Fig. 4.

7030B-C5 reduces PCSK9 expression transcriptionally in a HNF1α and FoxO3-responsive element-dependent manner. (a) The constructs of human PCSK9 promoter-luciferase reporters. Position +1 was designated as the nucleotide preceding the ATG start codon. Position −1 is the 3′ end of PCSK9 promoter inserts in common to all promoter-reporter constructs. The 5′ ends of the promoters in each promoter-reporter construct are marked by numbers at left, and the name of each construct is shown at right. (b) HepG2 cells were transfected with the PCSK9 promoter-luciferase reporter constructs PCSK9-P D1-D7 for 24 h and then treated with vehicle (0.1% DMSO) or 7030B-C5 for 24 h. (c and d) Deletion analysis of PCSK9 promoter suggested that HNF1/IRE and HINFP-bs might be the key cis-regulatory elements responsible for the regulation of PCSK9 by 7030B-C5. Mut-1 represents core nucleotide sequence of the HNF1/IRE site mutation, mut-2 represents core nucleotide sequence of the HINFP-bs site mutation. (e) HepG2 cells were treated with vehicle or 7030B-C5 in a series of concentrations for 24 h. Expression of HNF1α HINFP and FoxO3 protein were measured by Western blot. (f) HepG2 cells was transfected with siRNA negative control (siNC) or siRNA for the knockdown of FoxO3 (siFoxO3), HNF1α (siHNF1α), HINFP (siHINFP) and the level of FoxO3, HNF1α and HINFP protein was determined by Western blot analysis. (g) HepG2 cells were treated with 7030B-C5 (12.5 μM) for 24 h. Chromatin was isolated followed by immunoprecipitation with normal IgG, anti-HNF1α or anti-FoxO3 antibody. The PCR was conducted with primers for HNF1 or IRE in the PCSK9 promoter. (h) Regulation of PI3K/Akt pathway by 7030B-C5 in HepG2. Representative images are shown. Values are presented as means ± SEM (n = 3). *p < 0.05, ^#p < 0.05 vs. control in the corresponding group.