Hepatitis B virus inhibits the in vivo and in vitro synthesis and secretion of apolipoprotein C3

Abstract

Background: Hepatitis B virus (HBV) infection in the body can damage liver cells and cause disorders in blood lipid metabolism. Apolipoprotein C3 (ApoC3) plays an important role in the regulation of lipid metabolism, but no study on the HBV regulation of ApoC3 has been reported. This purpose of this study was to investigate the effect of HBV on ApoC3 expression and its regulatory mechanism.

Methods: The expression levels of ApoC3 mRNA and protein in the human hepatoma cell lines HepG2 and HepG2.2.15 were determined using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot. The HepG2 cells were co-transfected with the ApoC3 gene promoter and either HBV-infected clone pHBV1.3 or its individual genes. The changes in luciferase activity were assayed. The expression levels of ApoC3 mRNA and protein were determined using RT-qPCR and Western blot. The content of ApoC3 in the supernatant of the cultured cells was determined using an enzyme-linked immunosorbent assay (ELISA). The sera were collected from 149 patients with HBV infection and 102 healthy subjects at physical examination as the normal controls. The serological levels of ApoC3 in the HBV group and the normal control group were determined using ELISA. The contents of serum triglyceride (TG) and very-low-density lipoprotein (VLDL) in the HBV patients and the normal control were determined using an automatic biochemical analyser.

Results: The expression levels of ApoC3 mRNA and protein were lower in the HepG2.2.15 cells than in the HepG2 cells. pHBV1.3 and its X gene could inhibit the activity of the ApoC3 promoter and its mRNA and protein expression. The serum levels of ApoC3, VLDL and TG were 65.39 ± 7.48 μg/ml, 1.24 ± 0.49 mmol/L, and 0.46 ± 0.10 mmol/L in the HBV patients and 41.02 ± 6.88 μg/ml, 0.76 ± 0.21 mmol/L, 0.29 ± 0.05 mmol/L in the normal controls, respectively, statistical analysis revealed significantly lower serum levels of ApoC3, VLDL and TG in HBV patients than in the normal controls (P < 0.05).

Conclusion: HBV can inhibit the in vivo and in vitro synthesis and secretion of ApoC3.

Keywords: Apolipoprotein C3; Hepatitis B virus; Triglyceride; Very-low-density lipoprotein.

Fig. 1

Effect of HBV on ApoC3 mRNA and protein expression levels in HepG2 cells. a The total RNA of HepG2 and HepG2.2.15 cells was extracted, and the ApoC3 mRNA level was quantitatively determined by RT-qPCR; b The expression levels of ApoC3 protein in HepG2 and HepG2.2.15 were determined using Western blot; c The contents of ApoC3 in the supernatants of HepG2 and HepG2.2.15 cells were determined by ELISA. *P < 0.005

Fig. 2

Effect of pHBV1.3 on the promoter activity and mRNA and protein expression levels of ApoC3. a At 48 h after 0.6 μg of pHBV1.3 and its control plasmid pBlue-ks were each co-transfected with 0.2 μg of ApoC3 gene promoter pApoC3-Luc into HepG2 cells, the changes in luciferase activity were determined using a luminometer; each experiment was repeated three times; b at 48 h after 4 μg of pHBV1.3 and its control plasmid pBlue-ks were each transfected into HepG2 cells, the effect of HBV on the expression of ApoC3 mRNA was determined by RT-qPCR; c The expression levels of ApoC3 protein in HepG2 and HepG2.2.15 cells were determined by Western blot; d The content of ApoC3 in the supernatant of HepG2 cells was determined by ELISA. *P < 0.005

Fig. 3

HBV inhibited ApoC3 expression through its X gene. a HepG2 cells were transfected with pCMV-S, pCMV-E, pCMV-C, pCMV-X, pCMV-P and pCMV-tag2B, the changes in luciferase activity were determined using a luminometer at 48 h post-transfection, each experiment was repeated three times. b HepG2.2.15 cells were transfected with the HBX siRNA or the negative control siRNA(siRNA-NC) for 24 h. ApoC3 mRNA level was quantitatively determined by RT-qPCR; c The expression levels of ApoC3 protein in HepG2.2.15 cells were determined by Western blot; d The content of ApoC3 in the supernatant of HepG2.2.15 cells was determined by ELISA. *P < 0.005

Fig. 4

Measurement of ApoC3, TG and VLDL in HBV patients and normal controls. a Comparison of the serum levels of ApoC3 in HBV patients and normal controls; b Comparison of the serum levels of TG in HBV patients and normal controls; c Comparison of the serum levels of VLDL in HBV patients and normal controls. *P < 0.005