抑制usp18表达对人肝癌hep3b细胞抑制作用-inhibitory effect of usp 18 expression on human hepatoma hep3b cells.docxVIP

ABSTRACTPurpose:TodetecttheexpressionofUSP18inhepatomacarcinomacellsandinfluenceonbiologicalactivity(cellproliferation,cellclone,cellcycleandcellapoptosis)Methods:Inthefirstplace,ThemRNAandproteinlevelsofUSP18incellsofHepG2、Hep3B、HepG2.2.15、Huh7、SMMC-7721weredeterminedwithreversetranscription-PCRandwesternblottingandthehighestexpressedcellwasselectedforRNAiexperiment.Inaddition,threegroupsofspecificUSP18targetedsiRNAandnegativecontrolgroupweredesigned.Livercancercellswereinstantaneouslytransfectedusinglipofectaminemethod.Finally,wetransfectedthemtohepatomacarcinomacellanddetectedtheinterferingefficiencythroughreversetranscription-PCRandWestern-blot,andthencarriedoutthenexttests:MTTcellproliferationtest,flatplatecloneformationtest,cellcycletestandapoptosistest.Results:RT-PCRandWesternblotindicatedthatwashighexpressedinthefivelivercancerlines,andinterestinglyhigherexpressedinHBV-positivehepatomacellsthanHBV-negativeones.AfterinterferenceofUSP18,ExpressionofUSP18atthelevelofmRNAandproteininUSP18-siRNAgroupsweresignificantlyinhibitedComparedwiththenormalgroupandthenegativecontrolgroup,especiallyinUSP18-siRNA-1group,MTTassayandTabletcloningassayrevealedthatthenumberofcellproliferationandcloningefficienciessloweddownobviouslycomparedtothecontrolgroupandthenegativecontrolgroup,cellcyclewasblockedatG1phaseandthepercentageofapoptoticincreased.Conclusions:Hep3BwasthehighestexpressionofUSP18inthefivelivercancerlines.siRNA-targetingUSP18canmarkedlyinhibitedtheexpressionofUSP18inlivercancerHep3Bcells,anddecreasetheabilitiesofproliferationandcloningefficiencies,promotetheabilitiesofcellapoptosisandblockG1phaseofcellcycleoflivercancercells,especiallyinUSP18-siRNA-1group.Keywords:USP18;hepatomacarcinomacell;siRNA;biologicalactivity目录第1章引言1第2章材料与方法32.1研究所需材料32.1.1细胞株32.1.2主要试剂32.1.3自配试剂42.1.4主要仪器设备72.2实验方法82.2.1器材的准备82.2.2细胞培养82.2.3USP18高表达肝癌细胞株的筛选112.2.4细胞转染182.2.5USP18沉默效率检测192.2.6肿瘤生物学活性检测192.2.7统计学分析21第3章结果223.1筛选USP18高表达肝癌细胞株223.2转染233.2.1转染效果233.2.2沉默USP18基因对Hep3B细胞USP18mRNA表达