人卵巢癌相關(guān)候選基因HE4（WFDC2）轉(zhuǎn)錄調(diào)控機(jī)制，尤其是DNA甲基化在其中的作用.pdf

1、後旦大學(xué)丘洛布腫瘤研究所碩士研究生畢業(yè)論文THESISFORTHEMASTER’SDEGREE773130人卵巢癌相關(guān)候選基因HE4(WFDC2)轉(zhuǎn)錄調(diào)控機(jī)制尤其是繃甲基化在其中的作用TranscriptionalRegulationofthehumanoveriancarcinomaassociatedcandidategene：HE4(WFDC2)，theroleofDNAmethylationinparticular碩士研究生：趙

2、瑩瑤導(dǎo)師：朱景德研究員柬經(jīng)作者、導(dǎo)篩舄程勿金義公鏹!竺竺!堡!!!!型!!!!!!!!型絲g!!!壘!墅!苧曼!AbstractTheHE4(human_epididymisgeneproduct4gene，alsocalledWFDC2，shortfor盟AP—four一一disulfidescoreproteinDWaSfoundoverexpressedinamajorityofovariancarcinomatissuesbut

3、lowintheirnormalcounterpartsThepotentialoftheHE4proteinhasbeensuggestedasthedisgnosticbiomarkerforovariancarcinomasHoweverthetranscriptionalregulatorymechanismsunderlyingthesuggestedovariancancerspecifityofitspromoterhas

4、notbeeninvestigatedInthisstudywehaveengagedamoleculardissectionofthepromoteroftheHE4geneineitherunmethylatedormethylatedsituation，inadditionteassessingitsexpressionprofileTounveilthemolecularmechanismsunderlyingtranscrip

5、tioncontrolsoftheHE4gene，weusealistofmoleculargeneticapproachesbelow：1，Usingasemi—quantitativePCRmethodtodeterminetheexpressionprofileofHE4geneinapaneloftumorcelllines；2，Usingmoleculardissectiontoconstruct，baseduponthelu

6、ciferasereportergene，asetofthe5’，or3’deletionmutantsfromthefulllengthupstreamregion(1860／29)region，and10linker—scanningmutantsof(107／15)oftheHE4minimalpromoter；3，Viathetransienttransfection／dualreporterassa5thepromoterac

7、tivitiesoftheaboveconstructswerevigorouslytested；4，ViatheanalysescombiningbioinformaticsCO—transfectionandelectrophoresismobilityshmassay(EMSA)，theproteinsbindingspecifictOtheciselementswithinthekeyregion(71／一48)withsign

8、ificantfunctionalconsequencewasdetermined；5，Viatheimmunoprecipitationassa5theinteractionbetweentranscriptionfactorswereanalyzed；6，assessingthemethylationstateofthepromoterCpGislandofHE4geneusingmethylation—specificPCRmet

9、hodinconjunctionwithDNAsequencing；7，analysisoftherelationshipbetweentheCpGmethylationandpromoteractivitybyinvitromethylationfollowedbytransientCOtransfection／reporterassayandDNA—proteinbindingassayOurresultsshow：1，theHE4

10、genewasalsoexpressedinothercelllinesbesidesovariancancercelllinesanditsmRNAhasseveralsplicingisoform；2，theminimalpromoterofHE4genewasdefinedtOafta擘nentfrom107to15positionrelativetOthetranscriptionstartsite；3，theinteracti