alx4基因甲基化及其在肺癌发生中的作用分析word格式论文.docx

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alx4基因甲基化及其在肺癌发生中的作用分析word格式论文.docx

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alx4基因甲基化及其在肺癌发生中的作用分析word格式论文

MethylationofALX4geneanditsrolein CarcinogenesisoflungcancerAbstractObjective:LungcanceristhemostcommonmalignancyinChinaandworldwideamongbothmenandwomen.Todate,earlydetection,especiallymoleculardiagnosisrepresentsthemostpromisingstrategy.Withunderstandingofthemolecularmechanismoflungcancer, molecularmarkerscouldbeofgreatusetodetectlungcancerattheearlieststageofthis disease.Throughmethylation-sensitiverepresentationaldifferenceanalysis,wehaverecentlyidentifiedanovelpreferentiallymethylatedgene,ALX4(Aristaless-likeHomeobox4),whichishypermethylatedinlungcancer.SeveralstudiesalsoshowedthatALX4genehypermethylationfrequentlyoccurredincolorectalandgastriccancertissues.However,theroleandfunctionofALX4inlungcarcinogenesisremainslargelyunknown. Inthisstudy,weinvestigatedtheepigeneticregulation, biologicalfunctionofALX4inlungcancer.Methods:ThemethylationstatusofALX4wasanalyzedbyMSPinlungcancercelllines,non-tumorandtumorlungtissues.TheexpressionofALX4genewasdeterminedinlungcancercelllinesand5-Aza-dC-treatedlungcancercelllines.ThefunctionofALX4genewasanalyzedinover-expressionorknock-downlungcancercells by CCK-8 assay, colonyformationassay andflow cytometryanalysis.Results:ThemethylationstatusofALX4wasanalyzedbyMSPinsamplesfrom9lungcancercelllines,98lungcancercasesand20non-tumorcases.9celllinesshowed hypermethylationofALX4gene.ALX4hypermethylationwasfrequentlyinprimarylungcancertissues(52/98,53.1%),butnotoccurredin20normallungtissues,indicatingthatALX4methylationisfrequentandtumor-specific.ThehypermethylationofAlx4wasnegativelycorrelatedtothedifferentiation,butnotcorrelatedtoage,genderandsmoking.ALX4wasfoundtobedown-regulatedsignificantlyinprimarytumorspecimenswithALX4methylationbyRT-PCR.Aftertreatedthecelllinewith5-aza-dC,theexpressionofALX4restored.TheresultsshowedDNAhypermethylationregulatestheexpressionofALX4gene.Toexplorepotentialtumor-suppressorroleofALX4gene,wetransfectedGFP-ALX4into95DandH358cellsandgeneratedstabletransfectants.EctopicexpressionofALX4intheselungcancercell