Identification of targeting relationship between mouse lncRNA-H19 and miR-199 a-5 p by dual-luciferase reporter assay / 中国病理生理杂志
Chinese Journal of Pathophysiology
;
(12): 2256-2260, 2016.
Artigo
em Chinês
| WPRIM
| ID: wpr-506556
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 .
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Tipo de estudo:
Estudo diagnóstico
Idioma:
Chinês
Revista:
Chinese Journal of Pathophysiology
Ano de publicação:
2016
Tipo de documento:
Artigo
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