Mmu-miR-107与Cacna2d1基因3'UTR结合位点的预测及验证

Predict and confirm the Cacna2d1 gene 3'UTR binding sites for mmu-miR-107

[目的]预测并验证mmu-miR-107与Cacna2d1基因3'UTR的结合位点。[方法]生物信息学预测miR-107与小鼠Cacna2d1基因3'UTR的结合位点,将包含3个靶位点的3'UTR片段克隆至荧光素酶载体p GL3中,构建野生型p GL3-Cacna2d1-WT3'UTR载体;并用重叠延伸PCR技术构建分别含有单个突变位点的载体Mut1200-207、Mut2361-367、Mut3902-908和同时含有3个突变结合位点的载体Mut4plus。将各重组质粒与miRNA mimics共转染C2C12细胞,检测荧光素酶活性。[结果]与共转染p GL3-Cacna2d1-WT 3'UTR和miR-NC组相比,共转染p GL3-Cacna2d1-WT 3'UTR和mimics-miR-107组荧光素酶活性显著下降(P<0.01);与共转染对应的突变体和miR-NC组相比,转染突变体Mut2361-367/Mut3902-908和mimics-miR-107组的荧光素酶活性均下降(P<0.05);转染突变体Mut1200-2077/Mut4plus和mimics-miR-107组的荧光素酶活性下降不明显(P>0.05)。[结论]Cacna2d1基因3'UTR段双荧光素酶基因报告载体及突变体构建成功,初步证实miR-107与Cacna2d1 3'UTR的200-207位点结合。

[Objective]To predict and confirm the Cacna2d1 gene 3＇ UTR binding sites for mmu-miR-107. [Methods]Bio-information assay revealed that the 3＇UTR of Cacna2d1 harbors three putative binding sites for miR-107. Thus,the 3＇ UTR of Cacna2d1 gene was amplified by PCR,and inserted into p GL3 Luciferase reporter vector（made the wild-type plasmid p GL3-Cacna2d1-WT 3＇UTR）. Then three mutant plasmids containing single binding site（Mut1200-207,Mut2361-367 or Mut3902-908） and a mutant plasmid containing all the three binding sites（Mut4plus） were made by overlap extension PCR. Both recombinant plasmids and mimics NC（or miR-107） were transfected into C2C12 cells,and the activity of luciferas was detected by Luciferase Reporter Assay. [Results]Compared with the C2C12 cells co-transfected with p GL3-Cacna2d1-WT 3＇ UTR and miR-negetive control,the luciferase activity of C2C12 cells transfected with p GL3-Cacna2d1-WT 3＇UTR and mimics-miR-107 was decreased（P〈0.01）; Compared with the C2C12 cells co-transfected with mutant p GL3-Cacna2d1-WT 3＇ UTR and miR-negetive control,the luciferase activity of C2C12 cells co-transfected with Mut2361-367 and mimics-miR-107,co-transfected with Mut3902-908 and mimics-miR-107 were decreased（P〈0.05）; the luciferase activity of C2C12 cells co-transfected with Mut1200-207 and mimics-miR-107,co-transfected with Mut4 plusand mimics-miR-107 were not obviously decreased（P〉0.05）. [Conclusion]The luciferase reporter plasmids containing wild-type 3＇ UTR of Cacna2d1 and mutant 3＇UTR of Cacna2d1 were constructed successfully,the 3＇UTR of Cacna2d1 harbors the binding sites 200-207 for miR-107.