Identification of cellular genes showing differential expression associated with hepatitis B virus infection

Figure 1 Effects of short hairpin RNA targeting hepatitis B virus on huH-1 cells. A sequence of nineteen nucleotides that is shared by all four open reading frames of HBV was cloned into pSUPER.retro and named pSUPER.HB4, which was further mutated at the ninth A to G and designated pSUPER.HB4G. Stable transformants of each plasmid in huH-1 cells were established as huHpS, huHB4 and huHB4G, respectively. A: Representative morphologies (upper panel) and average doubling times (lower graph) of each transformant. Each stable transformant was maintained at 37 °C with 5% CO₂ in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and microscopically observed after 48 h (original magnification: 10x). The cells were counted on days 2 and 4 to calculate the average doubling times of each cell line from three independent cultures (40.9 ± 1.8 h, 46.8 ± 2.6 h and 45.1 ± 1.2 h in huHpS, huHB4G, and huHB4, respectively). The observed doubling times were not significantly different between huHpS and huHB4 but were different between huHpS and huHB4G (P = 0.022); B: The concentrations of HBsAg in the culture medium from huHpS, huHB4G, and huHB4 were 0.44 ± 0.046 IU/mL, 0.48 ± 0.091 IU/mL and 0.010 ± 0.0016 IU/mL on day 2 and 1.16 ± 0.11, 1.86 ± 0.26, and 0.050 ± 0.036 IU/mL on day 4, respectively. On both days, HBsAg was significantly reduced in huHB4 compared with the other two clones, but it was increased in huHB4G compared with huHpS (P = 0.0001 and P < 0.0001, respectively). C: Immunohistochemistry for HBx protein in HBx transgenic mouse liver 48 h after hydrodynamic gene delivery of pSUPER.retro, pSUPER.HB4G or pSUPER.HB4. The positive signals were remarkably reduced in mice that received pSUPER.HB4 compared with mice that received the other vectors, but were rather overrepresented in pSUPER.HB4G compared with pSUPER.retro (original magnification: 4x).