Figure 11.

HIRA is required for the maintenance of cccDNA transcriptional activity. (A) Experimental timeline for HIRA knock-down after HBV infection. HepG2^hNTCP cells were infected for 16 hours and then extensively washed and transfected with siHIRA or siCTL at 4 dpi. Cells were harvested for analysis at 7 dpi. (B) HIRA messenger RNA (mRNA) and protein expression after siRNA transfection was determined by real-time qPCR and Western blot. β-actin served as Western blot loading control. (C) cccDNA and (D) 3.5-kb RNA amount was measured by qPCR and Northern blot at 7 dpi. cccDNA quantification was normalized over β-globin quantity, while relative 3.5-kb RNA amount was normalized over the housekeeping gene GUSb expression. (E) HepG2^hNTCP cells were transfected with siRNA against HIRA according to the timeline shown in Figure 2B and inoculated for 16 hours with HBV at 250 viral genome equivalents/cell. The cells were harvested for analysis at 7 dpi. mRNA levels of hepatocyte nuclear factor-1α (HNF1α), hepatocyte nuclear factor-4α (HNF4α), signal transducer and activator of transcription 1 and 2 (STAT1, STAT2), nuclear receptor farnesoid X (FXR), specificity protein 1 (SP1), peroxisome proliferator activated receptor α (PPARα), CAMP responsive element binding protein 1 (CREB1), and CCAAT/enhancer-binding protein α (C/EBPα) were quantified by real-time qPCR assay and expressed as a percentage of TRA-treated cells after normalization over GUSb housekeeping gene expression. Data represent the means ± SEM of at least 3 independent experiments. Graphs represent the means ± SEM of at least 3 independent experiments. The 2-tailed P value was calculated for a risk threshold of .05 using the 2/K sample permutation test with Monte Carlo resampling approximation. ∗∗P < .01.