pak1基因3′-utr区荧光素酶报告基因载体de构建及鉴定【期刊】.pdfVIP

现代泌尿外科杂志 2015年 6月第 2O卷第 6期 415 · 基础研究 · PAK1基因3-UTR区荧光素酶报告基因载体的构建及鉴定 刘 伟 ，寇 博 ，贺大林，郭 鹏 (西安交通大学医学部第一附属医院泌尿外科 ，陕西西安 710061) ConstructionofPAK13％UTR luciferasereportervectorandidentificationofitsac— tivity LIU W ei，KOU Bo，HE Da一1in，GUO Peng (DepartmentofUrology，theFirstAffiliated HospitalofXi’anJiaotongUniversityHealthSciencecenter， Xi’an710061。China) · ABSTRACT：Objective Toconstructtheluciferasereportergenevectorcontaining3-untranslatedregion(3-UTR)of p21一activatedkinase一1 (PAK1)，which willprovidebasisforsubsequentfunction andmechanism research onPAK1andmi— croRNA一145．Methods The3-UTRofPAK1genewasamplifiedbyPCR，andinsertedintopOL3controlreporter~ector afterlinkandrestrictionenzymedigestion．Itwaspredictedbybioinformationmethod~hatmiR．145couldtargetthe3-UTRof PAK1．TherecombinantplasmidwithmicroRNA m imicsormicroRNA controlweretransfectedintoT24orJ82cellsusingLi· pofectamine2000 to detecttherelativeluciferaseactivity．Inaddition，theexpressionofPAK1wasanalysedbyreal·timePCR ． Results Therecombinantplasmidwasconstructedandconfirmedbyenzymedigestionandsequencereaction．Aftertransfec— tionwith theplasmidpGL3-PAK13-U TR W T，wefound thatcomparedwithT24 orJ82 cellstreatedwith miRNA con，the luciferaseactivityofT24orJ82cellstransfectedwith miRNA mim icswassignificantly decreasedwith statisticsignificance． Futhermore，theexpression ofPAK1 in miR一145mimicsgroup wasremarkably lowerthan thatin miR一145 controlgroup． Conclusions ThePAK13一UTR luciferasereportervectorwasconstructedsuccessfully，andtheluciferaseactivityofthere— combinantplasm idcouldbedramaticallyreducedbymiR一145．ItindicatedthatmiR一145coulddownregulatesPAK1expression bytargetingits3r_UTR ． KEY W ORDS：p21一activatedkinase．1；3p_untranslatedregionsluciferasereportergene；microRNA-145；bladdercancer 摘要 ：目的 针对人 p21小 GTP酶活化激酶 1(p21一activatedkinase-1，PAK1)基 因的3端非翻译区(