miR-23a-27a表达载体构建及功能初探

Construction of a miR-23a-27a cluster expression plasmid： a preliminary study of its function

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-23a-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—timePCR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Westernblot和real．timePCR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-步深入研究miR-23a和miR-27a的功能奠定了基础。

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 pre- miR-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 Sprouty2 gene was altered after transfection of pre-miR-27a-pcDNA3.1 plasmid while the RNA level remained unchanged. Conclusion Sprouty2 may be 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.