用双萤光素酶报告基因技术验证小鼠 lncRNA-H19与 miR-199a-5p 的靶向关系

摘要

目的：构建长链非编码RNA-H19（ lncRNA-H19）萤光素酶报告质粒，利用双萤光素酶报告基因技术验证小鼠lncRNA-H19与微小RNA-199a-5p（miR-199a-5p）的靶向关系。方法：通过生物信息学网站RegRNA 2.0预测获取小鼠lncRNA-H19与miR-199a-5p潜在的互补结合位点。将H19及其突变体克隆到萤光素酶载体psiCHECK-2中，构建H19野生型和突变型质粒，并采用酶切和测序方法鉴定psiCHECK-2-H19载体是否构建成功。将H19野生型和突变型质粒分别与miR-199a-5p模拟物、miR-199a-5p抑制剂、miR-199a-5p 模拟物阴性对照或miR-199a-5p抑制剂阴性对照在293T细胞中共转染。收集细胞后通过双萤光素酶报告系统检测不同组别的萤光素酶活性，从而对lncRNA-H19与miR-199a-5p的靶向调节关系进行验证。结果：构建的重组萤光素酶报告质粒经酶切及测序鉴定正确，双萤光素酶报告基因检测显示，与miR-199a-5p模拟物阴性对照组相比，miR-199a-5p模拟物组H19野生型报告基因的萤光素酶活性显著降低，下降约49％左右（P＜0.01），而miR-199a-5p 抑制剂组H19野生型报告基因的萤光素酶活性较miR-199a-5p模拟物组明显增高（P＜0.01）。 miR-199a-5p模拟物、miR-199a-5p抑制剂、miR-199a-5p模拟物阴性对照以及miR-199a-5p 抑制剂阴性对照对H19突变型的萤光素酶活性均无明显影响。结论：lncRNA-H19能够靶向结合miR-199a-5p，并在转录后水平对其有直接抑制作用。%AIM:To validate the association between long noncoding (lncRNA)-H19 and microRNA-199a-5p (miR-199a-5p) through the dual-luciferase reporter gene system by construction of a luciferase reporter vector containing the gene of lncRNA-H19.METHODS:The potential complementary binding sites of lncRNA-H19 and miR-199a-5p were predicted by RegRNA 2.0.The H19 gene or its mutant ( Mut) fragment was cloned into luciferase reporter vector psi-CHECK-2.Restriction enzyme analysis and sequence analysis were used to identify whether the recombinant plasmids of the H19 and H19-Mut were successfully constructed .miR-199a-5p mimics, miR-199a-5p inhibitor, miR-199a-5p mimics neg-ative control or miR-199a-5p inhibitor negative control was co-transfected into the 293T cells with the luciferase reporters containing H19 or H19-Mut.Dual-luciferase reporter assay was performed to detect the luciferase activity in different groups in order to verify the relationship between lncRNA-H19 and miR-199a-5p.RESULTS:The results of double enzyme diges-tion and DNA sequencing showed that the sequence of luciferase reporter vector was correct .The results of dual-luciferase reporter assay indicated that the H 19 reporter gene luciferase activity significantly decreased in miR-199a-5p mimics group by 49%(P<0.01), and the H19 reporter gene luciferase activity was obviously upregulated in miR-199a-5p inhibitor group compared with miR-199a-5p mimics group ( P<0.01).However, miR-199a-5p mimics, miR-199a-5p inhibitor, miR-199a-5p mimics negative control and miR-199a-5p inhibitor negative control showed no effect at H 19-Mut reporter gene.CONCLUSION:lncRNA-H19 binds to miR-199a-5p to exert an inhibitory effect at transcriptional level .