Identification of targeting relationship between mouse lncRNA-H19 and miR-199 a-5 p by dual-luciferase reporter assay
10.3969/j.issn.1000-4718.2016.12.022
- VernacularTitle: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
- Author:
Jingying HOU
;
Changqing ZHOU
;
Shaoxin ZHENG
;
Tianzhu GUO
;
Huibao LONG
;
Quanhua WU
;
Tingting ZHONG
;
Hao WU
;
Lei WANG
;
Tong WANG
- Keywords:
Long noncoding RNA-H19;
MicroRNA-199a-5p;
Dual-luciferase reporter gene
- From:
Chinese Journal of Pathophysiology
2016;32(12):2256-2260
- CountryChina
- Language:Chinese
-
Abstract:
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 .
