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[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 validate the binding site between 3’UTR of mmu-miR-107 and Cacna2d1 gene. [Methods] Bioinformatics predicted the binding site between miR-107 and mouse 3’UTR of Cacna2d1 gene. The 3’UTR fragment containing 3 target sites was cloned into luciferase vector pGL3 to construct wild-type pGL3 -Cacna2d1-WT3’UTR vector. Mutations of sites Mut1200-207, Mut2361-367, Mut3902-908 and Mut4plus containing three mutated sites were constructed by overlap extension PCR. C2C12 cells were co-transfected with the recombinant plasmids and miRNA mimics to detect the luciferase activity. [Results] Compared with co-transfection of pGL3-Cacna2d1-WT 3’UTR and miR-NC group, luciferase activity of pGL3-Cacna2d1-WT 3’UTR and mimics-miR-107 co- <0.01). The luciferase activity of Mut2361-367 / Mut3902-908 and mimics-miR-107 transfected mutants were lower than that of cotransfected mutants and miR-NC groups (P <0.05). The transfection mutant Mut1200-2077 / Mut4plus and mimics-miR-107 group decreased luciferase activity was not significant (P> 0.05). [Conclusion] The luciferase reporter gene and its mutants were successfully constructed in 3’UTR segment of Cacna2d1 gene. The results confirmed that miR-107 binds to site 200-207 of Cacna2d1 3’UTR.