPB1siRNAA549P53。hnRNPB1siRNAA549P-P53FE(P0.01),EF(P0.05)。2。2　hnRNPB1siRNAA549P53P-P53Table2　EffectsofhnRNPB1siRNAontheexpressionofP53andP-P53indifferentgroupsGroupnExpressionratioofP53proteinExpressionratioofP-P53proteinA60.98±0.090.87±0.06*D60.95±0.070.75±0.06*E60.96±0.080.30±0.08　F60.97±0.060.21±0.04　　　A,D,E,F:Denotesthesameasthoseintable1.*P0.05,vsEandFgroup3　p53,,P53,P53,、,。P53(6～20min),,100。[3-5],P53,P5355%,58%,Ⅰ、ⅡP53Ⅲ、Ⅳ,P53。A549P53,。hnRNP、30RNA,hnRNPA2、B1。Matsayama[6]4hnRNPB1,100%,94.4%75%,hnRNPB1。Fleischhaker[7]RT-PCR18hnRNPB1mRNA,hnRNPB1mRNA,14hnRNPB1mRNA。,hnRNPB1。,hnRNPB1。[8],hnRNPA2siRNAhnRNPA2Colo16HaCaT,p21BRCA1mRNA,p53。hnRNPA2hnRNPB1,7p15,[9]。hnRNPB1cDNAhnRNPA25′36bp,。hnRNPB1siRNAA549,p53mRNA,hnRNPB1p53。,hnRNPB1siRNAA549P-P53A549,hnRNPB1siRNAP53。DNA,DNA(DNA-dependentproteinkinase,DNA-PK),P53。[10]hnRNPB1DNA-PK,hnRNPB1DNA-PK,,hnRNPB1siRNADNA-PKP53。hnRNPB1DNA-PKP53,P53,,。1PinoI,PioR,ToledoG,etal.Alteredpatternsofexpressionofmembersoftheheterogeneousnuclearribonucleoprotein(hnRNP)familyinlungcancer.LungCancer,2003;41(2):131-143.2　,,　.HnRNPB1RNA.,2006;27(8):771-773.3LuoJC,ZehabR,AnttilaS,etal.DetectionofserumP53proteininlungcancerpatients.JOccupMed,1994;36(2):155-160.4KawasakiM,NakanishiY,KuwanoK,etal.TheutilityofP53immunostainingoftransbronchialbiopsyspecimensoflungcancer:P53overexpressionpredictspoorprognosisandchemoresistanceinadvancednon-smallcelllungcancer.ClinCancerRes,1997;3(7):1195-200.(835)814()2008395　,53.8%、48.8%41.2%,,92%,、。,,,。,CEA、CA125、CA199、CYFRA21-1NSE27.4%～60.0%,CYFRA21-1;91.5%～99.4%,CA199。,,,,CYFRA21-1,CA199。3、4,。54,。5,,,。,,,NSCLCCEA+CA125+CYFRA21-1,SCLCCEA+CYFRA21-1+NSE,CYFRA21-1,NSESCLC。,,、,、。1,..,2001;24(8):450-451.2　,　..,1996;23(2):98-103.3KulpaJ,WojcikE,ReinfussM,etal.Carcinoembryonicantigen,squamouscellcarcinomaantigen,CYFRA21-1,andneuronspecificenolaseinsquam