Abstract: Objective To construct dual-luciferase reporter plasmids containing the wild type and mutant rat extracellular signal-regulated kinase 1 (ERK1) gene 3ʹ untranslated regions (UTR) which were used to detect rno-miR-15b-5p's putative target gene. Methods The rat ERK1 gene 3ʹ UTR fragment was amplified by polymerase chain reaction (PCR) from PC12 cell cDNA and cloned into pmiR-RB-Report^TM vector. The mutant rat ERK1 gene 3ʹ UTR fragment was obtained by overlap PCR and inserted into pmiR-RB-Report^TM vector. Successful wild type and mutant recombinant plasmids were confirmed by DNA sequencing. PC12 cells were co-transfected with rno-miR-15b-5p mimic and pmiR-ERK1 3ʹ UTR or pmiR-ERK1-mut 3ʹ UTR and then analyzed by dual-luciferase reporter assay system. The achieved mutation sequence of the target site TGCTGCT was mutated to CGAACGT and GTACACG, respectively. Results The wild-type reporter vector pmiR-ERK1 3ʹ UTR and the mutant reporter vector pmiR-ERK1-mut 3ʹ UTR were successfully constructed. The rno-miR-15b-5p mimic decreased the activity of pmiR-ERK1 3ʹ UTR plasmid (P<0.001) but did not decrease the activity of pmiR-ERK1-mut 3ʹ UTR plasmid. Conclusion The recombinant pmiR-ERK1 3ʹ UTR and pmiR-ERK1-mut 3ʹ UTR plasmids were constructed successfully, and luciferase activities demonstrated that the 3ʹ UTR of ERK1 gene might be a potential target of rno-miR-15b-5p.

Key words: rno-miR-15b-5p, extracellular signal-regulated kinase 1, overlap polymerase chain reaction, pmiR-ERK1 3'UTR, pmiR-ERK1-mut 3'UTR, luciferase reporter assay