Fig. 5

Impact of HBx-targeted miRNAs on 3.5Kb/pgRNA transcription and HBV replication. a HepG2 cells are co-transfected with linear wild type HBV genomes (HBV) and the indicated pre-hsa-miRNAs. HBV 3.5Kb/pgRNA levels were determined by real-time qPCR using specific primers. Results were expressed as fold induction relative to mock controls (Ctl) after normalization towards endogenous human β-actin mRNAs. b rcHBV-DNA levels associated to cytoplasmatic core particles were determined by real-time qPCR after normalization to β-globin. Histograms represent the mean from 3 independent experiments; bars indicate SDs. P values were determined using the Student’s t test. *P < 0.05. c DNMT3A and p65 recruitment on miR224 promoter. ChIPed chromatin from mock and HBV-wt replicating HepG2 cells. Results are expressed as in Fig. 4a. d Left panel. Putative regulatory region (−2500/+1000 from TSS) and schematic representation of the luciferase construct containing the miR-224 promoter region. The sequence was analyzed by motif software (http://www.genome.jp/tools/motif/) using a cut off score of 85%. Black diamonds represent p53 binding sites; white circles represent NFkB consensus; white triangles represent AP1 consensus; black rectangles represent TCF/LEF consensus and grey star represents HNF-3β consensus. Right panel. HepG2 cells were transfected with 250 ng of the −1841/+1035 miR-224 luciferase construct together with 250 ng of HBx expression vector. Luciferase activity was assayed 30h after transfection and expressed as fold induction over the control. Histograms represent the mean of 3 independent experiments each performed in duplicate; bars indicate S.D. P-values: * 0,02 ≤ P < 0,05; ** 0,01 ≤ P < 0.02; *** 0,005 ≤ P < 0.01