|
|
|
| Construction of SOCS3 3′-UTR luciferase reporter vector and identification of its activity |
| (1.Department of Microbiology and Immunology, Xuzhou Medical College, Xuzhou Jiangsu 221002; 2.Department of Microbiology and Immunology, Nanjing Medical University, Nanjing Jiangsu 210029, China) |
|
|
|
|
Abstract [Abstract]Objective: To study the microRNA(miRNA) targeting to 3′untranslated region(3′UTR) of suppressors of cytokine signaling3(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 (pGL3Promoter), then the recombinant pGL3Promoter/SOCS3 was identified. The miRNAs targeting SOCS3 3′UTR was predicted by Target Scan5.2, RNAhybrid and FINDTAR3 softwares. Thereafter, pGL3Promoter/SOCS3 and miRNAs were cotransfected 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 pGL3Promoter reporter vector, which verified by Xba I digestion and DNA sequencing. The predicted miRNAs targeting SOCS3 3′UTR included miR203, miR291a5p, miR9, miR140 and miR130b. Compared with the control group, the luciferase activity of pGL3Promoter/SOCS3 treated with miR203, miR291a5p, or miR140 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 miR20, miR291a5p or miR140.
|
|
|
|
|
|
|
|
|
2012, Vol. 22 
