HCBP6 Modulates Triglyceride Homeostasis in Hepatocytes Via the SREBP1c/FASN Pathway.

Hypertriglyceridemia leads to liver steatosis, cardiovascular disease, and type 2 diabetes. Although HCBP6 (hepatitis C virus core-binding protein 6) was previously shown to be an HCV (hepatitis C virus) core-binding protein, its biological function remains unclear. Here, we demonstrate that HCBP6 negatively regulates intracellular triglyceride (TG) levels in hepatocytes. We found that bidirectional manipulation of hepatocyte HCBP6 expression by knockdown or overexpression results in increased or decreased TG accumulation, respectively. In addition, HCBP6 mRNA and protein levels exhibited significant time- and dose-dependent increases in a cellular model of lipid-overload hepatic steatosis. Furthermore, TG levels are regulated by HCBP6-sterol regulatory element binding protein 1c (SREBP1c)-mediated fatty acid synthase (FASN) expression. We also demonstrate that HCBP6 mRNA and protein expression is inhibited by microRNA-122 (miR-122), and miR-122 overexpression elicited more robust translational repression of luciferase activity driven by the full 3'-UTR of HCBP6. Taken together, our results provide new evidence that miR-122-regulated HCBP6 functions as a sensor protein to maintain intrahepatocyte TG levels.

MicroRNA-185-5p mediates regulation of SREBP2 expression by hepatitis C virus core protein.

AIM: To investigate the molecular mechanism for regulation of cholesterol metabolism by hepatitis C virus (HCV) core protein in HepG2 cells. METHODS: HCV genotype 1b core protein was cloned and expressed in HepG2 cells. The cholesterol content was determined after transfection. The expression of sterol regulatory element binding protein 2 (SREBP2) and the rate-limiting enzyme in cholesterol synthesis (HMGCR) was measured by quantitative real-time PCR and immunoblotting after transfection. The effects of core protein on the SREBP2 promoter and 3'-untranslated region were analyzed by luciferase assay. We used different target predictive algorithms, microRNA (miRNA) mimics/inhibitors, and site-directed mutation to identify a putative target of a particular miRNA. RESULTS: HCV core protein expression in HepG2 cells increased the total intracellular cholesterol level (4.05 ± 0.17 vs 6.47 ± 0.68, P = 0.001), and this increase corresponded to an increase in SREBP2 and HMGCR mRNA levels (P = 0.009 and 0.037, respectively) and protein expression. The molecular mechanism study revealed that the HCV core protein increased the expression of SREBP2 by enhancing its promoter activity (P = 0.004). In addition, miR-185-5p expression was tightly regulated by the HCV core protein (P = 0.041). Moreover, overexpression of miR-185-5p repressed the SREBP2 mRNA level (P = 0.022) and protein expression. In contrast, inhibition of miR-185-5p caused upregulation of SREBP2 protein expression. miR-185-5p was involved in the regulation of SREBP2 expression by HCV core protein. CONCLUSION: HCV core protein disturbs the cholesterol homeostasis in HepG2 cells via the SREBP2 pathway; miR-185-5p is involved in the regulation of SREBP2 by the core protein.

[Identification of HCV core protein binding proteins by yeast two-hybrid].

OBJECTIVE: To identify HCV core protein binding proteins. METHODS: The library was amplified, purified, and then were transformed into yeast strain Y187. The reconstructed plasmid pGBKT7-core was transformed into yeast strain AH109 and screened on the nutrient deficiency medium SD/-Trp. The transformed AH109 mated with Y187 containing the library plasmid. The diploid yeast cells were plated on nutrient deficiency medium SD/-Trp/-Leu/-His/-Ade and SD/-Trp/-Leu/-His/-Ade containing X-alpha-gal for selecting. The plasmids in diploid yeast cells were extracted and electrotransformed into E.coli DH5alpha. The plasmids in DH5alpha were extracted and sequenced. RESULTS: Eleven proteins, including chymotrypsinogen B1 precursor, carboxypeptidase A1, trypsinogen 2, chymotryptic peptide C, trypsin 1, carboxypeptidase B1, kinesin superfamily proteins 3B, trypsin 2, mitochondria protein gene, elastase 3A and colipase were found to be able to bind to HCV core protein. CONCLUSIONS: Proteins related with metabolism of glucose and lipid may bind to HCV core protein.

[Early prediction of lamivudine response in e antigen-negative chronic hepatitis B patients].

OBJECTIVE: To evaluate the effects of ALT, HBsAg and HBV DNA at the baseline, 4 and 12 weeks after lamivudine treatment on the long term (104 weeks) response in e antigen-negative chronic hepatitis B patients. METHODS: 127 adult e antigen-negative chronic hepatitis B patients were enrolled in this study. All patients received treatment on LAM 100 mg/d for at least 104 weeks. The liver function, serum HBV markers and HBV viral load were regularly checked during the treatment. The effects of ALT, HBsAg and HBV DNA at the baseline, 4 and 12 weeks after lamivudine treatment on the response at 104 weeks were statistically analyzed. RESULTS: The proportion of patients with serum HBV DNA less than 1000 copies / ml at 104 weeks after LAM treatment was 50.0% and 86.8% in patients with baseline ALT less than 5 ULN and ALT is more than or equal to 5 ULN, respectively (P less than 0.01). In patients with baseline HBsAg less than 2000 COI and HBsAg is more than or equal to 2000 COI, the proportion of patients with serum HBsAg less than 500 COI at 104 weeks after LAM treatment was 19.1% and 17.5%, respectively (P more than 0.05). the HBsAg serological conversion rates were respectively 2.1% and 2.5% , respectively (P more than 0.05), the proportion of patients with serum HBV DNA less than 1000 copies/ml was 61.7% and 67.5%, respectively (P more than 0.05). In patients with baseline HBV DNA less than 10(6) copies/ml and HBV DNA is more than or equal to 10(6) copies/ml, the proportion of patients with HBV DNA less than 1000 copies/ml were statistically different at 4 weeks and 12 weeks after treatment, however, the proportion of patients with HBV DNA less than 1000 copies/ml at 104 weeks after LAM treatment was 62.7% and 67.1%, respectively (P more than 0.05). In patients with HBV DNA less than 1000 copies/ml and HBV DNA is more than or equal to 1000 copies/ml at 4 weeks after treatment, the proportion of patients with HBV DNA less than 1000 copies/ml at 104 weeks after LAM treatment was 70.7% and 60.9%, respectively (P more than 0.05). In patients with HBV DNA less than 1000 copies/ml and HBV DNA is more than or equal to 1000 copies/ml at 12 weeks after treatment, the proportion of patients with HBV DNA less than 1000 copies/ml at 104 weeks after treatment was 78.8% and 38.1%, respectively (P less than 0.01). CONCLUSION: e antigen negative chronic hepatitis B patients with baseline ALT is more than or equal to 5 ULN and HBV DNA less than 1000 copies/ml at 12 weeks after treatment have better virological response at 104 weeks after LAM treatment. The baseline HBsAg and HBV DNA load are associated with the virological response at 104 weeks after LAM treatment.