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时间:2020-05-04
《miR-4483靶基因鉴定及其抗肝纤维化机制研究-论文.pdf》由会员上传分享,免费在线阅读,更多相关内容在应用文档-天天文库。
1、中国实用医刊2014年4月第41卷第7期ChineseJournalofPracticalMedicineAor.2014.V01.41.N0.7·41·miR-4483靶基因鉴定及其抗肝纤维化机制研究顿少志周言孙长宇【摘要】目的分析并鉴定miR-4483对TIMP一1表达水平的靶向调控作用,并探讨miR-4483的抗肝纤维化机制。方法应用生物信息学方法预测miR-4483靶基因为TIMP一1。采用重叠PCR技术将靶基因3’UTR片段克隆至pGL3·M载体构建野生型及突变型重组质粒,和pRL—TK、miRNA前体共转染入大鼠肝星状细胞系HSC
2、—T6,检测荧光素酶的表达。将miRNA前体转染HSC—T6后检测TIMP一1的mRNA和蛋白质表达水平。结果酶切和测序证实重组质粒构建成功,共转染miR-4483前体组可下调野生型质粒的荧光素酶活性(P<0.05),不影响突变型质粒的荧光素酶活性,而共转染miR-NC组,突变型质粒和野生型质粒荧光素酶活性比较差异无统计学意义(P>0.05);上调miR-4483水平后,TIMP-1mRNA和蛋白的表达水平与转染miR.NC和空白组比较差异有统计学意义(P<0.05)。结论TIMP-1是miR-4483的靶基因,miR-4483通过负向调控T
3、IMP一1表达而起到抗肝纤维化作用。【关键词】miR-4483;肝纤维化;TIMP.1;荧光素酶报告IdentificationofmiR-4483targetgeneandstudyonitsmechanismofantiliverfibrosisDUNShao—zhi,zHoUYan,sUNChang—yu.DepartmentofInfectiousDiseases,theFirstAffiliatedHospitalofZhengzhouUniversity,Zhengzhou450052.Cina【Abstract】0bjectiv
4、eToanalyzeandidentifythetargetregulationeffectofmiR-4483onTIMP-1expressionlevels.andstudytheantiliverfibrosismechanismofmiR-4483.MethodsThetargetgene0fmiR4483wasTIMP一1.predictedbybioinformaticsmethod.Thewild—typeandmutantrecombinantplasmids(pGL3一TIMP-1-wtandpGL3一TIMP一1一rout
5、)wereconstructedbyoverlapextensionPCRoftargetgene3’UTRfragmentcloningintothevectorpGL3.M.whichwereco.transfeetedintoHSC.T6cellswiththemiRNA-4483mimicsandpRL-TKfordetectionoflueiferaseexpession.Rea1.timeRT—PCRandWest—ernblotwereperformedtoexaminethemRNAandproteinexpressionle
6、velsofTIMP.1aftermiRNA-4483mimicstransfectedintoHSC.T6cells.ResultsTherecombinantplasmidwasconfirmedbyrestrictionenzymedigestionandsequencing.Theco—transfectedmiRNA_4483mimicscandown—regulatethelucifer-aseactivityofthewild。typeplasmids(P<0.05).butcannotinfluencetheluciferas
7、eactivityofthemu—tant.typeplasmids.Therewasmosignificantdifferenceinthelueiferaseactivitybetweenthewild.typeplasmidsandmutantplasmidsco—transfectedwithmiR-NC(P>0.05).Up—regulationofmiR-4483levelscandramaticallysuppresstheTIMP一1mRNAandproteinexpressionlevelsinHSC一r6cells(P<0
8、.05).ConclusionsTIMP—listhetargetgeneofmiR-4483.amdmiR-4483hasantiliverfibrosiseff
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