Role of STAT1 in the resistance of HBV to IFN-α

Abstract

The objective of the present study was to explore the mechanism of hepatitis B virus (HBV) resistance to interferon (IFN), and the role of signal transducer and activator of transcription 1 (STAT1). HepG2.2.15 cells were stimulated with a long-term (6-24 weeks) low-dose interferon (IFN)α-2b (10-70 IU/ml), so as to construct and screen a HepG2.2.15 cell model resistant to IFNα-2b. The changes of STAT1 and other proteins in the JAK-STAT signaling pathway, before and after drug resistance, were compared. The phosphorylation of STAT1 in HepG2.2.15 cells resistant to IFNα-2b was significantly decreased, and the expression level of 2',5'-oligoadenylate synthetase 1 was downregulated. Decreased phosphorylation of STAT1 in the JAK-STAT signaling pathway a contributor to the development of resistance to IFN-α in HBV.

Keywords: Hepatitis B virus; drug resistance; interferon α-2b; phosphorylation; signal transducer and activator of transcription 1.

Figure 1

Expression levels of STAT1 and p-STAT1 in HepG2.2.15 cells after long-term stimulation with a low dose of IFNα-2b. The control group, 12-week stimulated group and 24-week stimulated group were treated with (0, 250, 500 and 1,000 IU/ml) IFNα-2b for 72 h. The protein expression levels of STAT1 and p-STAT1 were detected via western blotting. Data were expressed as mean ± SD. Error bars were calculated from three independent experiments. Experiments were performed least in triplicate. The P-values were obtained using the One-way ANOVA followed by Tukey's post hoc test. ^**P<0.01 vs. control. p-, phosphorylated.

Figure 2

Effects of AG-490 and TSA on HBsAg, HBeAg and HBV DNA of IFNα-2b. The control and model groups were treated with 0, IFNα-2b (1,000 IU/ml), IFNα-2b (1,000 IU/ml) + AG-490 (1 µM), IFNα-2b (1,000 IU/ml) + TSA (30 nmol/ml), IFNα-2b (1,000 IU/ml) + AG-490 (1 µM) + TSA (30 nmol/ml) for 24 h. (A) The levels of HBsAg and HBeAg were detected using ELISA kits. (B) The amount of HBV DNA was detected by PCR-fluorescence probing. Data were expressed as the mean ± SD. Error bars were calculated from three independent experiments. Experiments were performed at least in triplicate. The P-values were obtained using one-way ANOVA followed by Tukey's post hoc test. ^*P<0.05; ^**P<0.01. TSA, Trichostatin A; HBsAg, hepatitis B surface antigen; HBeAg, hepatitis B e antigen; HBV, hepatitis B virus.

Figure 3

Effects of AG-490 and TSA on the expression levels of STAT1, p-STAT1 and OAS1 with IFNα-2b. The control and model groups were treated with 0, IFNα-2b (1,000 IU/ml), IFNα-2b (1,000 IU/ml) + AG-490 (1 µM), IFNα-2b (1,000 IU/ml) + TSA (30 nmol/ml), IFNα-2b (1,000 IU/ml) + AG-490(1 µM) + TSA (30 nmol/ml) for 24 h. Then, proteins were quantified via western blotting. Data were expressed as the mean ± SD. Error bars were calculated from three independent experiments. Samples were done at least in triplicate. The P-values were obtained using one-way ANOVA followed by Tukey's post hoc test. ^*P<0.05; ^**P<0.01. TSA, Trichostatin A; p-, phosphorylated-; OAS1, 2'-5'-oligoadenylate synthetase 1.

Figure 4

Effects of AG-490 and TSA on the mRNA expression level of STAT1, OAS1 and USP18. The control and model groups were treated with 0, IFNα-2b (1,000 IU/ml), IFNα-2b (1,000 IU/ml) + AG-490 (1 µM), IFNα-2b (1,000 IU/ml) + TSA (30 nmol/ml), IFNα-2b (1,000 IU/ml) + AG-490(1 µM) + TSA (30 nmol/ml) for 24 h. Then, the mRNA expression levels of STAT1, OAS1 and USP18 were quantified via reverse transcription-quantitative PCR. Data were expressed as the mean ± SD. Error bars were calculated from three independent experiments. Experiments were performed at least in triplicate. The P-values were obtained using one-way ANOVA followed by Tukey's post hoc test. ^**P<0.01. TSA, Trichostatin A; USP18, Ubiquitin-Specific Protease 18; OAS1, 2'-5'-oligoadenylate synthetase 1.