R Salpini1, M Surdo1, M F Cortese2, G A Palumbo3, L Carioti1, G Cappiello4, A Spanò4, P Trimoulet5, H Fleury5, J Vecchiet6, C Pasquazzi7, C Mirabelli8, R Scutari1, A Sacco1, M Alkhatib1, G Missale9, S Francioso10, L Sarmati11, M Andreoni11, M Angelico10, F Ceccherini-Silberstein1, M Levrero12, C F Perno13, L Belloni14, V Svicher15. 1. Department of Experimental Medicine and Surgery, University of Rome 'Tor Vergata' Rome, Italy. 2. Department of Experimental Medicine and Surgery, University of Rome 'Tor Vergata' Rome, Italy; Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain. 3. Department of Internal Medicine-DMISM, Sapienza University, Rome, Italy. 4. 'S. Pertini Hospital', Rome, Italy. 5. Hôpital Pellegrin Tripode, Bordeaux, France. 6. 'SS Annunziata' Hospital, Chieti, Italy. 7. 'S Andrea' Hospital, Rome, Italy. 8. Department of Experimental Medicine and Surgery, University of Rome 'Tor Vergata' Rome, Italy; University of Michigan Medical School, Ann Arbor, MI, USA. 9. Hospital of Parma, Parma, Italy. 10. Hepatology Unit, Tor Vergata University Hospital, Rome, Italy. 11. Infectious Diseases Unit, Tor Vergata University Hospital, Rome, Italy. 12. Department of Internal Medicine-DMISM, Sapienza University, Rome, Italy; INSERM U1052 - Cancer Research Centre of Lyon, 69008 Lyon, France. 13. Department of Experimental Medicine and Surgery, University of Rome 'Tor Vergata' Rome, Italy; Haematology and Oncohaematology, University of Milan, Italy. 14. Department of Internal Medicine-DMISM, Sapienza University, Rome, Italy; Centre for Life NanoSciences, IIT-Sapienza, Rome, Italy. 15. Department of Experimental Medicine and Surgery, University of Rome 'Tor Vergata' Rome, Italy. Electronic address: valentina.svicher@uniroma2.it.
Abstract
OBJECTIVE: We aimed to investigate HBx genetic elements correlated with hepatitis B virus (HBV) -related hepatocellular carcinoma (HCC) and their impact on (a) HBV replicative efficiency, (b) HBx binding to circular covalently closed DNA (cccDNA), (c) apoptosis and cell-cycle progression, and (d) HBx structural stability. METHODS: This study included 123 individuals chronically infected with HBV: 27 with HCC (77.9% (21/27) genotype D; 22.1% (6/27) genotype A) and 96 without HCC (75% (72/96) genotype D; 25.0% (24/96) genotype A). HepG2 cells were transfected by wild-type or mutated linear HBV genome to assess pre-genomic RNA (pgRNA) and core-associated HBV-DNA levels, HBx-binding onto cccDNA by chromatin immunoprecipitation-based quantitative assay, and rate of apoptosis and cell-cycle progression by cytofluorimetry. RESULTS: F30V was the only HBx mutation correlated with HCC (18.5% (5/27) in HCC patients versus 1.0% (1/96) in non-HCC patients, p 0.002); a result confirmed by multivariate analysis. In vitro, F30V determined a 40% and 60% reduction in pgRNA and core-associated HBV-DNA compared with wild-type (p <0.05), in parallel with a significant decrease of HBx binding to cccDNA and decreased HBx stability. F30V also decreased the percentage of apoptotic cells compared with wild-type (14.8 ± 6.8% versus 19.1 ± 10.1%, p <0.01, without affecting cell-cycle progression) and increased the probability of HBx-Ser-31 being phosphorylated by PI3K-Akt kinase (known to promote anti-apoptotic activity). CONCLUSIONS: F30V was closely correlated with HBV-induced HCC in vivo, reduced HBV replicative efficiency by affecting HBx-binding to cccDNA and increased anti-apoptotic HBx activity in vitro. This suggests that F30V (although hampering HBV's replicative capacity) may promote hepatocyte survival, so potentially allowing persistent production of viral progeny and initiating HBV-driven hepatocarcinogenesis. Investigation of viral genetic markers associated with HCC is crucial to identify those patients at higher risk of HCC, who hence deserve intensive liver monitoring and/or early anti-HBV therapy.
OBJECTIVE: We aimed to investigate HBx genetic elements correlated with hepatitis B virus (HBV) -related hepatocellular carcinoma (HCC) and their impact on (a) HBV replicative efficiency, (b) HBx binding to circular covalently closed DNA (cccDNA), (c) apoptosis and cell-cycle progression, and (d) HBx structural stability. METHODS: This study included 123 individuals chronically infected with HBV: 27 with HCC (77.9% (21/27) genotype D; 22.1% (6/27) genotype A) and 96 without HCC (75% (72/96) genotype D; 25.0% (24/96) genotype A). HepG2 cells were transfected by wild-type or mutated linear HBV genome to assess pre-genomic RNA (pgRNA) and core-associated HBV-DNA levels, HBx-binding onto cccDNA by chromatin immunoprecipitation-based quantitative assay, and rate of apoptosis and cell-cycle progression by cytofluorimetry. RESULTS:F30V was the only HBx mutation correlated with HCC (18.5% (5/27) in HCC patients versus 1.0% (1/96) in non-HCC patients, p 0.002); a result confirmed by multivariate analysis. In vitro, F30V determined a 40% and 60% reduction in pgRNA and core-associated HBV-DNA compared with wild-type (p <0.05), in parallel with a significant decrease of HBx binding to cccDNA and decreased HBx stability. F30V also decreased the percentage of apoptotic cells compared with wild-type (14.8 ± 6.8% versus 19.1 ± 10.1%, p <0.01, without affecting cell-cycle progression) and increased the probability of HBx-Ser-31 being phosphorylated by PI3K-Akt kinase (known to promote anti-apoptotic activity). CONCLUSIONS:F30V was closely correlated with HBV-induced HCC in vivo, reduced HBV replicative efficiency by affecting HBx-binding to cccDNA and increased anti-apoptotic HBx activity in vitro. This suggests that F30V (although hampering HBV's replicative capacity) may promote hepatocyte survival, so potentially allowing persistent production of viral progeny and initiating HBV-driven hepatocarcinogenesis. Investigation of viral genetic markers associated with HCC is crucial to identify those patients at higher risk of HCC, who hence deserve intensive liver monitoring and/or early anti-HBV therapy.