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

ABSTRACT

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.