大鼠细胞外信号调节激酶1基因3'非编码区双荧光素酶报告载体的构建及rno-miR-15b-5p对其活性的影响

Construction of Rat Extracellular Signal- regulated Kinase 1 Gene 3' Untranslated Regions Dual-luciferase Reporter Plasmids and Effect of rno-miR-15b-5p on Its Activitiy

目的以大鼠细胞外信号调节激酶1(ERK1)基因3'非编码区(UTR)为研究对象,构建含有ERK1基因3'UTR区的野生型以及突变型重组双荧光素酶报告载体,通过分析双荧光素酶报告载体的活性,明确rno-miR-15b-5p对报告载体的调节作用。方法采用miRNeasy Mini Kit提取大鼠肾上腺嗜铬细胞瘤PC12细胞的总RNA,以PC12细胞的c DNA为模板,利用聚合酶链式反应(PCR)将ERK1基因3'UTR克隆到pmiR-RB-Report^(TM) vector中。利用重叠延伸PCR,将ERK1基因3'UTR中的rno-miR-15b-5p潜在的靶序列TGCTGCT分别突变成CGAACGT和GTACACG,并将突变的ERK1基因3'UTR克隆到pmiR-RB-Report^(TM) vector中。结果成功构建了包含ERK1基因3'UTR区的野生型报告载体pmiR-ERK1 3'UTR和突变型报告载体pmiR-ERK1-mut 3'UTR。利用荧光素酶活性检测系统,发现rno-miR-15b-5p mimic可以降低野生型报告载体的活性(P<0.001),但不影响突变型报告载体的活性。结论成功构建ERK1基因3'UTR区野生型和突变型双荧光素酶报告载体,初步证明ERK1基因3'UTR区是rno-miR-15b-5p的结合靶点。

Objective To construct dual-luciferase reporter plasmids containing the wild type and mutant rat extracellular signal-regulated 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 c DNA and cloned into pmiR-RB-Report（TM） vector. The mutant rat ERK1 gene 3＇ 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 3＇ 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-ERK1 3＇ UTR and the mutant reporter vector pmiR-ERK1-mut 3＇ UTR were successfully constructed. The rno-miR-15b-5p mimic decreased the activity of pmiR-ERK1 3＇ UTR plasmid（P〈0.001） but did not decrease the activity of pmiR-ERK1-mut 3＇ UTR plasmid. Conclusion The recombinant pmiR- ERK1 3＇ 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.