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A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells
Published on: June 16, 2022
[The Targeted Regulating Role of Has-miR-577 and Has-miR-583 on Gene FGF-21 Detected by the Dual Luciferase Reporter
Mei Zhang1, He He1, Hao-Lan Song1
1Department of Laboratory Medicine/Clinical Research Center of Laboratory Medicine,West China Hospital,Sichuan University,Chengdu 610041,China.
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.
