miR-4483靶基因鉴定及其抗肝纤维化机制研究-论文.pdf

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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