Construction of SOCS3 3′-UTR luciferase reporter vector and identification of its activity

Objective: To study the microRNA(miRNA) targeting to 3′-untranslated region(3′-UTR) of suppressors of cytokine signaling-3(SOCS3) gene,a SOCS3 3′-UTR luciferase reporter vector was constructed and to analyse the miRNAs regulated SOCS3 expression through detecting the luciferase activity of SOCS3 3′-UTR.Methods: The 3′-UTR fragment of SOCS3 gene was amplified by PCR from genomic DNA of primary astrocytes of mouse and cloned into luciferase reporter vector(pGL3-Promoter),then the recombinant pGL3-Promoter/SOCS3 was identified.The miRNAs targeting SOCS3 3′-UTR was predicted by Target Scan5.2,RNAhybrid and FINDTAR3 softwares.Thereafter,pGL3-Promoter/SOCS3 and miRNAs were co-transfected into HEK 293T cells and the luciferase activity of SOCS3 3′-UTR was measured.Results: 3′-UTR fragment of SOCS3 gene was successfully cloned into the pGL3-Promoter reporter vector,which verified by Xba I digestion and DNA sequencing.The predicted miRNAs targeting SOCS3 3′-UTR included miR-203,miR-291a-5p,miR-9,miR-140 and miR-130b.Compared with the control group,the luciferase activity of pGL3-Promoter/SOCS3 treated with miR-203,miR-291a-5p,or miR-140 was remarkably decreased,respectively.Conclusion: The SOCS3 3′-UTR luciferase reporter vector was constructed successfully,and the luciferase activity of the recombinant vector can be suppressed significantly by miR-20,miR-291a-5p or miR-140.