Figure 5

HBx promotes CREB-mediated activation of miR-3188 and downregulates ZHX2. (a) The results indicated that HBx and miR-3188 expression was upregulated and ZHX2 expression was downregulated in 150 HCC tissues, and HBx was strongly associated with miR-3188 (R² =0.819, P<0.01) and ZHX2 expression (R²=0.703, P<0.01) (b) Western blot analysis of HBx and ZHX2 expression in four pairs of HCC tumor tissues (T) and their corresponding adjacent nontumor liver tissues (N). (c) The miR-3188 expression of liver tissues were significant higher in HBV transgenic mice than those in C57BL/6 mice (n=3 each group). (d) qRT-PCR analysis for miR-3188 and western blotting analysis for ZHX2 in HepG2 and HepG2.2.15 cells with or without HBx inhibition. (e) qRT-PCR analysis for miR-3188 and western blotting analysis for ZHX2 in HepG2 and SMMC-7721 cell lines transfected with HBx at different doses and controls. (f) HepG2-HBx and SMMC7721-HBx cells were transfected with siCREB or control siRNA and analyzed for miR-3188 expression by qRT-PCR and for ZHX2 expression by western blotting. (g) HepG2 and SMMC7721 cells were transfected with CREB and analyzed for miR-3188 expression by qRT-PCR and for ZHX2 expression by western blotting. (h) Luciferase assay of the putative miR-3188 promoter in HepG2 and SMMC7721 cells transfected with HBx and controls. (i) ChIP analysis showed that HBx promoted CREB occupying on the putative miR-3188 promoter in HepG2 and SMMC7721 cells. All experiments were performed in triplicate. *P<0.05, **P<0.01