[The Targeted Regulating Role of Has-miR-577 and Has-miR-583 on Gene FGF -21 Detected by the Dual Luciferase Reporter System.]

OBJECTIVES: To determine the targeted regulating role of has-miR-577 and has-miR-583 on the expression of fibroblast growth factor 21 ( FGF -21) based on a constructed luciferase reporter FGF -21 gene vector.

METHODS: The site of has-miR-577 and has-miR-583 target genes FGF -21 were predicted by the bioinformatics analyzing tools online. FGF -21 gene fragments,combined with has-miR-577 or has-miR-583 sequences and mutant sequences,were designed and synthesized.The wild type (psiCHECK2- FGF -21) and mutant (psiCHECK2- FGF -21-mut) luciferase reporter gene carriers were constructed.The relevant plasmids [hsa-miR-577mimics,hsa-miR-583 mimics or miR negative control (miR-NC)] and luciferase reporter gene carrier (wild type or mutant ) were co-transfected into 293T cells.The luciferase reporter system was used to detect the luciferase activity.The effects of has-miR-577 and has-miR-583 on the expression of FGF -21 were observed.

RESULTS: The double enzyme electrophoresis and sequencing results showed that the gene fragment size and sequences of the wild type (psiCHECK2- FGF -21) and mutant (psiCHECK2- FGF -21-mut) carriers met expectations of the experiment.The luciferase assays revealed that has-miR-577 and has-miR-583 significantly diminished luciferase activity from the reporter vector containing 3'UTR of FGF -21 ( P <0.05),whereas no suppression of luciferase activity was found in the mutant (psiCHECK2- FGF -21-mut).

CONCLUSIONS: FGF -21 gene can be targeted by has-miR-577 and has-miR-583.