ABSTRACT
Objective To verify whether early growth response-3(EGR3) gene is targeted by microRNA-181b using molecular biology methods so as to provide guidance for the subsequent study on microRNA-181b`s role in the molecular mechanisms of schizophrenia.Methods Bioinformatic methods predicted that EGR3 gene is targeted by microRNA-181b.PCR methods amplified the fragment in EGR3 gene 3`UTR including the putative microRNA-181b binding site.Then the sequence was cloned into the pmirGLO luciferase vector.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-EGR3 vector.Finally, the pmirGLO vector, the pmirGLO-EGR3 vector, microRNA-181b mimics and negative control (NC) were divided into 5 groups and transfected into HEK393T cells;the luciferase activity was tested by dual luciferase reporter gene assay.Results The results of restriction enzyme digestion and sequencing demonstrated that the PCR fragmentwas successfully cloned into pmirGLO vector.The transfection results showed that the recombinant plasmid was successful transfected into HEK293T under the fluorescence microscope, with transfection efficiency being about 90％.The results of dual luciferase activity assay demonstrated that microRNA-181b significantly decreased the reporter gene`s activity compared with the NC.Conclusion At the cellular level, the schizophrenia susceptibility gene EGR3 was verified to be targeted by micorRNA-181b, which provides a new clue for the subsequent study on microRNA-181b`s role in the molecular mechanisms of schizophrenia.