Abstract:

Objective  To construct a series of neprilysin (NEP) gene promoter deletion mutant-luciferase reporter plasmids and explore sites in the NEP promoter region that are related to the regulation of the NEP promoter activity by ginsenoside Rb1. Methods  The NEP promoter-luciferase reporter gene plasmid pGL3-nep2.4 was constructed by using a 2.4kb DNA fragment upstream of the NEP gene and the luciferase reporter vector pGL3-basic. A series of 5′ NEP promoter deletion mutant luciferase reporter plasmids derived from pGL3-nep2.4 were obtained via Erase-a-Base System. Human neuroblastoma SH-SY5Y cells transfected with the mutants were treated with Rb1, and the NEP promoter activities in them were determined by dual-luciferase reporter assay. Results  The recombinant pGL3-nep2.4 plasmid containing the NEP promoter was successfully constructed. Dual-luciferase reporter assay showed that the luciferse activity in SH-SY5Y cells transfected with pGL3-nep2.4 was 13.1 times of that in the cells transfected with pGL3-basic(P<0.01). After SH-SY5Y cells transfected with pGL3-nep2.4 were treated with Rb1, the luciferase activity in them was 2.9 times of that in the control cells (P<0.01). Dual-luciferase reporter assay also showed that the NEP promoter activity decreased to 29% (P<0.01) or 25%  (P<0.01) of that in the corresponding control group after the -894  to -857 bp (Region Ⅰ) or -100  to -82bp (Region Ⅳ) region upstream of the NEP gene was deleted, whereas it increased to 5.12 (P<0.01) or 1.81 times (P<0.01) of that in the corresponding control group after the -559  to -534bp (Region Ⅱ) or -223  to -179bp (Region Ⅲ) region upstream of the NEP gene was deleted, respectively. After Rb1 treatment, the luciferase activity in SH-SY5Y cells transfected with the mutant deleting Regions Ⅰ, Ⅱ or Ⅲ was no different from that in the control cells (P>0.05), and in SH-SY5Y cells transfected with the mutant pGL3-244 containing Region Ⅳ, it was 1.61 times of that in the control cells (P<0.01) while in SH-SY5Y cells transfected with the mutant pGL3-226 containing no Region Ⅳ, it was different from that in the control cells (P>0.05). Conclusion  The 2.4kb DNA fragment upstream of the NEP gene has the strong promoter activity that could be increased by Rb1. Two positive (Region Ⅰ and Ⅳ) and two negative (Region Ⅱ and Ⅲ) regulatory regions are identified in the NEP promoter, and the increase of the NEP promoter activity caused by Rb1 is related to Region IV.

Key words: Ginsenoside Rb1; Neprilysin; Promoter