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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RegRNA2.0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psi CHECK-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psi CHECK-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。
OBJECTIVE: To construct long-chain non-coding lncRNA-H19 luciferase reporter plasmids and to validate the relationship between lncRNA-H19 and microRNA-199a-5p (miR-199a-5p ) Targeting relationship. Methods: The potential complementary binding sites of mouse lncRNA-H19 and miR-199a-5p were predicted by bioinformatics website RegRNA2.0. H19 and its mutants were cloned into the luciferase vector psi CHECK-2 to construct H19 wild-type and mutant plasmids and digested and sequenced to identify whether the psi CHECK-2-H19 vector was successfully constructed. The H19 wild type and mutant plasmids were co-transformed in 293T cells with miR-199a-5p mimic, miR-199a-5p inhibitor, miR-199a-5p mock negative or miR-199a-5p inhibitor negative dye. The luciferase activity of different groups was detected by dual luciferase reporter system after collecting the cells, and the regulation of the relationship between lncRNA-H19 and miR-199a-5p was verified. Results: The constructed recombinant luciferase reporter plasmid was identified by restriction enzyme digestion and sequencing. The dual luciferase reporter assay showed that the miR-199a-5p mimic The luciferase activity of H19 wild-type reporter gene was significantly decreased by about 49% (P <0.01), while the luciferase activity of H19 wild-type reporter gene in miR-199a-5p inhibitor group was lower than that of miR-199a -5p mimics group was significantly higher (P <0.01). The miR-199a-5p mimics, miR-199a-5p inhibitor, miR-199a-5p mimic negative control and miR-199a-5p inhibitor negative control had no significant effect on the H19 mutant luciferase activity. Conclusion: lncRNA-H19 can target and bind to miR-199a-5p directly and directly at the post-transcriptional level.