ABSTRACT
Objective: To verify whether EFNB2 gene is targeted by miRNA-497 using molecular biology methods. Methods: Bioinformatic method predicted that EFNB2 gene is targeted by miRNA-497. PCR methods amplified the fragment in EFNB2 gene 3'-UTR including the putative miRNA-497 binding site. Then the sequences of wide type (WT) and mutant (MUT) were cloned into the pmirGLO luciferase vector, respectively. The DNA sequences of the amplified fragments were identified by restriction enzyme digestion and sequencing, and were consistent with the reference sequence from UCSC. This constructed vector was marked as pmirGLO-EFNB2 vector. Finally, the pmirGLO vector, the pmirGLO-EFNB2 vector, miRNA-497 mimics and negative control (NC) were divided into five groups and transfected into HEK393T cells, and the luciferase activity was tested after 24 h and 48 h by dual luciferase reporter gene assay. Results: The results of DNA sequencing demonstrated that the PCR fragment was successfully cloned into pmirGLO vector. The transfection results showed that the recombinant plasmid of WT and MUT was successfully transfected into HEK293T. The results of dual luciferase activity assay demonstrated that miRNA-497 significantly decreased the reporter gene activity compared with the NC. Conclusion: At the cellular level, the schizophrenia susceptibility gene EFNB2 was verified to be targeted by miRNA-497, which provides a new idea and new clue for subsequent studies on miRNA-497 function in the molecular mechanism of schizophrenia.