let-7a微小rna在黑素瘤中的表达及其对a375细胞增殖的影响

《let-7a微小rna在黑素瘤中的表达及其对a375细胞增殖的影响》由会员上传分享，免费在线阅读，更多相关内容在教育资源-天天文库。

1、35820l0年5月第43Mav2O1O，Vo1．43，No．5·论著·let一7a微小RNA在黑素瘤中的表达及其对A375细胞增殖的影响王震英王焱薛燕宁徐秀莲曾学思孙建方【摘要】目的探讨甲醛固定石蜡包埋的黑素瘤组织中le卜7a微小RNA(microRNA)的表达情况及其对黑素瘤A375细胞增殖的影响。方法选取黑素瘤蜡块13份，先天性色素痣蜡块10份，抽提microRNA，采用茎环引物逆转录为cDNA，然后采用TaqmanMGB探针，相应的引物，进行实时荧光定量PCR。继而采用cy3标记的siRNA阴性对照物转染A375细胞，荧光显微镜下观察转

2、染效率，最后将let一7a的模拟物转染入A375细胞，MTT实验检测转染后细胞增殖的变化。结果从石蜡包埋组织中成功抽提出microRNA，而且定量PCR结果显示let一7a在黑素瘤中的表达明显低于色素痣组织(t=2．364，P<0．05)。荧光显微镜下观察siRNA转染效率约80％一90％，hsa—let一7a转染A375细胞后，使其增殖受抑，转染后24h抑制率达32．7％。结论let一7a在黑素瘤中表达下降，过表达let一7a可使A375细胞增殖受到抑制，提示其在黑素瘤中可能起抑癌基因样作用。【关键词】黑色素瘤；微RNAs；let一7a；转染

3、；细胞增殖ExpressionofmicroRNAlet-7ainmelanomaanditsefectontheproliferationofA375humanmelanomacel、lineWANGZhen．ying,WANGran．XUEYah—ning,XUXiu．1ian,zENGXire—si．SUNJian-fang．InstituteofDermatology,ChineseAcademyofMedicalSciencesandPekingUnionMedicalCollege，Naniing210O2．ChinaCorresp

4、ondingauthor：sUNJian-fang,Email：faugrnin5758@yahoo．con．cn【Abstract】ObjectiveToinvestigatetheexpressionofmicroRNA1et一7ainformalin—fixedparaffin—embedded(FFPE)melanomatissueanditseffectontheproliferationofA375celllines．MethodsTissuesampleswereobtainedfrom13patientswithmalignan

5、tmelanomaand10withcongenitalpigmentednevus，thenfixedwithformalinandembededwithparaffin．microRNAswereisolatedfromthesesamplesandreverselytranscriptedtocDNAwithstem—loopprimer．Then．realtimequantitativePCRwasperformedwithTaqmanMGBprobeandmatchedprimerstomeasuretheexpressionofmi

6、croRNAlet一7a．cy3一LabelednegativecontrolsiRNAwastransfectedintoA375cellsfollowedbytheexaminationoftransfectionefficiencyunderfluo—rescensemicroscopy．Subsequently．hsa1et一7amimicsweretransfectedintoA375cellstoobservetheireffectontheproliferationofthesecells．ResultsmicroRNAswere

7、successfullyisolatedfromFFPEtissues．andquantitativePCRshowedasignificantreductionintheexpressionofmicroRNAlet．7ainmelanomatissuecomparedwithnevustissue(t=2．364P<0．05)．Thetransfectionemciencywasf0undtobeabout80％～90％．Aftertransfectionwithhsa—let一7amimics．theproliferationofA375

8、cellswasinhibitedandtheinhibitionrateamountedto32．7％at24hours．ConclusionsTh