Construction of Rat Extracellular Signal-regulated Kinase 1 Gene 3' Untranslated Regions Dual-luciferase Re-porter Plasmids and Effect of rno-miR-15b-5p on Its Activitiy
LUO Han-jiang
XU Yun-feng
LI Xiao-xiao
YANG Yu-tao
XU Zhi-qing
Abstract：Objective To construct dual-luciferase reporter plasmids containing the wild type and mutant rat extracellular signal-regulat-ed kinase 1 (ERK1) gene 3' untranslated regions (UTR) which were used to detect rno-miR-15b-5p's putative target gene. Methods The rat ERK1 gene 3' UTR fragment was amplified by polymerase chain reaction (PCR) from PC12 cell cDNA and cloned into pmiR-RB-ReportTM vector. The mutant rat ERK1 gene 3' UTR fragment was obtained by overlap PCR and inserted into pmiR-RB-ReportTM vector. Successful wild type and mutant recombinant plasmids were confirmed by DNA sequencing. PC12 cells were co-transfected with rno-miR-15b-5p mim-ic and pmiR-ERK13' UTR or pmiR-ERK1-mut 3' 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-ERK13' UTR and the mutant reporter vector pmiR-ERK1-mut 3' UTR were successfully constructed. The rno-miR-15b-5p mimic de-creased the activity of pmiR-ERK13' UTR plasmid (P<0.001) but did not decrease the activity of pmiR-ERK1-mut 3' UTR plasmid. Conclu-sion The recombinant pmiR-ERK13' UTR and pmiR-ERK1-mut 3' UTR plasmids were constructed successfully, and luciferase activities demonstrated that the 3' UTR of ERK1 gene might be a potential target of rno-miR-15b-5p.
Keywords：rno-miR-15b-5pextracellular signal-regulated kinase 1overlap polymerase chain reactionpmiR-ERK13' UTRpmiR-ERK1-mut 3' UTRluciferase reporter assay
Publication Date：2017-01-01
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：7( 166-172 )