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。
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 psiCHECK-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 negative control or miR-199a-5p inhibitor negative control was co-transfected into the 293 T 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 digestion and DNA sequencing showed that the sequence of luciferase reporter vector was correct.The results of dual-luciferase reporter assay indicated that the H19 reporter gene luciferase activity significantly decreased in miR-199a-5p mimics groupby 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 H19-Mut reporter gene.CONCLUSION：lncRNA-H19 binds to miR-199a-5p to exert an inhibitory effect at transcriptional level.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2016年第12期2256-2260,共5页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.81270213
No.81070125
No.81670306)
广东省科技计划(No.2010B031600032
No.2014A020211002)
高校基本科研业务费中山大学青年教师重点培育项目(No.13ykzd16)
广东省医学科研基金资助项目(No.A2016264)
