摘要目的 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.

abstractsObjective 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.