中华病理学杂志
中華病理學雜誌
중화병이학잡지
Chinese Journal of Pathology
2012年
7期
470-474
,共5页
马怡晖%于双妮%卢朝辉%陈杰
馬怡暉%于雙妮%盧朝輝%陳傑
마이휘%우쌍니%로조휘%진걸
微RNAs%靶基因修复%遗传载体%细胞系,肿瘤
微RNAs%靶基因脩複%遺傳載體%細胞繫,腫瘤
미RNAs%파기인수복%유전재체%세포계,종류
MicroRNAs%Targeted gene repair%Genetic vectors%Cell line,tumor
目的 构建miR-23a-27a簇真核表达载体并初步探讨该簇的靶基因及其功能.方法 应用分子克隆的方法构建联合表达pre-miR-23 a-27a簇及单独表达pre-miR-23a、pre-miR-27a的真核载体,应用双荧光素酶报告基因试验和real-time PCR验证该载体表达的有效性,应用microRNA靶基因预测软件和双荧光素酶报告基因试验寻找及鉴定pre-miR-23a-27a的靶基因,应用Western blot和real-time PCR的方法在乳腺癌细胞MCF-7中初步探讨miR-27a的功能.结果 (1)pre-miR-23a-27a-pcDNA3.1、pre-miR-23a-pcDNA3.1、pre-miR-27a-pcDNA3.1重组质粒能够在真核细胞HEK293T中有效转录加工出相应的miR-23a和miR-27a;(2)miR-23a和miR-27a能够同连有Sprouty2基因MRE序列的pRL-TK重组质粒作用,但是只有miR-27a能够同连有Sprouty2基因3'-非翻译区(UTR)全长序列的pRL-TK重组质粒作用,而将Sprouty2基因3'-UTR中同miR-27a结合位点定点突变后,miR-27a无法同其作用;(3)在人乳腺癌细胞MCF-7中转染pre-miR-27a-pcDNA3.1真核表达载体,能够在蛋白水平显著影响Sprouty2基因的表达,但是对其RNA水平没有显著影响.结论 Sprouty2可能是miR-27a的功能靶基因,pre-miR-23a-27a-pcDNA3.1、pre-miR-23a-pcDNA3.1、pre-miR-27a-pcDNA3.1载体的构建为下一步深入研究miR-23a和miR-27a的功能奠定了基础.
目的 構建miR-23a-27a簇真覈錶達載體併初步探討該簇的靶基因及其功能.方法 應用分子剋隆的方法構建聯閤錶達pre-miR-23 a-27a簇及單獨錶達pre-miR-23a、pre-miR-27a的真覈載體,應用雙熒光素酶報告基因試驗和real-time PCR驗證該載體錶達的有效性,應用microRNA靶基因預測軟件和雙熒光素酶報告基因試驗尋找及鑒定pre-miR-23a-27a的靶基因,應用Western blot和real-time PCR的方法在乳腺癌細胞MCF-7中初步探討miR-27a的功能.結果 (1)pre-miR-23a-27a-pcDNA3.1、pre-miR-23a-pcDNA3.1、pre-miR-27a-pcDNA3.1重組質粒能夠在真覈細胞HEK293T中有效轉錄加工齣相應的miR-23a和miR-27a;(2)miR-23a和miR-27a能夠同連有Sprouty2基因MRE序列的pRL-TK重組質粒作用,但是隻有miR-27a能夠同連有Sprouty2基因3'-非翻譯區(UTR)全長序列的pRL-TK重組質粒作用,而將Sprouty2基因3'-UTR中同miR-27a結閤位點定點突變後,miR-27a無法同其作用;(3)在人乳腺癌細胞MCF-7中轉染pre-miR-27a-pcDNA3.1真覈錶達載體,能夠在蛋白水平顯著影響Sprouty2基因的錶達,但是對其RNA水平沒有顯著影響.結論 Sprouty2可能是miR-27a的功能靶基因,pre-miR-23a-27a-pcDNA3.1、pre-miR-23a-pcDNA3.1、pre-miR-27a-pcDNA3.1載體的構建為下一步深入研究miR-23a和miR-27a的功能奠定瞭基礎.
목적 구건miR-23a-27a족진핵표체재체병초보탐토해족적파기인급기공능.방법 응용분자극륭적방법구건연합표체pre-miR-23 a-27a족급단독표체pre-miR-23a、pre-miR-27a적진핵재체,응용쌍형광소매보고기인시험화real-time PCR험증해재체표체적유효성,응용microRNA파기인예측연건화쌍형광소매보고기인시험심조급감정pre-miR-23a-27a적파기인,응용Western blot화real-time PCR적방법재유선암세포MCF-7중초보탐토miR-27a적공능.결과 (1)pre-miR-23a-27a-pcDNA3.1、pre-miR-23a-pcDNA3.1、pre-miR-27a-pcDNA3.1중조질립능구재진핵세포HEK293T중유효전록가공출상응적miR-23a화miR-27a;(2)miR-23a화miR-27a능구동련유Sprouty2기인MRE서렬적pRL-TK중조질립작용,단시지유miR-27a능구동련유Sprouty2기인3'-비번역구(UTR)전장서렬적pRL-TK중조질립작용,이장Sprouty2기인3'-UTR중동miR-27a결합위점정점돌변후,miR-27a무법동기작용;(3)재인유선암세포MCF-7중전염pre-miR-27a-pcDNA3.1진핵표체재체,능구재단백수평현저영향Sprouty2기인적표체,단시대기RNA수평몰유현저영향.결론 Sprouty2가능시miR-27a적공능파기인,pre-miR-23a-27a-pcDNA3.1、pre-miR-23a-pcDNA3.1、pre-miR-27a-pcDNA3.1재체적구건위하일보심입연구miR-23a화miR-27a적공능전정료기출.
Objective To construct a miR-23a-27a cluster expression plasmid and to explore the target genes and function of the cluster.Methods The pre-miR-23a-27a-pcDNA3.1,pre-miR-23a and premiR-27a plasmids were cloned by molecular biology method,and their expression efficiency was tested by dual luciferase reporter gene assay and real-time PCR.Several possible target genes of miR-23a and miR-27a were chosen using softwares and further tested by dual luciferase reporter gene assay.Finally,the function of miR-27a was analyzed in MCF-7 cell by Western blot and real-time PCR Results miR-23a and miR-27a were transcribed from pre-miR-23a-27a-pcDNA3.1,pre-miR-23a and pre-miR-27a plasmids in HEK293T cells,and both influenced the MRE of Sprouty2 gene in pRL-TK vector,and only miR-27a influenced the 3'-untranslated regions(UTR) full length of Sprouty2 gene while miR-27a did not influence the 3'-UTR of Sprouty2 gene with the sited-mutation in the MRE.The protein expression level of Spronty2 gene was altered after transfection of pre-miR-27a-pcDNA3.1 plasmid while the RNA level remained unchanged.Conclusion Sprouty2 may he the functional target gene of miR-27a,and the construction of plasmids in the study may provide a fundamental basis for the further functional investigation of miR-23a and miR-27a.
