rno-miR-129-5p对大鼠大麻素Ⅰ型受体基因3′-非编码区双荧光素酶活性的影响

Construction of rat cannabinoid receptors 1 gene 3′-untranslated regions dual-luciferase reporter plasmids and effect of rno-miR-129-5p on its activity

[目的]探究rno-miR-129-5p对含有大鼠大麻素Ⅰ型受体(CB1R)基因3′-非编码区(UTR)双荧光素酶报告载体的调控作用。[方法]以大鼠前额叶皮层脑区和海马脑区cDNA为模板,利用聚合酶链式反应(PCR)扩增出含有rno-miR-129-5p与CB1R基因3′-UTR结合位点的目的片段。利用重叠延伸的方法将两个靶序列CAAAAA分别突变为CAGGCC,并将CB1R 3′-UTR和CB1R-mut 3′-UTR插入到pmiR-RB-Report^(TM) vector载体中。将rno-miR-129-5p mimic或其Negative control(NC)与野生型和突变型双荧光素酶报告载体共转染至PC12细胞后,检测其荧光素酶活性。[结果]成功构建了包含CB1R基因3′-UTR野生型双荧光素酶报告载体pmiR-CB1R 3′-UTR和突变型双荧光素酶报告载体pmiR-CB1R-mut 3′-UTR。荧光素酶活性检测发现rno-miR-129-5p mimic可以下调野生型报告载体的活性(P<0.05),而对突变型没有影响。[结论]初步证明CB1R基因3′-UTR是rno-miR-129-5p的作用靶点。

[Objective]To explore the effect of rno-miR-129-5 p on dual luciferase reporter vector containing 3′-untranslated regions(UTR)of rat cannabinoid type 1 receptor(CB1 R)gene.[Method]The rat CB1 R gene 3′-UTR was amplified by polymerase chain reaction(PCR).Two target sequences CAAAAA were mutated into CAGGCC by overlap extension,and CB1 R 3′-UTR and CB1 R mut 3′-UTR were inserted into pmiR-RB-Report^(TM) vector.Rno-miR-129-5 p mimic or its negative control(NC)was co-transfected with wild type or mutant reporter vectors into PC12 cells,and the luciferase activities were measured.[Result]The wild type reporter vector pmiR-CB1 R 3′-UTR and the mutant reporter vector pmiR-CB1 R-mut 3′-UTR were successfully constructed.Luciferase activities showed that rno-miR-129-5 p mimic could decrease the activity of wild type reporter vector(P<0.05),but had no effect on mutant dual reporter vector.[Conclusion]The rat CB1 R gene 3′-UTR might be a potential target of rno-miR-129-5 p.