Screening And Identification Of Genes Involved In Control Of HBV Replication In Patients With Chronic Hepatitis B Virus (HBV) Infection

Part Ⅰ Objective The objective of the current study was to identify the differential intrahepatic host genes influence on the regulating of HBV replication in the liver of chronically HBV-infected (CHB) patients. Methods:Affymetrix RNA chip analysis was used to analyse the gene expression profiles of different clinical stages of chronic HBV infection in liver tissue.83CHB patients were included in the present study and divided into5groups:immune tolerant phase (n=22), immune clearance phase (HBeAg-positive chronic hepatitis B)(n=25), immune activation (HBeAg negative chronic hepatitis B)(n=25), inactive HBsAg carrier (n=11) and healthy control(n=6). Diagnostic criteria in accordance with the Chinese Medical Association in2005about "Chinese hepatitis B prevention and treatment guidelines in the diagnosis chronic HBV infection". In order to exclude the influence of immune factors, we mainly concentrate on the gene expression profiles between immune tolerant group and inactive carrier group. Results:There were statistically109differentially expressed genes in inactive carrier group compared with the immune tolerant phase (p<0.01); among them,54genes significantly up-regulated and55genes were down-regulated respectively. Conclusion:109cellular genes expression might correlate with HBV replication and reflect the immune response to HBV infection. The analysis of different clinical stages of CHB with liver RNA chip results showed that the expression of109genes were significantly different.Part Ⅱ The differentially expressed genes effect on HBV replication and gene expression were validated in vitro in hepatoma cell lines by specific siRNAs knock down. Methods:HepG2.2.15in hepatoma cell lines stably expressing HBV were transfected with109gene-specific small interfering RNA, and southern blot detect the intracellular HBV replication intermediates, the CMIA assay detect the HBV antigen secretion in the supernatant of cells. Moreover, Huh7hepatoma cell line were transiently contransfected with specific siRNA and replication competent HBV clone, and southern blot test the HBV relication, CMIA assay detect the HBsAg and HBeAg antigen secretion. Results:HBV replication in HepG2.2.15or Huh7were significantly enhanced or inhibited after silencing some of these genes. Conclusion:Knock down some of these109genes with specific small interfering RNA could influence the HBV replication markedly in hepatoma cell lines in vitro. More importantly, some of selected genes effect on HBV replication were identified for the first time.Part Ⅲ We selected4genes named FAM176A, HIGD1A, MARVELD3and TOMM34which showed strong effect on the HBV replication for further study and verified their effect on the HBV replication or antigen secretion and the possible mechanism on the control of HBV replication in details. Methods:The siRNA targeting the FAM176A, HIGD1A, MARVELD3and TOMM34were transfected to HepG2.2.15cells. Additionally, over-expression of these genes with plasmids cotranfected with HBV replication competent clone in Huh7cells. Intracellular HBV replication intermediates were detected by southern blot. The expression of HBsAg and HBeAg antigens were detected by CMIA test. Intracellular HBcAg protein expression after siRNA transfection was detected by western blot. Knock down of these genes effect on HBV promoter activities were evaluated by luciferse reporter assay. Results: The intrahepatic FAM176A、 HIGD1A and TOMM34level in CHB patients had a negative correlation with the patients HBV DNA titers, but the MARVELD3level in the liver was positive correlated with HBV DNA titers. It was noted that these genes knocking down resulted in a marked increase of HBV replication, accompanied with up-regulated antigen expression, and progeny secretion in HepG2.2.15cells. Consistently, Overexpression of these genes could significantly inhibit the HBV replication or antigen secretion in HBV plasmid co-transfected Huh7cells. Knocking down of MARVELD3by specific siRNA could enhance the HBV core and X promoter activity in HepG2.2.15hepatoma cells. Conclusion:We found that the4genes intrahepatic expression level in CHB patients had a significant correlation with the serum HBV DNA titers. Our results demonstrated that these differentially expressed intrahepatic genes might control HBV replication and their expression level might reflect the different outcomes of HBV infection. It will be useful for studying host-virus interactions in the pathogenesis of HBV infection and providing novel perspectives on development of therapeutic strategies in counteracting chronic HBV infection.