Construction and identification of human human runt-related transcription factor 2 P2 promoter luciferase reporter gene vector

Objective To construct a luciferase reporter gene vector containing human human runt-related transcription factor 2 (RUNX2) P2 promoter region and test its expression in human aortic valve interstitial cells,and to study the effect of bone morphogenic protein-7 (BMP-7) on RUNX2 promoter,for further investigation of RUNX2 expression regulation in human aortic valve calcification.Methods The RUNX2 P2 promoter region was cloned from human genomic DNAs using polymerase chain reaction.Products of PCR were digested by KpnⅠ and NheⅠ,and inserted into a luciferase reporter pGL3-Basic vector.The correctness of the recombinant plasmid pGL3-RUNX2-P2 was confirmed by sequencing.Human aortic valve interstitial cells were transfected with pGL3-RUNX2-P2 and treated with BMP-7.The relative luciferase activity was detected.Results A period of 1.5 kbp of RUNX2 P2 promoter region were successfully cloned into pGL3-Basic,which was confirmed by DNA sequencing.The relative luciferase activity was significantly higher in the cells transfected with pGL3-RUNX2-P2 than that in the cells transfected with the empty vector pGL3-Basic (0.273 ± 0.032 vs.0.028 ± 0.005,P ＜ 0.05).BMP-7 upregulated the luciferase activity (0.474 ± 0.059 vs.0.273 ± 0.032,P ＜ 0.05).Conclusion A luciferase reporter gene vector containing human RUNX2 P2 promoter region was successfully constructed,providing a powerful tool for further investigation of RUNX2 expression regulation in human aortic valve calcification.BMP-7 upregulates RUNX2 promoter activity. Key words: Human runt-related transcription factor 2 gene ;  Bone morphogenic protein-7 ;  Luciferase ;  Reporter vector