Interaction between miRNAs and Thyroid Hormone Receptor TRβ in Bastard Halibut Paralichthys olivaceus
LIU Suping, FU Yuanshuai, YU Jie, ZHANG Junling, SHI Zhiyi
Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture and Rural Affairs, Shanghai Engineering Research Center of Aquaculture, National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China
Abstract：Thyroid hormone (TH) plays an important role in regulating bastard halibut Paralichthys olivaceus metamorphosis through binding to its receptors (TRs), which is one of the key members of TRs, while miRNAs regulate the expression of target genes at the post-transcriptional level by binding to the 3'-untranslated region (3'-UTR) of target genes. We predicted that the binding site of multiple miRNAs might is in the 3'-UTR of TRβ gene in bastard halibut by bioinformatics. In order to further explore the relationship between miRNAs and TRβ, in this study, total RNAs from bastard halibut adults were extracted by the Trizol method. The 3'-UTR sequence of TRβ gene was cloned from the total RNA by 3'-RACE method; then the PCR primers for the desired restriction sites SacⅠ and XbaⅠ required for the recombinant plasmid containing dual-luciferase reporter gene were designed and synthesized according to the cloned 3'-UTR sequence of TRβ gene. After PCR amplification, the target 3'-UTR cDNA sequence of TRβ gene containing specific restriction enzyme site was obtained, whose gene fragment and pmirGLO vector were digested by double enzymes. The ligation reaction was performed with T4 DNA Ligase and transformed into DH5α competent cells. The recombinant plasmid was extracted, and then double enzyme digestion, agarose gel electrophoresis and sequencing validation were performed. The results showed that the dual-luciferase recombinant reporter vector was successfully constructed and named as pmirGLO-TRβ-3'-UTR; finally five predicted miRNAs (pol-miR-125a, pol-miR-214, pol-miR-460-5p, pol-miR-138 and pol-miR-125a*) were selected to examine the interaction with TRβ, five miRNA mimics and the recombinant plasmid pmirGLO-TRβ-3'-UTR constructed above were transfected into 293T cells, and the luciferase reporter gene system was used to detect the luminescence value. The analysis revealed that bastard halibut TRβ is target gene of pol-miR-125a, pol-miR-214, pol-miR-460-5p and pol-miR-138, but it is not a target gene of pol-miR-125a*. These findings provide the basis for the further study on the function and mechanism of miRNAs and TRβ in the metamorphosis of bastard halibut.
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2020, Vol. 39 
